http://snltranscripts.jt.org/76/76ocarter.phtml
Saturday Night Live Transcripts
Season 2: Episode 15
76o: Sissy Spacek / Richard Baskin Ask President Carter Walter Cronkite.....Bill MurrayMrs. Edward Horbath (on phone).....Jane CurtinPresident Jimmy Carter.....Dan AykroydDr. Midnight (on phone).....Garrett MorrisPeter (on phone).....Tom Davis
Announcer: And now, live from the White House, "Ask President Carter". Walter Cronkite: Good evening, and welcome to #10 of the CBS radio series "Ask President Carter", continuing an experiment in presidential communication with the American people. Mr. President, our first call this week comes Mrs. Edward Hobrath, or maybe it's Horbraithe?Mrs. Edward Horbath (on phone): [ echoing ] That's Horbath.Walter Cronkite: Mrs. Edward Horbath - excuse me - of Maple Trace, Kansas!President Jimmy Carter: Hello, Mrs. Horbath.Mrs. Edward Horbath (on phone): [ echoing ] Hello, Mr. President, how are you today?President Jimmy Carter: Very fine. Could you turn your radio down, please?Mrs. Edward Horbath (on phone): [ echoing ] Oh my.. yes..Walter Cronkite: Mrs. Horbath, do you have a question for the President?Mrs. Edward Horbath (on phone): Yes, sir. I'm an employee of the U.S. Postal Service in Kansas. Last year they installed an automated letter sorting system called the Marvex-3000, here in our branch..President Jimmy Carter: Yes.Mrs. Edward Horbath (on phone): ..but the system doesn't work too good. Letters keep getting clogged in the first-level sorting grid. Is there anything that can be done about this?President Jimmy Carter: Well, Mrs. Horbath, Vice-President Mondale and I were just talking about the Morvex-3000 this morning.. uh.. I do have a suggestion - you know the caliber poised on the first grid sliding armiture?Mrs. Edward Horbath (on phone): Yes.President Jimmy Carter: Okay, there's a three-digit setting there, where the post and the armiture meet. Now, when the system was installed, the angle of cross-slide was put at a maximum setting of 1.. if you reset it at the three-mark like it says in the assembly instructions, I think it will solve any clogging problems in the machine.Mrs. Edward Horbath (on phone): Oh, thanks, Mr. President! Oh, by the way, I think you're doing a great job!Walter Cronkite: Looks like you've been doing your homework, Mr. President. I'd like to take this opportunity to say that none of these calls are screened, that the people of America are talking directly with the President. Our next call comes from a man who calls himself Dr. Midnight.President Jimmy Carter: Hello, Dr. Midnight.Dr. Midnight (on phone): Is Rosalyn there! I really like her!Walter Cronkite: Alright, sir, thank you very much for calling..Dr. Midnight (on phone): Hey, Cronkite! You stupid motherfu..[ Carter jumps out of his chair to disconnect Dr. Midnight's call ]Walter Cronkite: Thank you, Mr. President, ha ha! Our next call is Peter Elkin of Westbrook, Oregan, whom I am told is 17 years of age.Peter (on phone): Hello? Hello?President Jimmy Carter: Yes. Hello, Peter?Peter (on phone): Is this the President?President Jimmy Carter: Yes, it is.Walter Cronkite: Do you have a question for the President?Peter (on phone): Uh.. I, uh.. I took some acid.. I'm afraid to leave my apartment, and I can't wear any clothes.. and the ceiling is dripping, and uh.. I, uh..Walter Cronkite: Well, thank you very much for calling, sir..President Jimmy Carter: Just a minute, Walter, this guy's in trouble. I think I better try to talk him down. Peter?Peter (on phone): Yeah..?President Jimmy Carter: Peter, what did the acid look like?Peter (on phone): They were these little orange pills. President Jimmy Carter: Were they barrel shaped?Peter (on phone): Uh.. yes. President Jimmy Carter: Okay, right, you did some orange sunshine, Peter. Peter (on phone): Very good of you to know that, sir. President Jimmy Carter: How long ago did you take it, Peter? Peter (on phone): Uh.. I don't know. I can't read my watch. President Jimmy Carter: Alright, Peter, just listen. Everything is going to be fine. You're very high right now. You will probably be that way for about five more hours. Try taking some vitamin B complex, vitamin C complex.. if you have a beer, go ahead and drink it..Peter (on phone): Okay..President Jimmy Carter: Just remember you're a living organism on this planet, and you're very safe. You've just taken a heavy drug. Relax, stay inside and listen to some music, Okay? Do you have any Allman Brothers?Peter (on phone): Yes, I do, sir. Everything is okay, huh Jimmy?President Jimmy Carter: It sure is, Peter. You know, I'm against drug use myself, but I'm not going to lay that on you right now. Just mellow out the best you can, okay?Peter (on phone): Okay..!President Jimmy Carter: Okay.Walter Cronkite: Well, thank you, Mr. President. Our time is up for this week, but let me remind you that it is now time to buy your tickets for the first annual "I Slept At The White House" lottery, on sale at federal office buildings everywhere.President Jimmy Carter: I figure there's no harm in trying, Walt. The tickets are only a dollar, and maybe someone out there will win an all-expense paid trip to spend the night here with us in Washington. Good night!Walter Cronkite: Good night, thank you very much.
Wednesday
Scientific Proof That Clubbing Rats Have Lousy Sex Lives
http://www.synthtopia.com/content/2008/12/22/scientific-proof-that-clubbing-rats-have-lousy-sex-lives/
22Dec08
Scientific Proof That Clubbing Rats Have Lousy Sex Lives(Synthtopia)
Earlier in the year, we reported on Italians scientists that were studying the clubbing behavior rats on ecstasy.
At the time, we thought that Italy was the place to be if you got reincarnated as a lab rat.
But those Italians scientists didn't stop with giving rats ecstasy and exposing them to music at club noise levels.
Somehow, they got the funds to do additional research and this time they studied the sex lives of clubbing rats.
And it turns out that raving rats, at least in Italy, have pretty lousy sex lives. In fact, a single dose of ecstasy, in combination with loud music, "notably impaired copulatory behavior of sexually experienced male rats".
In fact, "combined treatment of MDMA and music stimulation did not fully restore normal sexual behavior as the animals reaching ejaculation still showed a marked reduction of copulatory efficiency."
So, if you're worried about your copulatory efficiency, and place any weight on scientific studies like this, you'll want to avoid the "combined treatment of MDMA and music stimulation."
Here's the details of the study:
----
http://www.ncbi.nlm.nih.gov/pubmed/19024211?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
Eur Rev Med Pharmacol Sci. 2008 Sep-Oct;12(5):285-92.
Effects on rat sexual behaviour of acute MDMA (ecstasy) alone or in combination with loud music.
Cagiano R, Bera I, Sabatini R, Flace P, Vermesan D, Vermesan H, Dragulescu SI, Bottalico L, Santacroce L.
Abstract
The effects on sexual behaviour of acute low doses of methylendioxymethamphetamine (MDMA) (0.3, 1, 3 mg/kg/i.p.), alone or in combination with exposure to loud music (1 h stimulation), were investigated in Wistar rats. Results indicate that acute MDMA, at dose of 3 mg/kg, notably impaired copulatory behavior of sexually experienced male rats.
In particular, MDMA-exposed animals exhibited a significant increase in intromission and ejaculation latencies as well as a significant decrease in percentage of rats displaying copulatory activity (one intromission at least).
Surprisingly, one hour exposure to loud music, which per se resulted ineffective, antagonized the suppressive effect of MDMA by increasing the percent of animals displaying sexual activity.
However, combined treatment of MDMA and music stimulation did not fully restore normal sexual behavior as the animals reaching ejaculation still showed a marked reduction of copulatory efficiency.
These findings demonstrate that the systemic administration of a single low dose of MDMA, alone or in combination with loud music, which is commonly present in certain environments such as rave parties, notably impairs copulatory activity of male rats.
22Dec08
Scientific Proof That Clubbing Rats Have Lousy Sex Lives(Synthtopia)
Earlier in the year, we reported on Italians scientists that were studying the clubbing behavior rats on ecstasy.
At the time, we thought that Italy was the place to be if you got reincarnated as a lab rat.
But those Italians scientists didn't stop with giving rats ecstasy and exposing them to music at club noise levels.
Somehow, they got the funds to do additional research and this time they studied the sex lives of clubbing rats.
And it turns out that raving rats, at least in Italy, have pretty lousy sex lives. In fact, a single dose of ecstasy, in combination with loud music, "notably impaired copulatory behavior of sexually experienced male rats".
In fact, "combined treatment of MDMA and music stimulation did not fully restore normal sexual behavior as the animals reaching ejaculation still showed a marked reduction of copulatory efficiency."
So, if you're worried about your copulatory efficiency, and place any weight on scientific studies like this, you'll want to avoid the "combined treatment of MDMA and music stimulation."
Here's the details of the study:
----
http://www.ncbi.nlm.nih.gov/pubmed/19024211?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
Eur Rev Med Pharmacol Sci. 2008 Sep-Oct;12(5):285-92.
Effects on rat sexual behaviour of acute MDMA (ecstasy) alone or in combination with loud music.
Cagiano R, Bera I, Sabatini R, Flace P, Vermesan D, Vermesan H, Dragulescu SI, Bottalico L, Santacroce L.
Abstract
The effects on sexual behaviour of acute low doses of methylendioxymethamphetamine (MDMA) (0.3, 1, 3 mg/kg/i.p.), alone or in combination with exposure to loud music (1 h stimulation), were investigated in Wistar rats. Results indicate that acute MDMA, at dose of 3 mg/kg, notably impaired copulatory behavior of sexually experienced male rats.
In particular, MDMA-exposed animals exhibited a significant increase in intromission and ejaculation latencies as well as a significant decrease in percentage of rats displaying copulatory activity (one intromission at least).
Surprisingly, one hour exposure to loud music, which per se resulted ineffective, antagonized the suppressive effect of MDMA by increasing the percent of animals displaying sexual activity.
However, combined treatment of MDMA and music stimulation did not fully restore normal sexual behavior as the animals reaching ejaculation still showed a marked reduction of copulatory efficiency.
These findings demonstrate that the systemic administration of a single low dose of MDMA, alone or in combination with loud music, which is commonly present in certain environments such as rave parties, notably impairs copulatory activity of male rats.
Brain Boosters: How should we deal with cognitive-enhancing drugs?
http://www.stanfordalumni.org/news/magazine/2009/marapr/farm/news/greely.html
GREELY: Enhancement is no dirty word.
Courtesy Henry Greely
Here's a good way to stir people up: publish a commentary in a prominent science journal supporting the use—with safeguards—of prescription drugs such as Ritalin and Adderall as a way for healthy adults to improve brain functions, including concentration and memory. (Those two drugs are central nervous system stimulants commonly prescribed for attention deficit and hyperactivity disorders.)Law School professor Henry Greely was first author among the seven professors and scientists who collaborated on the article for Nature. He has gotten an earful since its December appearance, and he sat down with STANFORD for a question-and-answer follow-up. An abridged version of this Q&A appeared in the print version of the magazine.You’ve provoked quite a bit of attention. What’s your take on the overall reaction so far? Part of me thinks that any attention is good attention, because we think this is a really important issue. I have to admit I also sort of cringe sometimes when I look at my e-mail. The two most common reactions I’ve gotten are: “How much crack were you were smoking when you wrote this?” and “How much money did your friends in the pharma industry pay you to write this?”But at the same time, I’ve gotten some very thoughtful responses, including some pretty negative responses, from a couple of my friends and colleagues here at the University. It’s an issue that people care about. One of the reasons that we wrote this piece is we think people care about it more than they’ve thought about it. The other big reason is we think there’s more and more of this coming down the road, and we’re just not prepared as a society yet for how we should deal with good, safe, cognitively enhancing drugs that will almost certainly be available in the next 10 to 20 years.You acknowledge that a great deal is unknown about the benefits and the risks of cognitive-enhancing drugs, so why advocate for their wider use before more research is done?One of the things that has troubled me most about the reaction is that so many people have read that article as saying, “Let’s freely use Adderall and Ritalin.” That’s not our goal, that’s not our position, that’s not what we want. We do think that safety is a major issue, and particularly Adderall and Ritalin are not benign drugs. They may be useful; they are useful to people with certain illnesses. But they’ve got really dangerous side effects. One of the things we think most important is further research on what the effects of these kinds of drugs will be.And an important regulatory note here: the FDA rules drugs as safe and effective, so Adderall, Ritalin, Provigil (Modafinil) have all been declared by the FDA safe and effective. But it’s safe and effective for a particular use. So Adderall and Ritalin are safe and effective for whatever the FDA has approved them for—say ADHD—but the FDA has never said they’re safe and effective for various off-label uses. Yet once a drug is approved by the FDA for one use, a physician can prescribe it for any use whatsoever. If you’re talking about enhancement uses, you really want a better safety profile than if you’re talking about a drug being used for a serious disease.So, for example, metastatic pancreatic cancer, about the most serious disease you can have: 99 percent of people with that diagnosis are dead within a year. If we came up with a drug that killed half of them instantly and cured the other half, that’s a miracle drug. That’s safe and effective. If it also cures teenage acne, it’s not a safe and effective drug for teenage acne. If it improves your memory, it’s not a safe and effective drug for improving your memory. So, our regulatory system isn’t set up to look at or think about the enhancement uses of drugs that are approved for medical conditions. We think there needs to be more research and more work on safety. More drugs are going to come along, and some of them will have the characteristic that they not only make sick people better but they make healthy people still better. These drugs will be there, we need to think about how we should deal with them. One strong reaction is a knee-jerk “We should ban them”: that they’re cheating, that they’re unnatural, that they’re somehow wrong. The main point of this article is that we don’t agree with that. Enhancement is not a dirty word. I’m a teacher—my job is to enhance people. I’m a parent—my job, until they became teenagers, was to enhance my kids.We do enhancement all the time. Education is enhancement. And as a law professor, I’m not only teaching my students facts that are important to them, but ways of manipulating those facts, ways of dealing with them. That’s cognitive enhancement. And it only works if I actually change their brains. If you remember tomorrow anything I’ve said today, it will be because I’ve made physical or electro-chemical changes in the cells of your brain. It’s kind of a weird thought, but true. So why is it that we do enhancement by so many other ways, but if you start talking about doing it through drugs, suddenly it becomes evil?‘We like to think that if people are smarter and working better—their brains are working better—the world will be a better place.’Well, I think part of it is, drug is a dirty word. Somebody’s talked about our pharmacological Puritanism. It’s a very love-hate relationship, as all of us who enjoy a glass of wine know. I mean, our society is probably one of the biggest users of drugs that change mental states, and also one of the most negative toward them in this odd sort of way. Well, there’s some good reasons to be worried about drugs, and we’ve laid some of them out, particularly enhancing drugs: safeness, coercion and fairness. And those are appropriate concerns, but they’re not knee-jerk concerns. They’re not, “All enhancing drugs are bad in all circumstances at all times.” Right now, to the extent the public has thought about this issue at all, it’s kind of the knee-jerk “drugs are bad, enhancement is bad, let’s ignore it.” Not a good solution.And we have a pretty good idea that at Stanford, as at other universities, we’ve got kids out there who are buying, borrowing or stealing Adderall or Ritalin and using it to study. We’re largely ignoring that. What we should do about it is a tricky, difficult issue, which is in part why we’re ignoring it. But as more and more of these drugs come along, the ostrich strategy is going to be worse and worse.What sparked your initial interest in this topic and what have you been doing to investigate it? I’ve been working on ethical, legal and social issues in biosciences since about 1991, 92, starting with genetics. (I) started doing neuroscience work in 2002, and that’s where I plan to spend most of the rest of my career. I think it’s really fascinating, really hard, not going to be figured out in my lifetime and more important than genetics. Once I started doing neuroscience work, I started thinking about enhancement as one of the important issues. And I should note with the neuroscience side of things, there are really two different, icky, knee-jerk-producing kinds of enhancements. Drugs is one. The other one is neuroelectronic interfaces—plugging your brains into computers. Which I think has equally eerie, and maybe in the long run, bigger implications in terms of enhancement in the brain than the drugs do.You’re arguing that cognitive-enhancing drug use can’t be judged by the same standards as physically or sports-enhancing drug use. But won’t the concerns overlap and wouldn’t cognitive drugs substantially benefit many athletes?That’s a really good question that nobody else has ever asked. So what drug has been most abused—apart from alcohol—for the longest period of time in major league baseball? Yes, amphetamines. And that is mainly a mental thing, not mainly a physical thing at all.We didn’t want to get into the sports issue in part because we didn’t want to deal with all that baggage in this article. My own view is that sports enhancement should be looked at pretty much the same way I think cognitive enhancement should be looked at. There are issues of fairness, safety and coercion. These people who go on about the integrity of the game—you know, God didn’t say the bases are going to be 90 feet apart. These are rules; we change the rules from time to time. I’m old enough to remember when the pitching mound was lowered because the pitchers were too good. Carl Yastrzemski had the lowest league-leading batting average in history at .301. Same year that Bob Gibson had the lowest all-time ERA. And then they lowered the pitching mound. And in recent years they’ve brought in the fences. There’s not a platonic ideal despite what true lovers of baseball might believe about what baseball is. Look at the old gloves. Fielding is much better, in part because they’ve got better equipment. We enhance bats. We have rules about what you can enhance and what you can’t. And I think it is cheating to violate the rules—at least more than whatever the standards of the game are about how much you’re allowed to violate the rules. Holding happens on every play in football. Water polo apparently is incredibly dirty under the water. Baseball pitchers will do what they can. But I’m not advocating violating the rules. The reaction against drug-related enhancement in sports is strong and deep. I think it’s not entirely rational. It threatens to color the reaction to cognitive enhancement. Cognitive enhancement is different in a way that we state in the article. The world isn’t particularly better off if the weightlifting record is 402 kilograms instead of 400 kilograms. Or if the Turk wins the gold medal instead of the Iranian. Maybe it’s an academic bias, but we like to think that if people are smarter and working better—their brains are working better—the world will be a better place. We tried to avoid the analogy with sports because we don’t want people’s views of this to be so strongly colored by their views of sports. But I do think one can distinguish between the two in a number of different ways, the most important being that there’s the potential for real personal and social benefits from cognitive enhancement that is much, much weaker in terms of sports enhancement.As you weighed the pros and cons of your recommendations, what was most persuasive to you about the potential individual or social advantages?For me personally it’s the sense of inevitability. We’re going to have to confront this issue, and we haven’t even begun to think about it.In terms of the drugs themselves, I personally have a bit of this drug Puritanism. And yet, if this can help people do their job or lives their lives better, it’s very hard for me to see why we should try to stop them. We’ve got these scary scenarios out there—people using them so that they can be 24-7 machines. There’s also the possibility of the person who’s struggling in school, who has always wanted to graduate or always wanted to be a doctor, always wanted to be something else, who maybe wasn’t born with as much talent, or didn’t grow up with a family that strongly supported education or had to work early in addition to schooling. And we should be very reluctant, I think, to condemn without good reason people’s efforts to improve themselves, even if they involve drugs.The legal obstacles to implementing your recommendations are formidable, yes? Law’s a very practical subject. It really eschews seeking the best and is in reality a practice of muddling through and making do. And we could, I think, with fairly minor adjustments to the food and drug laws, muddle through dealing with enhancements. I think the FDA would have to modify its approach in drug approvals. I’d like to see some increased safety requirements and regulatory requirements for safety evidence. I think in an ideal world the main changes would be to the drug approval laws and to the scheduled substance laws. Podcast: Hank Greely on the Ethics of Cognitive-enhancing Drugs The second part makes it very complicated because the first part’s only federal. The second part is not just federal but every state’s laws, as we see with the continuing medical marijuana problems. California says it’s good, and the Feds say it’s bad. So, if given free rein, I would rewrite the regulatory regime in a way to make the regulation of these kinds of drugs, when used for enhancing purposes, more rational. It’s always a lot easier to get an administrative agency to change its regulations than to get Congress to change a statute.But I do think that some changes will be needed. I’m ultimately not pessimistic about whether we’ll get those changes. I certainly don’t believe we’ll get the legal changes we need as soon as we need them in perfect form. But we’re pretty good at muddling through, and if in fact—and this is of course the big if behind the whole subject—we produce drugs that have a significant cognitive-enhancing effect, we won’t be able to ignore these issues. And then the laws will have to adapt to them one way or the other. It is an article of faith more than anything proven by experience that we’re likely to adapt better the more we’ve talked about it and thought about it.
GREELY: Enhancement is no dirty word.
Courtesy Henry Greely
Here's a good way to stir people up: publish a commentary in a prominent science journal supporting the use—with safeguards—of prescription drugs such as Ritalin and Adderall as a way for healthy adults to improve brain functions, including concentration and memory. (Those two drugs are central nervous system stimulants commonly prescribed for attention deficit and hyperactivity disorders.)Law School professor Henry Greely was first author among the seven professors and scientists who collaborated on the article for Nature. He has gotten an earful since its December appearance, and he sat down with STANFORD for a question-and-answer follow-up. An abridged version of this Q&A appeared in the print version of the magazine.You’ve provoked quite a bit of attention. What’s your take on the overall reaction so far? Part of me thinks that any attention is good attention, because we think this is a really important issue. I have to admit I also sort of cringe sometimes when I look at my e-mail. The two most common reactions I’ve gotten are: “How much crack were you were smoking when you wrote this?” and “How much money did your friends in the pharma industry pay you to write this?”But at the same time, I’ve gotten some very thoughtful responses, including some pretty negative responses, from a couple of my friends and colleagues here at the University. It’s an issue that people care about. One of the reasons that we wrote this piece is we think people care about it more than they’ve thought about it. The other big reason is we think there’s more and more of this coming down the road, and we’re just not prepared as a society yet for how we should deal with good, safe, cognitively enhancing drugs that will almost certainly be available in the next 10 to 20 years.You acknowledge that a great deal is unknown about the benefits and the risks of cognitive-enhancing drugs, so why advocate for their wider use before more research is done?One of the things that has troubled me most about the reaction is that so many people have read that article as saying, “Let’s freely use Adderall and Ritalin.” That’s not our goal, that’s not our position, that’s not what we want. We do think that safety is a major issue, and particularly Adderall and Ritalin are not benign drugs. They may be useful; they are useful to people with certain illnesses. But they’ve got really dangerous side effects. One of the things we think most important is further research on what the effects of these kinds of drugs will be.And an important regulatory note here: the FDA rules drugs as safe and effective, so Adderall, Ritalin, Provigil (Modafinil) have all been declared by the FDA safe and effective. But it’s safe and effective for a particular use. So Adderall and Ritalin are safe and effective for whatever the FDA has approved them for—say ADHD—but the FDA has never said they’re safe and effective for various off-label uses. Yet once a drug is approved by the FDA for one use, a physician can prescribe it for any use whatsoever. If you’re talking about enhancement uses, you really want a better safety profile than if you’re talking about a drug being used for a serious disease.So, for example, metastatic pancreatic cancer, about the most serious disease you can have: 99 percent of people with that diagnosis are dead within a year. If we came up with a drug that killed half of them instantly and cured the other half, that’s a miracle drug. That’s safe and effective. If it also cures teenage acne, it’s not a safe and effective drug for teenage acne. If it improves your memory, it’s not a safe and effective drug for improving your memory. So, our regulatory system isn’t set up to look at or think about the enhancement uses of drugs that are approved for medical conditions. We think there needs to be more research and more work on safety. More drugs are going to come along, and some of them will have the characteristic that they not only make sick people better but they make healthy people still better. These drugs will be there, we need to think about how we should deal with them. One strong reaction is a knee-jerk “We should ban them”: that they’re cheating, that they’re unnatural, that they’re somehow wrong. The main point of this article is that we don’t agree with that. Enhancement is not a dirty word. I’m a teacher—my job is to enhance people. I’m a parent—my job, until they became teenagers, was to enhance my kids.We do enhancement all the time. Education is enhancement. And as a law professor, I’m not only teaching my students facts that are important to them, but ways of manipulating those facts, ways of dealing with them. That’s cognitive enhancement. And it only works if I actually change their brains. If you remember tomorrow anything I’ve said today, it will be because I’ve made physical or electro-chemical changes in the cells of your brain. It’s kind of a weird thought, but true. So why is it that we do enhancement by so many other ways, but if you start talking about doing it through drugs, suddenly it becomes evil?‘We like to think that if people are smarter and working better—their brains are working better—the world will be a better place.’Well, I think part of it is, drug is a dirty word. Somebody’s talked about our pharmacological Puritanism. It’s a very love-hate relationship, as all of us who enjoy a glass of wine know. I mean, our society is probably one of the biggest users of drugs that change mental states, and also one of the most negative toward them in this odd sort of way. Well, there’s some good reasons to be worried about drugs, and we’ve laid some of them out, particularly enhancing drugs: safeness, coercion and fairness. And those are appropriate concerns, but they’re not knee-jerk concerns. They’re not, “All enhancing drugs are bad in all circumstances at all times.” Right now, to the extent the public has thought about this issue at all, it’s kind of the knee-jerk “drugs are bad, enhancement is bad, let’s ignore it.” Not a good solution.And we have a pretty good idea that at Stanford, as at other universities, we’ve got kids out there who are buying, borrowing or stealing Adderall or Ritalin and using it to study. We’re largely ignoring that. What we should do about it is a tricky, difficult issue, which is in part why we’re ignoring it. But as more and more of these drugs come along, the ostrich strategy is going to be worse and worse.What sparked your initial interest in this topic and what have you been doing to investigate it? I’ve been working on ethical, legal and social issues in biosciences since about 1991, 92, starting with genetics. (I) started doing neuroscience work in 2002, and that’s where I plan to spend most of the rest of my career. I think it’s really fascinating, really hard, not going to be figured out in my lifetime and more important than genetics. Once I started doing neuroscience work, I started thinking about enhancement as one of the important issues. And I should note with the neuroscience side of things, there are really two different, icky, knee-jerk-producing kinds of enhancements. Drugs is one. The other one is neuroelectronic interfaces—plugging your brains into computers. Which I think has equally eerie, and maybe in the long run, bigger implications in terms of enhancement in the brain than the drugs do.You’re arguing that cognitive-enhancing drug use can’t be judged by the same standards as physically or sports-enhancing drug use. But won’t the concerns overlap and wouldn’t cognitive drugs substantially benefit many athletes?That’s a really good question that nobody else has ever asked. So what drug has been most abused—apart from alcohol—for the longest period of time in major league baseball? Yes, amphetamines. And that is mainly a mental thing, not mainly a physical thing at all.We didn’t want to get into the sports issue in part because we didn’t want to deal with all that baggage in this article. My own view is that sports enhancement should be looked at pretty much the same way I think cognitive enhancement should be looked at. There are issues of fairness, safety and coercion. These people who go on about the integrity of the game—you know, God didn’t say the bases are going to be 90 feet apart. These are rules; we change the rules from time to time. I’m old enough to remember when the pitching mound was lowered because the pitchers were too good. Carl Yastrzemski had the lowest league-leading batting average in history at .301. Same year that Bob Gibson had the lowest all-time ERA. And then they lowered the pitching mound. And in recent years they’ve brought in the fences. There’s not a platonic ideal despite what true lovers of baseball might believe about what baseball is. Look at the old gloves. Fielding is much better, in part because they’ve got better equipment. We enhance bats. We have rules about what you can enhance and what you can’t. And I think it is cheating to violate the rules—at least more than whatever the standards of the game are about how much you’re allowed to violate the rules. Holding happens on every play in football. Water polo apparently is incredibly dirty under the water. Baseball pitchers will do what they can. But I’m not advocating violating the rules. The reaction against drug-related enhancement in sports is strong and deep. I think it’s not entirely rational. It threatens to color the reaction to cognitive enhancement. Cognitive enhancement is different in a way that we state in the article. The world isn’t particularly better off if the weightlifting record is 402 kilograms instead of 400 kilograms. Or if the Turk wins the gold medal instead of the Iranian. Maybe it’s an academic bias, but we like to think that if people are smarter and working better—their brains are working better—the world will be a better place. We tried to avoid the analogy with sports because we don’t want people’s views of this to be so strongly colored by their views of sports. But I do think one can distinguish between the two in a number of different ways, the most important being that there’s the potential for real personal and social benefits from cognitive enhancement that is much, much weaker in terms of sports enhancement.As you weighed the pros and cons of your recommendations, what was most persuasive to you about the potential individual or social advantages?For me personally it’s the sense of inevitability. We’re going to have to confront this issue, and we haven’t even begun to think about it.In terms of the drugs themselves, I personally have a bit of this drug Puritanism. And yet, if this can help people do their job or lives their lives better, it’s very hard for me to see why we should try to stop them. We’ve got these scary scenarios out there—people using them so that they can be 24-7 machines. There’s also the possibility of the person who’s struggling in school, who has always wanted to graduate or always wanted to be a doctor, always wanted to be something else, who maybe wasn’t born with as much talent, or didn’t grow up with a family that strongly supported education or had to work early in addition to schooling. And we should be very reluctant, I think, to condemn without good reason people’s efforts to improve themselves, even if they involve drugs.The legal obstacles to implementing your recommendations are formidable, yes? Law’s a very practical subject. It really eschews seeking the best and is in reality a practice of muddling through and making do. And we could, I think, with fairly minor adjustments to the food and drug laws, muddle through dealing with enhancements. I think the FDA would have to modify its approach in drug approvals. I’d like to see some increased safety requirements and regulatory requirements for safety evidence. I think in an ideal world the main changes would be to the drug approval laws and to the scheduled substance laws. Podcast: Hank Greely on the Ethics of Cognitive-enhancing Drugs The second part makes it very complicated because the first part’s only federal. The second part is not just federal but every state’s laws, as we see with the continuing medical marijuana problems. California says it’s good, and the Feds say it’s bad. So, if given free rein, I would rewrite the regulatory regime in a way to make the regulation of these kinds of drugs, when used for enhancing purposes, more rational. It’s always a lot easier to get an administrative agency to change its regulations than to get Congress to change a statute.But I do think that some changes will be needed. I’m ultimately not pessimistic about whether we’ll get those changes. I certainly don’t believe we’ll get the legal changes we need as soon as we need them in perfect form. But we’re pretty good at muddling through, and if in fact—and this is of course the big if behind the whole subject—we produce drugs that have a significant cognitive-enhancing effect, we won’t be able to ignore these issues. And then the laws will have to adapt to them one way or the other. It is an article of faith more than anything proven by experience that we’re likely to adapt better the more we’ve talked about it and thought about it.
Reduced Hospitalizations for Acute Myocardial Infarction After Implementation of a Smoke-Free Ordinance
http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5751a1.htm (for data and editorial comments)
Reduced Hospitalizations for Acute Myocardial Infarction After Implementation of a Smoke-Free Ordinance --- City of Pueblo, Colorado, 2002--2006
Please note: An erratum has been published for this article. To view the erratum, please click here.
Exposure to secondhand smoke (SHS) has immediate adverse cardiovascular effects, and prolonged exposure can cause coronary heart disease (1). Nine studies have reported that laws making indoor workplaces and public places smoke-free were associated with rapid, sizeable reductions in hospitalizations for acute myocardial infarction (AMI) (2--7). However, most studies examined hospitalizations for 1 year or less after laws were implemented; thus, whether the observed effect was sustained over time was unknown. The Pueblo Heart Study examined the impact of a municipal smoke-free ordinance in the city of Pueblo, Colorado, that took effect on July 1, 2003 (3). The rate of AMI hospitalizations for city residents decreased 27%, from 257 per 100,000 person-years during the 18 months before the ordinance's implementation to 187 during the 18 months after it (the Phase I post-implementation period).* This report extends that analysis for an additional 18 months through June 30, 2006 (the Phase II post-implementation period). The rate of AMI hospitalizations among city residents continued to decrease to 152 per 100,000 person-years, a decline of 19% and 41% from the Phase I post-implementation and pre-implementation period, respectively. No significant changes were observed in two comparison areas. These findings suggest that smoke-free policies can result in reductions in AMI hospitalizations that are sustained over a 3-year period and that these policies are important in preventing morbidity and mortality associated with heart disease. This effect likely is mediated through reduced SHS exposure among nonsmokers and reduced smoking, with the former making the larger contribution (4,6,7).
Two control sites were selected for comparison with the city of Pueblo: 1) the area of Pueblo County outside the city of Pueblo limits and 2) El Paso County, including Colorado Springs, the most populous city in this county. The city of Pueblo and Colorado Springs are located approximately 45 miles apart (Figure 1). Neither of the control sites had smoke-free laws in place before or during the study periods. Based on data from the Behavioral Risk Factor Surveillance System, the adult smoking prevalence for Pueblo County (including the city of Pueblo) and El Paso County during 2002--2003 was 25.9% (95% confidence interval [CI] = 20.2%--31.6%) and 17.4% (CI = 14.5%--20.2%), respectively. The corresponding prevalences for 2004--2005 were 20.6% (CI = 15.4%--25.8%) and 22.3% (CI = 19.3%--25.4%). Separate smoking prevalence estimates were not available for the city of Pueblo.
Persons with recognized AMIs that occur in the city of Pueblo and Pueblo County receive care at two hospitals, Parkview Medical Center and St. Mary-Corwin Medical Center, both located within the city of Pueblo. Persons with recognized AMIs that occur in El Paso County receive care at two other hospitals, Penrose Hospital and Memorial Hospital, both located in Colorado Springs. Data on AMI hospitalizations were drawn from electronic Colorado Hospital Association administrative data. These data included admission date, primary diagnosis code (based on International Classification of Diseases, Ninth Revision codes 410.0--410.9), sex, age, postal code of residence, and hospital name. No other patient-level data, including smoking status, were available. U.S. Census Bureau population data for 2006 were used as denominators in calculating AMI hospitalization rates. A more extensive description of the study's methodology has been published previously (3). AMI hospitalization rates among residents of the city of Pueblo, the area of Pueblo County outside the city of Pueblo limits, and El Paso County were compared across three periods: 0--18 months before the smoke-free law took effect (pre-implementation period), 0--18 months after this date (Phase I, post-implementation period), and 19--36 months after this date (Phase II, post-implementation period), for a total of 54 months. Rates were compared between periods using a chi-square test. Relative rates (RRs) were calculated as the ratios of AMI rates between two periods. Data presented in this report were not adjusted for seasonality because a season-adjusted analysis of Phase I versus the pre-implementation period found that the adjustment did not significantly change the findings (3).
During Phase II, AMI hospitalizations among residents of the city of Pueblo continued to decrease (Figure 2). AMI hospitalization rates differed significantly across all three periods within the city of Pueblo (p<0.001). ci =" 0.67--0.96)" href="http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5751a1.htm#tab">Table). In contrast, no significant change was observed for residents of the area of Pueblo County outside the city of Pueblo limits (139 per 100,000 person-years versus 115 per 100,000 person-years; RR = 1.21 [CI = 0.80--1.62]) or for residents of El Paso County (149 per 100,000 person-years versus 150 per 100,000 person-years; RR = 0.99 [CI = 0.91--1.08]) during the same period. The RR for AMI hospitalizations in the city of Pueblo in the Phase II post-implementation period compared with the pre-implementation period (rate = 257 per 100,000 person-years) was 0.59 (CI = 0.49--0.70). In contrast, RRs for the area of Pueblo County outside the city of Pueblo limits and for El Paso County for the same period were 1.03 (CI = 0.68--1.39) and 0.95 (CI = 0.87--1.03), respectively; the pre-implementation period rates were 135 per 100,000 person-years and 157 per 100,000 person-years, respectively. Within each site, the distribution of AMI patients by age and sex was unchanged over time.
To further examine whether the change in AMI rates could be attributed to pre-existing secular trends, AMI rates were examined for all three sites for three 18-month periods immediately preceding the pre-implementation phase. No statistically significant secular trend occurred in any of the three sites before July 1, 2003.
To ensure that the observed change in the city of Pueblo was not attributable to undercounting fatal AMIs post-implementation, the number of AMI deaths for the city of Pueblo were obtained from the Health Statistics Section of the Colorado Department of Public Health and Environment. After accounting for AMI deaths in a conservative manner (by assuming that all fatal AMIs occurred in patients who failed to reach the hospital) and adding these numbers to the hospital AMI admission data, the RR for the city of Pueblo remained statistically significant at 0.82 (CI = 0.64--0.97) from the Phase II to Phase I post-implementation periods and at 0.66 (CI = 0.55--0.77) from Phase II post-implementation to the pre-implementation period.
Reported by: RN Alsever, MD, Parkview Medical Center; WM Thomas, PhD, St. Mary-Corwin Medical Center; C Nevin-Woods, DO, R Beauvais, S Dennison, R Bueno, Pueblo City-County Health Dept; L Chang, PhD, Colorado State Univ-Pueblo; CE Bartecchi, MD, Univ of Colorado School of Medicine. S Babb, MPH, A Trosclair, MS, M Engstrom, MS, T Pechacek, PhD, R Kaufmann, PhD, Office on Smoking and Health, National Center for Chronic Disease Prevention and Health Promotion, CDC.
Reduced Hospitalizations for Acute Myocardial Infarction After Implementation of a Smoke-Free Ordinance --- City of Pueblo, Colorado, 2002--2006
Please note: An erratum has been published for this article. To view the erratum, please click here.
Exposure to secondhand smoke (SHS) has immediate adverse cardiovascular effects, and prolonged exposure can cause coronary heart disease (1). Nine studies have reported that laws making indoor workplaces and public places smoke-free were associated with rapid, sizeable reductions in hospitalizations for acute myocardial infarction (AMI) (2--7). However, most studies examined hospitalizations for 1 year or less after laws were implemented; thus, whether the observed effect was sustained over time was unknown. The Pueblo Heart Study examined the impact of a municipal smoke-free ordinance in the city of Pueblo, Colorado, that took effect on July 1, 2003 (3). The rate of AMI hospitalizations for city residents decreased 27%, from 257 per 100,000 person-years during the 18 months before the ordinance's implementation to 187 during the 18 months after it (the Phase I post-implementation period).* This report extends that analysis for an additional 18 months through June 30, 2006 (the Phase II post-implementation period). The rate of AMI hospitalizations among city residents continued to decrease to 152 per 100,000 person-years, a decline of 19% and 41% from the Phase I post-implementation and pre-implementation period, respectively. No significant changes were observed in two comparison areas. These findings suggest that smoke-free policies can result in reductions in AMI hospitalizations that are sustained over a 3-year period and that these policies are important in preventing morbidity and mortality associated with heart disease. This effect likely is mediated through reduced SHS exposure among nonsmokers and reduced smoking, with the former making the larger contribution (4,6,7).
Two control sites were selected for comparison with the city of Pueblo: 1) the area of Pueblo County outside the city of Pueblo limits and 2) El Paso County, including Colorado Springs, the most populous city in this county. The city of Pueblo and Colorado Springs are located approximately 45 miles apart (Figure 1). Neither of the control sites had smoke-free laws in place before or during the study periods. Based on data from the Behavioral Risk Factor Surveillance System, the adult smoking prevalence for Pueblo County (including the city of Pueblo) and El Paso County during 2002--2003 was 25.9% (95% confidence interval [CI] = 20.2%--31.6%) and 17.4% (CI = 14.5%--20.2%), respectively. The corresponding prevalences for 2004--2005 were 20.6% (CI = 15.4%--25.8%) and 22.3% (CI = 19.3%--25.4%). Separate smoking prevalence estimates were not available for the city of Pueblo.
Persons with recognized AMIs that occur in the city of Pueblo and Pueblo County receive care at two hospitals, Parkview Medical Center and St. Mary-Corwin Medical Center, both located within the city of Pueblo. Persons with recognized AMIs that occur in El Paso County receive care at two other hospitals, Penrose Hospital and Memorial Hospital, both located in Colorado Springs. Data on AMI hospitalizations were drawn from electronic Colorado Hospital Association administrative data. These data included admission date, primary diagnosis code (based on International Classification of Diseases, Ninth Revision codes 410.0--410.9), sex, age, postal code of residence, and hospital name. No other patient-level data, including smoking status, were available. U.S. Census Bureau population data for 2006 were used as denominators in calculating AMI hospitalization rates. A more extensive description of the study's methodology has been published previously (3). AMI hospitalization rates among residents of the city of Pueblo, the area of Pueblo County outside the city of Pueblo limits, and El Paso County were compared across three periods: 0--18 months before the smoke-free law took effect (pre-implementation period), 0--18 months after this date (Phase I, post-implementation period), and 19--36 months after this date (Phase II, post-implementation period), for a total of 54 months. Rates were compared between periods using a chi-square test. Relative rates (RRs) were calculated as the ratios of AMI rates between two periods. Data presented in this report were not adjusted for seasonality because a season-adjusted analysis of Phase I versus the pre-implementation period found that the adjustment did not significantly change the findings (3).
During Phase II, AMI hospitalizations among residents of the city of Pueblo continued to decrease (Figure 2). AMI hospitalization rates differed significantly across all three periods within the city of Pueblo (p<0.001). ci =" 0.67--0.96)" href="http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5751a1.htm#tab">Table). In contrast, no significant change was observed for residents of the area of Pueblo County outside the city of Pueblo limits (139 per 100,000 person-years versus 115 per 100,000 person-years; RR = 1.21 [CI = 0.80--1.62]) or for residents of El Paso County (149 per 100,000 person-years versus 150 per 100,000 person-years; RR = 0.99 [CI = 0.91--1.08]) during the same period. The RR for AMI hospitalizations in the city of Pueblo in the Phase II post-implementation period compared with the pre-implementation period (rate = 257 per 100,000 person-years) was 0.59 (CI = 0.49--0.70). In contrast, RRs for the area of Pueblo County outside the city of Pueblo limits and for El Paso County for the same period were 1.03 (CI = 0.68--1.39) and 0.95 (CI = 0.87--1.03), respectively; the pre-implementation period rates were 135 per 100,000 person-years and 157 per 100,000 person-years, respectively. Within each site, the distribution of AMI patients by age and sex was unchanged over time.
To further examine whether the change in AMI rates could be attributed to pre-existing secular trends, AMI rates were examined for all three sites for three 18-month periods immediately preceding the pre-implementation phase. No statistically significant secular trend occurred in any of the three sites before July 1, 2003.
To ensure that the observed change in the city of Pueblo was not attributable to undercounting fatal AMIs post-implementation, the number of AMI deaths for the city of Pueblo were obtained from the Health Statistics Section of the Colorado Department of Public Health and Environment. After accounting for AMI deaths in a conservative manner (by assuming that all fatal AMIs occurred in patients who failed to reach the hospital) and adding these numbers to the hospital AMI admission data, the RR for the city of Pueblo remained statistically significant at 0.82 (CI = 0.64--0.97) from the Phase II to Phase I post-implementation periods and at 0.66 (CI = 0.55--0.77) from Phase II post-implementation to the pre-implementation period.
Reported by: RN Alsever, MD, Parkview Medical Center; WM Thomas, PhD, St. Mary-Corwin Medical Center; C Nevin-Woods, DO, R Beauvais, S Dennison, R Bueno, Pueblo City-County Health Dept; L Chang, PhD, Colorado State Univ-Pueblo; CE Bartecchi, MD, Univ of Colorado School of Medicine. S Babb, MPH, A Trosclair, MS, M Engstrom, MS, T Pechacek, PhD, R Kaufmann, PhD, Office on Smoking and Health, National Center for Chronic Disease Prevention and Health Promotion, CDC.
Ozone Shots As Effective As Surgery for Back Pain
http://www.medpagetoday.com/MeetingCoverage/SIR/13206?utm_source=WC&utm_medium=email&utm_campaign=Meeting_Roundup_SIR
Ozone Shots As Effective As Surgery for Back Pain
By Crystal Phend, Staff Writer, MedPage
-- Ozone gas injected into herniated disks may relieve back-related pain and disability without the risks of surgery, researchers said. The improvements were well above clinical significance thresholds and comparable to surgical discectomy, Kieran J. Murphy, M.D., of the University of Toronto, and colleagues reported to the Society of Interventional Radiology here. In the meta-analysis, 79.7% of ozone injection patients improved at least one category on the modified McNab composite pain and functional scale. The investigational treatment, appears to be a gentler way to take pressure off nerves pinched by the bulging disk, Dr. Murphy said.
Action Points --->
Explain to interested patients that ozone injections are not FDA approved for treatment of back pain and that further study is needed to confirm efficacy.
Note that these studies were published as abstracts and presented orally at a conference. These data and conclusions should be considered to be preliminary until published in a peer-reviewed journal.
In the group's experimental and animal studies, also reported here, a tiny drop in disk volume (0.3 cc), which occurred when ozone broke protein down into water and carbon dioxide, eliminated one-third of pressure on the disk (10 psi).
"If open surgery is one option and doing nothing is another option, this fits in the gap between," Dr. Murphy said.
Although Dr. Murphy recently invented a device to deliver a consistent dose (2% ozone-to-oxygen) to the spine with a 22-gauge needle, Italian researchers had pioneered the technique.
Finding the European results difficult to believe, Dr. Murphy said he started to investigate for himself.
In pigs, his group's experiments showed that muscle was unaffected by any level of ozone, whereas a 2% ozone-in-oxygen injection appeared to be optimal both for cytokine levels and effect on disk volume.
Chinese hamster ovary cell lines confirmed that ozone, not oxygen, broke down proteins found in the nucleus pulposus of the disk.
To determine ozone's effect on human spines, the group reviewed the literature and pooled data from 12 studies with a total of more than 8,000 patients who had been treated with ozone for back pain at multiple European centers.
The average improvement -- with study results weighted by study quality -- was 3.9 on the 10-cm Visual Analog Scale for pain (95% CI 3.2 to 4.5) and 25.7 on the 50-point Oswestry Disability Index (95% CI 18.8 to 32.6).
The procedure was "extremely safe," added Dr. Murphy. The complication risk with ozone injection was 0.003% (95% confidence interval 0.000% to 0.024%), much lower than surgical discectomy, he said. No serious complications were reported.
"In addition, the recovery time is significantly shorter for the oxygen/ozone injection than for the discectomy," Dr. Murphy added.
The meta-analysis did reveal significant bias on the order of less than 10% in the pain and disability findings, but the smaller studies also appeared to underestimate the treatment effect.
"The estimated improvement in pain and function is impressive in light of the broad patient inclusion criteria that included patients ranging in age from 13 to 94 with all types of disk herniations," he said.
These promising findings need confirmation in U.S. as well, commented SIR president Brian Stainken, M.D., of Roger Williams Hospital in Providence, R.I.
"The solutions we have out there for this aren't very good," he said at the press conference he moderated. "We need minimally invasive targeted therapies."
Dr. Murphy said his group is negotiating with the FDA over the design of the first such North American human trial, which he expects to begin in the next six months.
Dr. Murphy reported being inventor of an ozone spinal delivery device. Dr. Stainken reported no conflicts of interest.
Primary source: Society of Interventional RadiologySource reference:Steppan J, et al "A meta-analysis of the effectiveness and safety of ozone treatments for herniated lumbar discs" SIR 2009; Abstract 37. Additional source: Society of Interventional RadiologySource reference:Steppan J, et al "Ozone's mechanisms of action for relieving pain associated with herniated intervertebral discs" SIR 2009; Abstract 38. Related Article(s):
Surgery Eases Pain of Spinal Stenoses and Slipped Vertebra, but Sciatica Outcomes Similar
Ozone Shots As Effective As Surgery for Back Pain
By Crystal Phend, Staff Writer, MedPage
-- Ozone gas injected into herniated disks may relieve back-related pain and disability without the risks of surgery, researchers said. The improvements were well above clinical significance thresholds and comparable to surgical discectomy, Kieran J. Murphy, M.D., of the University of Toronto, and colleagues reported to the Society of Interventional Radiology here. In the meta-analysis, 79.7% of ozone injection patients improved at least one category on the modified McNab composite pain and functional scale. The investigational treatment, appears to be a gentler way to take pressure off nerves pinched by the bulging disk, Dr. Murphy said.
Action Points --->
Explain to interested patients that ozone injections are not FDA approved for treatment of back pain and that further study is needed to confirm efficacy.
Note that these studies were published as abstracts and presented orally at a conference. These data and conclusions should be considered to be preliminary until published in a peer-reviewed journal.
In the group's experimental and animal studies, also reported here, a tiny drop in disk volume (0.3 cc), which occurred when ozone broke protein down into water and carbon dioxide, eliminated one-third of pressure on the disk (10 psi).
"If open surgery is one option and doing nothing is another option, this fits in the gap between," Dr. Murphy said.
Although Dr. Murphy recently invented a device to deliver a consistent dose (2% ozone-to-oxygen) to the spine with a 22-gauge needle, Italian researchers had pioneered the technique.
Finding the European results difficult to believe, Dr. Murphy said he started to investigate for himself.
In pigs, his group's experiments showed that muscle was unaffected by any level of ozone, whereas a 2% ozone-in-oxygen injection appeared to be optimal both for cytokine levels and effect on disk volume.
Chinese hamster ovary cell lines confirmed that ozone, not oxygen, broke down proteins found in the nucleus pulposus of the disk.
To determine ozone's effect on human spines, the group reviewed the literature and pooled data from 12 studies with a total of more than 8,000 patients who had been treated with ozone for back pain at multiple European centers.
The average improvement -- with study results weighted by study quality -- was 3.9 on the 10-cm Visual Analog Scale for pain (95% CI 3.2 to 4.5) and 25.7 on the 50-point Oswestry Disability Index (95% CI 18.8 to 32.6).
The procedure was "extremely safe," added Dr. Murphy. The complication risk with ozone injection was 0.003% (95% confidence interval 0.000% to 0.024%), much lower than surgical discectomy, he said. No serious complications were reported.
"In addition, the recovery time is significantly shorter for the oxygen/ozone injection than for the discectomy," Dr. Murphy added.
The meta-analysis did reveal significant bias on the order of less than 10% in the pain and disability findings, but the smaller studies also appeared to underestimate the treatment effect.
"The estimated improvement in pain and function is impressive in light of the broad patient inclusion criteria that included patients ranging in age from 13 to 94 with all types of disk herniations," he said.
These promising findings need confirmation in U.S. as well, commented SIR president Brian Stainken, M.D., of Roger Williams Hospital in Providence, R.I.
"The solutions we have out there for this aren't very good," he said at the press conference he moderated. "We need minimally invasive targeted therapies."
Dr. Murphy said his group is negotiating with the FDA over the design of the first such North American human trial, which he expects to begin in the next six months.
Dr. Murphy reported being inventor of an ozone spinal delivery device. Dr. Stainken reported no conflicts of interest.
Primary source: Society of Interventional RadiologySource reference:Steppan J, et al "A meta-analysis of the effectiveness and safety of ozone treatments for herniated lumbar discs" SIR 2009; Abstract 37. Additional source: Society of Interventional RadiologySource reference:Steppan J, et al "Ozone's mechanisms of action for relieving pain associated with herniated intervertebral discs" SIR 2009; Abstract 38. Related Article(s):
Surgery Eases Pain of Spinal Stenoses and Slipped Vertebra, but Sciatica Outcomes Similar
Mice Get Hooked on Light
http://www.technologyreview.com/biomedicine/22313/
Mice Get Hooked on Light:
Hybrid proteins that respond to light could reveal the biochemistry of addiction.
By Jocelyn Rice, Technology Review
A newly created set of light-sensitive proteins grants scientists unprecedented control over the brain's biochemistry, potentially shedding light on addiction and other complex neural processes. To demonstrate the potential of this novel molecular toolbox, researchers from Stanford University engineered mice to carry light-sensitive proteins in the brain's reward center, which responds to drugs of abuse. Using pulses of light delivered directly to the brain, researchers were able to induce a druglike state, ultimately conditioning the mice to behave like drug-addicted animals.
"Drug addiction is one of the leading causes of disability worldwide, and it all funnels through the reward system," says senior researcher Karl Deisseroth, a bioengineer and psychiatrist at Stanford, who frequently works with drug-addicted patients. Addiction is immensely difficult to treat, in part because drugs create such potent and stable changes in the brain's reward system. "If we can understand better what those internal states are and how they become so stable--which we started to scratch the surface of in this paper--maybe we can develop more effective and potent therapies for substance abuse," he says.
Researchers created hybrid proteins by fusing the gene for a light-sensing pigment normally found in the eye to the genes for various members of a family of receptor proteins. The hybrid proteins sit in the cell membranes of neurons, with the light-sensitive portion protruding from the cell; when it absorbs photons of a certain wavelength, it changes shape, triggering the intracellular portion of the protein to launch a cascade of biochemical reactions inside the cell.
Deisseroth's team had previously engineered light-sensitive proteins that triggered neurons to fire. But this new system is more specific: rather than stimulating the entire neuron, it targets individual signaling pathways within the cell. The researchers can control different pathways by selecting the intracellular domain from a diverse palette of existing signaling proteins and pairing it with the light-sensitive proteins. "This allows us now to access a whole new dimension of cell states and brain states that we couldn't before," says Deisseroth.
Researchers used a virus to deliver genes encoding the hybrid proteins, dubbed optoXRs, to the animals' nucleus accumbens, a brain region that responds to pleasurable stimuli, such as food, sex, and drugs. Each mouse was allowed to roam freely through a series of adjoining chambers, one of which was preselected as a reward room. Whenever the mouse entered the reward room, a researcher fired a pulse of light through a fiber-optic cable into its nucleus accumbens, setting off whatever biochemical cascade corresponded to the particular optoXR it carried.
This setup is commonly used to study addiction: when a drug such as cocaine or amphetamine is consistently administered in the reward room, a mouse learns to associate that room with the reward and later chooses to spend most of its time there.
"What we found, very strikingly, was that this worked," says Deisseroth. One of the optoXRs, built from a receptor protein that normally responds to adrenaline and noradrenaline, produced results much like those seen with drug-based rewards. When loosed after training, mice with this optoXR strongly preferred to spend time in the reward room, where they had received light pulses activating their nucleus accumbens. The results of the study were published this week in Nature.
Many of the same proteins that Deisseroth's team activated with light can be targeted by drugs. But light has a number of advantages over drugs, says Michael Häusser, a professor of neuroscience at University College London, who was not involved in the research. While drugs take time to work and linger after administration, light allows for exquisite control over timing. And while drugs don't pick and choose which cells to affect, optoXRs can be genetically engineered to be expressed in only a specific type of cell.
The hybrid proteins represent a new molecular toolbox with applications beyond drug addiction and the brain's reward system. "There are all kinds of really cool games you can play with these new molecular tools to look at aspects of signaling pathways and how they interact," says Häusser. For example, they open the door to studying more mysterious receptor proteins, which can't be activated pharmaceutically and whose function is not well understood. "For some of them," says Häusser, "our toolbox for manipulating them is limited. So this gives us a fantastic new handle on these receptor classes, and allows us to manipulate them in a really powerful way."
Mice Get Hooked on Light:
Hybrid proteins that respond to light could reveal the biochemistry of addiction.
By Jocelyn Rice, Technology Review
A newly created set of light-sensitive proteins grants scientists unprecedented control over the brain's biochemistry, potentially shedding light on addiction and other complex neural processes. To demonstrate the potential of this novel molecular toolbox, researchers from Stanford University engineered mice to carry light-sensitive proteins in the brain's reward center, which responds to drugs of abuse. Using pulses of light delivered directly to the brain, researchers were able to induce a druglike state, ultimately conditioning the mice to behave like drug-addicted animals.
"Drug addiction is one of the leading causes of disability worldwide, and it all funnels through the reward system," says senior researcher Karl Deisseroth, a bioengineer and psychiatrist at Stanford, who frequently works with drug-addicted patients. Addiction is immensely difficult to treat, in part because drugs create such potent and stable changes in the brain's reward system. "If we can understand better what those internal states are and how they become so stable--which we started to scratch the surface of in this paper--maybe we can develop more effective and potent therapies for substance abuse," he says.
Researchers created hybrid proteins by fusing the gene for a light-sensing pigment normally found in the eye to the genes for various members of a family of receptor proteins. The hybrid proteins sit in the cell membranes of neurons, with the light-sensitive portion protruding from the cell; when it absorbs photons of a certain wavelength, it changes shape, triggering the intracellular portion of the protein to launch a cascade of biochemical reactions inside the cell.
Deisseroth's team had previously engineered light-sensitive proteins that triggered neurons to fire. But this new system is more specific: rather than stimulating the entire neuron, it targets individual signaling pathways within the cell. The researchers can control different pathways by selecting the intracellular domain from a diverse palette of existing signaling proteins and pairing it with the light-sensitive proteins. "This allows us now to access a whole new dimension of cell states and brain states that we couldn't before," says Deisseroth.
Researchers used a virus to deliver genes encoding the hybrid proteins, dubbed optoXRs, to the animals' nucleus accumbens, a brain region that responds to pleasurable stimuli, such as food, sex, and drugs. Each mouse was allowed to roam freely through a series of adjoining chambers, one of which was preselected as a reward room. Whenever the mouse entered the reward room, a researcher fired a pulse of light through a fiber-optic cable into its nucleus accumbens, setting off whatever biochemical cascade corresponded to the particular optoXR it carried.
This setup is commonly used to study addiction: when a drug such as cocaine or amphetamine is consistently administered in the reward room, a mouse learns to associate that room with the reward and later chooses to spend most of its time there.
"What we found, very strikingly, was that this worked," says Deisseroth. One of the optoXRs, built from a receptor protein that normally responds to adrenaline and noradrenaline, produced results much like those seen with drug-based rewards. When loosed after training, mice with this optoXR strongly preferred to spend time in the reward room, where they had received light pulses activating their nucleus accumbens. The results of the study were published this week in Nature.
Many of the same proteins that Deisseroth's team activated with light can be targeted by drugs. But light has a number of advantages over drugs, says Michael Häusser, a professor of neuroscience at University College London, who was not involved in the research. While drugs take time to work and linger after administration, light allows for exquisite control over timing. And while drugs don't pick and choose which cells to affect, optoXRs can be genetically engineered to be expressed in only a specific type of cell.
The hybrid proteins represent a new molecular toolbox with applications beyond drug addiction and the brain's reward system. "There are all kinds of really cool games you can play with these new molecular tools to look at aspects of signaling pathways and how they interact," says Häusser. For example, they open the door to studying more mysterious receptor proteins, which can't be activated pharmaceutically and whose function is not well understood. "For some of them," says Häusser, "our toolbox for manipulating them is limited. So this gives us a fantastic new handle on these receptor classes, and allows us to manipulate them in a really powerful way."
Effects of Beverage Alcohol Price and Tax Levels on Drinking
http://www.rwjf.org/pr/product.jsp?id=37409
Effects of Beverage Alcohol Price and Tax Levels on Drinking
This resource summarizes the results of 112 studies that examined the effect of raising alcohol taxes on beverage consumption. The meta-analysis found that raising the cost of alcohol effectively reduces drinking across broad populations of drinkers.
Effects of Beverage Alcohol Price and Tax Levels on Drinking
This resource summarizes the results of 112 studies that examined the effect of raising alcohol taxes on beverage consumption. The meta-analysis found that raising the cost of alcohol effectively reduces drinking across broad populations of drinkers.
Drinking and Biting in Adult Humans
http://www.jointogether.org/news/research/summaries/2007/drinking-and-biting.html
<> 86 percent of human-bite cases involve alcohol, and that men are bitten 12 times more often than women. Most bites occur on the face -- particularly the ears, nose and cheek -- as well as the fingers and forearm. Sixty-five percent of all bites involve the ear.
Drinking and BitingJune 21, 2007
Research Summary
It's a problem Mike Tyson knows well: fights that escalate into men biting other men. And researchers say that most human-bite cases involve males who have been drinking, the CanWest News Service reported June 20.
Irish researchers say that 86 percent of human-bite cases involve alcohol, and that men are bitten 12 times more often than women. Most bites occur on the face -- particularly the ears, nose and cheek -- as well as the fingers and forearm. Sixty-five percent of all bites involve the ear.
"I think a lot of people wouldn't know this happens, or to the extent that it happens," said study co-author Patricia Eadie, a plastic surgeon at St. James's Hospital in Dublin. "There's a lot of person-on-person violence that can be due to alcohol and drugs." Many such incidents go unreported because victims don't seek medical attention, she added.
The study was published in the July 2007 issue of the Emergency Medicine Journal.-->
<
Drinking and BitingJune 21, 2007
Research Summary
It's a problem Mike Tyson knows well: fights that escalate into men biting other men. And researchers say that most human-bite cases involve males who have been drinking, the CanWest News Service reported June 20.
Irish researchers say that 86 percent of human-bite cases involve alcohol, and that men are bitten 12 times more often than women. Most bites occur on the face -- particularly the ears, nose and cheek -- as well as the fingers and forearm. Sixty-five percent of all bites involve the ear.
"I think a lot of people wouldn't know this happens, or to the extent that it happens," said study co-author Patricia Eadie, a plastic surgeon at St. James's Hospital in Dublin. "There's a lot of person-on-person violence that can be due to alcohol and drugs." Many such incidents go unreported because victims don't seek medical attention, she added.
The study was published in the July 2007 issue of the Emergency Medicine Journal.-->
Trends in Adolescent Inhalant Use: 2002 to 2007
http://oas.samhsa.gov/2k9/inhalantTrends/inhalantTrends.cfm
Trends in Adolescent Inhalant Use: 2002 to 2007
The NSDUH Report - - Trends in Adolescent Inhalant Use: 2002 to 2007
HTML format (508 compliant version - -also contains the data table that was used to construct each figure; this data table is not found in printed or PDF version)
PDF format (260 KB, recommended for printing)
Highlights:
SAMHSA's National Survey on Drug Use and Health (NSDUH) defines inhalants as "liquids, sprays, and gases that people sniff or inhale to get high or to make them feel good." In 2007, almost one million youth used inhalants in the past year.
The percentage of youths aged 12 to 17 who used inhalants in the past year was lower in 2007 (3.9%) than in 2003 (4.5%), 2004 (4.6%), and 2005 (4.5%).
Among youth who used inhalants for the first time in the past year, the rate of the use of nitrous oxide or "whippets" declined between 2002 and 2007 among both males (40.2% to 20.2%) and females (22.3% to 12.2%).
In 2007, 17.2% of youth who initiated illicit drug use during the past year indicated that inhalants were the first drug that they used; this rate remained relatively stable between 2002 and 2007.
Past year dependence on or abuse of inhalants remained relatively stable between 2002 and 2007 with around 99,000 youth meeting the criteria for dependence or abuse in 2007.
Reports on youth
Reports on inhalants
Other drugs
Other topics
Other OAS publications and services
This Short Report, The NSDUH Report - - Trends in Adolescent Inhalant Use: 2002 to 2007, is based on SAMHSA's National Survey on Drug Use and Health conducted by the Office of Applied Studies (OAS) in the Substance Abuse and Mental Health Services Administration (SAMHSA). SAMHSA's National Survey on Drug Use and Health (NSDUH) is the primary source of information on the prevalence, patterns, and consequences of drug and alcohol use and abuse and for selected mental health measures in the general U.S. civilian non institutionalized population, age 12 and older. SAMHSA's National Survey on Drug Use & Health also provides estimates for drug use and for selected mental health measures by State.
This page has been accessed 2380 times since 3/16/09.
This page was last updated on March 16, 2009.
SAMHSA, an agency in the Department of Health and Human Services, is the Federal Government's lead agency for improving the quality and availability of substance abuse prevention, addiction treatment, and mental health services in the United States.
Santo Diame wins in District Court
http://www.erowid.org/chemicals/ayahuasca/ayahuasca_law24.shtml
Santo Diame wins in District Court
On March 18, 2009, a U.S. District Court judge, Owen Panner, found that the
U.S. Regligious Freedom Restoration Act (RFRA) protects the Santo Daime's use
of DMT-containing ayahuasca as part of their sincere religious practices.
This U.S. District Court (the lowest type of U.S. federal court) based its
decision on a U.S. Supreme Court decision from February 2006 in a case where
the UDV (another ayahuasca-using church) sought a preliminary injunction to
stop the DEA from seizing the ayahuasca they import into the United States.
The Supreme Court granted that preliminary injunction in favor of the UDV.
Judge Panner writes, "Guided by the unanimous decision of the United States
Supreme Court in a very similar case, Gonzales v. O Centro Espirita
Beneficente Uniao de Vegetal, 546 U.S. 418 (2006), I conclude that the
Religious Freedom Restoration Act, 42 U.S.C. ¤ ¤ 2000bb to 2000bb-4, requires
that plaintiffs be allowed to import and drink Daime tea for their religious
ceremonies, subject to reasonable restrictions."
". . .Plaintiffs have established their prima facie claim by more than a
preponderance of the evidence. Plaintiffs have established that they are
sincere in their religious beliefs, and that the ceremonial use ofthe Daime
tea is essential to their religion. It is obvious that prohibiting the use of
Daime tea would substantially burden the exercise of plaintiffs'
religion. . ."
". . .The government has failed to show that outright prohibition of the Daime
tea is the least restrictive means of furthering its interests. . ."
". . .Plaintiffs are entitled to relief under RFRA. Judgment will be entered
for plaintiffs in accordance with this opinion."
Unless a higher court overturns this decision in the future, this ruling
allows the Santo Daime church to legally import and use ayahuasca tea in
their religious practices without being in violation of federal law.
(Full court decision also available on this site)
Santo Diame wins in District Court
On March 18, 2009, a U.S. District Court judge, Owen Panner, found that the
U.S. Regligious Freedom Restoration Act (RFRA) protects the Santo Daime's use
of DMT-containing ayahuasca as part of their sincere religious practices.
This U.S. District Court (the lowest type of U.S. federal court) based its
decision on a U.S. Supreme Court decision from February 2006 in a case where
the UDV (another ayahuasca-using church) sought a preliminary injunction to
stop the DEA from seizing the ayahuasca they import into the United States.
The Supreme Court granted that preliminary injunction in favor of the UDV.
Judge Panner writes, "Guided by the unanimous decision of the United States
Supreme Court in a very similar case, Gonzales v. O Centro Espirita
Beneficente Uniao de Vegetal, 546 U.S. 418 (2006), I conclude that the
Religious Freedom Restoration Act, 42 U.S.C. ¤ ¤ 2000bb to 2000bb-4, requires
that plaintiffs be allowed to import and drink Daime tea for their religious
ceremonies, subject to reasonable restrictions."
". . .Plaintiffs have established their prima facie claim by more than a
preponderance of the evidence. Plaintiffs have established that they are
sincere in their religious beliefs, and that the ceremonial use ofthe Daime
tea is essential to their religion. It is obvious that prohibiting the use of
Daime tea would substantially burden the exercise of plaintiffs'
religion. . ."
". . .The government has failed to show that outright prohibition of the Daime
tea is the least restrictive means of furthering its interests. . ."
". . .Plaintiffs are entitled to relief under RFRA. Judgment will be entered
for plaintiffs in accordance with this opinion."
Unless a higher court overturns this decision in the future, this ruling
allows the Santo Daime church to legally import and use ayahuasca tea in
their religious practices without being in violation of federal law.
(Full court decision also available on this site)
Environmental Enrichment During Early Stages of Life Reduces the Behavioral, Neurochemical, and Molecular Effects of
Neuropsychopharmacology (2009) 34, 1102–1111; doi:10.1038/npp.2008.51; published online 7 May 2008
Environmental Enrichment During Early Stages of Life Reduces the Behavioral, Neurochemical, and Molecular Effects of Cocaine
Marcello Solinas1, Nathalie Thiriet1, Rana El Rawas1, Virginie Lardeux1 and Mohamed Jaber1
1Institut de Physiologie et Biologie Cellulaires, University of Poitiers, CNRS, Poitiers, France
Correspondence: Professor M Jaber, Institut de Physiologie et Biologie Cellulaires, University of Poitiers, CNRS-6187, 40 avenue du recteur Pineau, Poitiers 86022, France. Tel: +33 (0)5 49 453985; Fax: +33 (0)5 49 454014; E-mail: mohamed.jaber@univ-poitiers.fr
Received 25 January 2008; Revised 4 March 2008; Accepted 5 March 2008; Published online 7 May 2008.
Abstract
It is known that negative environmental conditions increase vulnerability to drugs, whereas little is known on whether positive environmental conditions such as enriched environments (EE) have protective effects against addiction. We have previously found that EE consisting of bigger cages containing several toys that were changed once per week reduce cocaine-induced increases in locomotor activity. Here, we also show that the rewarding effects of cocaine are blunted in mice reared from weaning to adulthood in EE compared to mice reared in standard environments (SE). In addition, although both EE and SE mice develop behavioral sensitization to cocaine, EE mice show less activation in response to repeated administration of cocaine injections and reduced responses to cocaine challenges. In vivo microdialysis experiments demonstrate that the protective effects of EE do not depend on reduced cocaine-induced increases in the dopamine levels in the ventral or dorsal striatum. On the other hand, they were associated with reduced cocaine-induced expression of the immediate early gene zif-268 in the nucleus accumbens (shell and core) of EE mice. Finally, basal levels of Delta-Fos B, a transcription factor known to be increased by sustained activation of striatal neurons, are higher in the striatum of EE compared to SE mice and repeated administration of cocaine increases Delta-Fos B levels in SE mice but decreases them in EE mice. Altogether our results demonstrate that exposure to complex environments during early stages of life produce dramatic changes in the striatum that result in reduced reactivity to drugs of abuse.
Keywords:
addiction, enrichment, behavior, dopamine, nucleus accumbens, molecular biology
INTRODUCTION
Of the many people who experience psychoactive drugs, only a small percentage develops drug addiction. It is believed that environmental factors play a central role in determining sensitivity to the rewarding effects of drugs and vulnerability to develop addiction (Sinha, 2001; Goeders, 2002; Marinelli and Piazza, 2002; Kreek et al, 2005). For example, several studies have demonstrated that environmental stressors increase the unconditioned, conditioned, and reinforcing effects of drugs such as amphetamine, cocaine, and heroin (Sinha, 2001; Goeders, 2002; Marinelli and Piazza, 2002; Kreek et al, 2005). Preclinical studies have also provided important information on the mechanisms underlying this enhanced response to the effects of drugs and have shown that the dopamine system is implicated in this excessive reaction to drugs (Rouge-Pont et al, 1995; Goeders, 2002; Marinelli and Piazza, 2002).
The investigation of negative factors exacerbating addiction has been extensive, whereas considerably less attention has been dedicated to environmental manipulations that may mimic positive life experiences such as enrichment environments (EE) and may provide protection against drugs' effects. EE have been shown to increase learning and memory (van Praag et al, 2000), to facilitate recovery from brain injuries (Will et al, 2004), to reduce the insurgence of neurodegenerative diseases (Nithianantharajah and Hannan, 2006) and some authors have suggested that EE represent more natural and healthy environment than current standard housing conditions (Wurbel, 2001). In addition, some studies have shown that the reinforcing effects of amphetamine and nicotine are reduced in rats reared in EE compared to rats reared in social isolation (Bowling et al, 1993; Bardo et al, 2001; Green et al, 2002, 2003).
In a previous study, we have shown that mice reared in EE show less locomotor activity and a different pattern of c-fos activation in the striatum in response to an acute injection of cocaine compared to mice reared in a standard environments (SE) (Bezard et al, 2003). We also found that EE mice had less dopaminergic neurons in the substantia nigra pars compacta, lower levels of the dopamine transporter (DAT), the molecular target of cocaine, and higher levels of neurotrophin BDNF in the striatum (Bezard et al, 2003). These findings provided an initial demonstration that EE may be crucial in determining resistance to drugs of abuse such as cocaine. However, little information is available on the effects of EE on cocaine's rewarding properties, which are considered directly responsible for the development of drug addiction and on the behavioral and molecular consequences of repeated administration of cocaine.
In this study, we housed mice either in EE or SE from weaning to adulthood and we used (1) conditioned place preferences (Cunningham et al, 2006) to assess the rewarding effects of cocaine; (2) behavioral sensitization, an animal model for the intensification of drug craving and relapse believed to underlie addiction in humans (Vanderschuren and Kalivas, 2000; Robinson and Berridge, 2003; Bradberry, 2007), to assess the consequences of repeated exposure to cocaine; (3) in vivo microdialysis techniques to assess the response to cocaine of the dopamine mesolimbic system at the presynaptic level (Di Chiara and Bassareo, 2007); (4) in situ hybridization techniques to assess the reactivity to cocaine of the dopamine mesolimbic system at the postsynaptic level by measuring expression of immediate early genes (Hope, 1998; Berke and Hyman, 2000; Gerfen, 2000) and (5) western blot techniques to assess whether the differences observed in the behavioral effects of cocaine could be due to changes in the levels of Delta-Fos B, a transcription factor that is implicated in long-term neuroadaptations associated with drug addiction (McClung et al, 2004).
MATERIALS AND METHODS
Subjects
Male C57Bl/6J mice (Janvier, France) were housed in a temperature-controlled environment on a 12-h light/dark cycle with the lights on from 0700 to 1900 hours and had ad libitum access to food and water. All experimentation was conducted during the light period. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for the care of laboratory animals.
Housing Environmental Conditions
After weaning (3 weeks of age), mice were randomly divided in two different housing environmental conditions: SE or EE. SE consisted of common cage housing (25 20 15 cm). The EE consisted of larger (60 38 20 cm) cages containing a constantly running wheel and a small house and four-five toys that were changed once a week with new toys of different shape and color. For both SE and EE conditions, mice were housed in groups of four (each mouse from a different sibling) for at least 2 months and maximum 3 months before the start of the experiments. All the experiments were conducted at adulthood.
Conditioned Place Preference
Conditioned place preference experiments were performed in four identical boxes (IMETRONIC, Pessac, France) formed by two lateral chambers (15 15 20 cm) connected by a central alley (5 15 20 cm). Two sliding doors separated the alley from the chambers. In each chamber, two Plexiglas prisms with triangular bases (5 7 19 cm) were arranged to form different patterns (always covering the same surface of the chamber) and were used as conditioned stimuli. Two different metallic grids, one with large (1 cm) squares and the other with small (0.5 cm) circles were also used as conditioned stimuli. Behavioral data were collected by an Imetronic interface connected to a PC. Two infrared photocells were present in each compartment and detected the presence and movements of mice. Conditioned place preference procedure consisted of three phases: preconditioning, conditioning, and test. For each manipulation, mice were brought to the experimental room 60 min before the start of the experiment to allow for habituation and to reduce stress. Preconditioning was performed on day 1. Mice were placed in the central compartment with the doors closed. After 15 s, the doors were opened and mice were free to explore the entire two-compartment apparatus for 20 min. The time spent in each compartment was recorded and was considered a measure of spontaneous preference. Conditioning sessions were performed on days 2, 3, and 4, twice per day with morning and afternoon sessions separated by at least 6 h. During these sessions, mice were injected with either saline or cocaine (10 and 20 mg/kg) and immediately confined to one of the pairing compartments for 30 min. The order of treatments (saline or cocaine), the time of cocaine injection (morning or afternoon), and the compartment (right or left) were counterbalanced in a semirandom order in both SE and EE mice. Control mice received saline on both sides of the cage. A total of three cocaine- and three saline-conditioned sessions were performed. Test sessions for conditioned place preference were conducted on day 6 and were similar to preconditioning sessions with animals placed in the central compartment for 15 s and then left free to choose a compartment for 20 min. The time spent in each compartment was measured and compared to the time spent in the same compartment during the preconditioning session. Preference scores, which served as a measure of rewarding effects of cocaine, were calculated by subtracting the time in seconds spent during the pretest from the time spent during the test day in the compartment paired to cocaine injections. For saline–saline groups, given that there was not a natural preference for either compartment, we arbitrarily chose to calculate preference score in the left compartment. We also verified that results would not have been different if we had chosen to calculate preference scores for the right compartment.
Locomotor Activity and Behavioral Sensitization Procedure
Motor activity was measured in Plexiglas cages (19 11 14 cm) placed in frames mounted with computer-monitored photocell beams (IMETRONIC). Horizontal locomotion was measured by the number of cage crossings. Behavioral data were collected by an Imetronic interface connected to a PC. Mice were first habituated to locomotor chambers for 60 min. Then, they were injected intraperitoneally with drug or saline and immediately placed back into the chamber.
Behavioral sensitization consisted of two phases: development and expression. For development of behavioral sensitization six injections of cocaine (15 mg/kg intraperitoneally (i.p.)) or saline were administered every second day. Expression of behavioral sensitization was measured 30 days after the last injection (withdrawal day 30). A total of eight groups were obtained (N=8 per group): SE sal–sal; sal–coc; coc–sal and coc–coc and EE sal–sal; sal–coc; coc–sal and coc–coc. Sal–sal mice received saline both during development and expression of behavioral sensitization; sal–coc mice received saline during development and cocaine for the expression of behavioral sensitization; coc–sal mice received cocaine during development and saline for the expression of behavioral sensitization; coc–coc mice received cocaine both during development and expression of behavioral sensitization. Locomotor activity was measured for 90 min for cocaine during the development of behavioral sensitization. On withdrawal day 30, locomotor activity was measured for only 45 min to allow the killing of mice and for obtaining brains to perform in situ hybridization studies.
In Vivo Microdialysis
Two groups of SE and two groups of EE mice (N=5–6 per group) were used for in vivo microdialysis experiments. Concentric dialysis probes design was similar to the one previously described for rats (Solinas et al, 2006), but sizes were adapted for mice. The exposed dialyzing surface of the probes was limited to the lowest 1.0 mm portion of the probes. Probe diameter was 0.250 mm. Mice were deeply anaesthetized with Avertin (0.4–0.75 mg/g) and placed in a stereotaxic apparatus (Stoelting Co., Illinois, USA). Coordinates for the nucleus accumbens (NAc) (A=1.5; L=0.6; V=5.0) and caudate putamen (A=0.0; L=1.8; V=4.0) were in accordance with the mouse brain atlas (Paxinos and Franklin, 2001). Each mouse was implanted with a single probe in the left hemisphere. About 24 h after probe implant, experiments were performed in freely moving mice. Ringer's solution (147.0 mM NaCl, 2.2 mM CaCl2, and 4.0 mM KCl) was delivered through the dialysis probes at a constant flow rate of 1 l/min. Dialysate samples (20 l) were collected every 20 min and 10 l were immediately injected for analysis by high-performance liquid chromatography (5200a Coulochem III, ESA, Chelmsford, MA, USA). The mobile phase contained 100 mM NaH2PO4, 0.1 mM Na2EDTA, 0.5 mM n-octyl sulfate, and 18% (v/v) methanol (pH adjusted to 5.5 with Na2HPO4). After stable dopamine level values (less than 10% variability) were obtained for at least three consecutive samples (typically after about 3 h), treatments started. Mice were first injected with saline and dopamine levels monitored for 60 min, then with 10 mg/kg of cocaine and dopamine levels monitored for 120 min and, finally, with 20 mg/kg of cocaine and dopamine levels monitored for 120 min. Assay sensitivity for dopamine was 2 fmoles per sample. At the end of the microdialysis experiments, mice were euthanized by dislocation and brains were removed and left to fix in 4% formaldehyde in saline solution. Brains were then frozen and cut on a cryostat in serial coronal slices. Slices were colored with cresyl violet to identify the location of the probes (see Supplementary Figures S1 and S2). Only data from mice with correct probe location were considered for statistical analysis. Dopamine levels are expressed as a percentage of basal dopamine values. Basal dopamine values were calculated as the mean of three consecutive samples (differing no more than 10%) immediately preceding the first drug or vehicle injection.
In Situ Hybridization
In situ hybridization experiments were performed on the brain of mice sensitized as described above. The same eight experimental groups were analyzed: SE sal–sal; sal–coc; coc–sal and coc–coc and EE sal–sal; sal–coc; coc–sal and coc–coc (N=6 per group). Mice were killed 45 min after the last injection of cocaine at day 30 of withdrawal. This postmortem time was chosen because levels of immediate early genes peak within an hour after cocaine injections (Yano and Steiner, 2005). Brains were removed, frozen in isopentane at -40°C, and then stored at -80°C. Coronal tissue sections (14 m thick) were thaw mounted onto SUPERFROST slides (VWR International, Fontenay sous Bois, France) and stored at -80°C.
In situ hybridization was performed with [35S] (UTP)-labeled RNA for zif-268 (Thiriet et al, 2002). Sections were delipidated, acetylated (0.25% acetic anhydride in 0.1 M triethanolamine (pH 8)), prehybridized for 10 min at 60°C in 50% formamide/SSC 1 (150 mM NaC1 and 15 mM sodium citrate, pH 7), dehydrated and air-dried. Thirty microliters of the labeled probes diluted to 25 000 cpm/l with hybridization buffer (50% formamide, 10% dextran sulfate, SSC 4 , 10 mM dithiothreitol) was placed on tissue sections and covered with coverslips and left overnight at 52°C. Post-hybridization was then carried out as previously described (Thiriet et al, 2002). The sections were exposed to X-ray film (Kodak, biomax-MR) for 4 days, then 7 days to Storm phosphorimager system for quantitative analysis using Image Quant software. Optical densities for each area (see Supplementary Figure S3) were calculated by subtracting to all areas the background level determined between sections of each slide. A total of two slides per animal (four sections per slide) were used for each hybridization experiment. All experiments were duplicated.
Western Blot
To measure Delta-Fos B (35–37 kDa) protein levels in the striatum, mice were exposed to the same regimen for the development of cocaine sensitization used for behavioral and in situ hybridization experiments. A total of four groups were used: SE sal, SE coc, EE sal and EE coc (N=12 per group). Sal groups received six injections of saline, whereas coc groups received six injections of 15 mg/kg of cocaine every second day. Mice were killed by decapitation 18 h after the last injection because the levels of Delta-Fos B are maximal, whereas other Fos proteins are absent at this time point (McClung et al, 2004). Brains were immediately removed and striata (NAc and caudate putamen) were immediately dissected. Proteins were extracted using a slightly modified version of the protocol described by Brenhouse and Stellar (2006). Briefly, brain tissue was homogenized in boiling lysis buffer (350 l for 25 mg) containing 50 mM Tris–HCl pH 6.8, 100 mM DTT, 2% SDS, with 0.05U aprotinin A, 1 M pepstatin, 1 M leupeptin, 0.1 mM benzamidine, 10 nM chloroquin, 10 nM Soybean Trypsin inhibitor, 0.1 mM N-Tosyl-L-lysine chloromethyl ketone hydrochloride, 100 g/ml N-p-Tosyl-L-arginine methyl ester hydrochloride, and 0.1 mM PMSF protease inhibitors. After homogenization using ultra-turrax for 15 s (IMLAB Laboratories technology, France), samples were then boiled for 3 min and centrifuged at full speed for 10 min. Supernatant were collected and then stored at -20°C until analysis. Protein samples (about 30 g) were subjected to SDS–polyacrylamide gel electrophoresis (10% acrylamide/0.27% N,N'-methylenebisacrylamide resolving gel) and proteins were transferred to PVDF membrane. Blots were blocked for 1 h in 5% nonfat dry milk in TBS-Tween 0.1%. Primary antibody incubation was then performed in blocking buffer for 120 min at room temperature, using rabbit Fos-related antigen (FRA, dilution 1:500), which recognizes the Delta-Fos B isoforms (kindly provided by Dr Iadarola, National Institute of Dental and Craniofacial Research, National Institute of Health, USA). The blots were then washed 3 10 min in blocking buffer and were subsequently incubated for 60 min in a 1 : 1000 dilution of goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad Laboratories, France) in blocking buffer. The blots were then washed again 2 10 min in blocking buffer and then for 10 min in TBS, and developed with an enhanced chemiluminescence system (Pierce, France) before being exposed to Kodak MS Film (VWR, France) for 1 min. The blots were subsequently incubated with an anti--tubulin antibody (1/2000, Sigma-Aldrich, France). Levels of band density were quantified by densitometry with the VisioLab 2000 image analyser (Biocom, Paris, FRANCE), and for each sample the results are expressed as level of delta-Fos B over level of -tubulin. The experiment was repeated twice (each time with N=6 for each group). Care was taken to assure that quantification was performed within a linear range (Jaber et al, 1999). As relative values among experimental groups did not vary, data were pooled together by normalizing data to the mean value of each SE saline-treated group.
Drugs
Cocaine HCl (15 mg/kg, 10 mg/kg) was obtained from the Research Triangle Institute (Research Triangle Park, NC, USA). It was dissolved in sterile saline solutions (0.9%) and administered i.p. in a volume of 1 ml/100 g.
Statistics
All results are presented as group meansSEM. Differences in behavioral scores, in the levels of extracellular dopamine, mRNA and protein expression between groups were assessed by one, two or three way analysis of variance ANOVA. Results showing significant overall changes were subjected to Student–Newman–Keuls post-hoc test. Differences were considered significant when p<0.05.
RESULTS
EE Mice are Less Sensitive than SE Mice to the Reinforcing Effects of Cocaine
Neither SE nor EE mice showed significant natural preferences for either conditioned place preference compartments during pretests or developed preferences for one or the other compartment when saline was given in both compartments (Figure 1a and b). On the other hand, SE mice developed significant place preferences to cocaine at the dose of 10 and 20 mg/kg, whereas EE mice did not develop significant preferences at either doses (Figure 1b) (two-way ANOVA, environment effect, F(1, 67)=5.93, p<0.05;>0.05).
Figure 1.
Rewarding effects of cocaine in mice reared in standard (SE) or enriched (EE) environments. EE and SE mice were tested in the conditioned place preference paradigm after conditioning to cocaine (10 and 20 mg/kg i.p.) or saline. EE showed less conditioned place preference to cocaine than SE mice. (a) Shows time in sec spent in the cocaine-paired compartments before and after conditioning, whereas (b) shows preference scores. Preference score was calculated by the time spent in the cocaine-paired compartment during the test session minus the time spent in the same compartment during the pretest session. Post-hoc Student–Newman–Keuls's test: **p<0.01 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f1.html#figure-title" target="_blank">Full figure and legend (43K)
EE Mice are Less Sensitive than SE Mice to the Stimulant Effects of Repeated Administration of Cocaine
As previously reported (Bezard et al, 2003), the first injection of cocaine (15 mg/kg i.p.) produces a higher activation in SE mice as compared to EE mice (Figure 2a and b). With repeated administrations, both groups developed sensitization, ie, for both groups the effects of cocaine on day 6 were higher than on day 1 (Figure 2a). However, the effects of cocaine were consistently lower (about 50%) in EE compared to SE mice (Figure 2a) (three-way ANOVA, environment effect, F(1, 59)=3772, p<0.0001;>0.05; treatment day interaction, F(5, 354)=4.04, p<0.01;>0.05). In addition, sensitization was maintained in the two groups after 30 days of withdrawal from cocaine as demonstrated by the sensitized response to 10 mg/kg of cocaine (Figure 2b). However, once again, the amplitude of cocaine's effects was higher in SE than in EE mice (Figure 2b) (three-way ANOVA, environment effect, F(1, 55)=25.55, p<0.0001;>0.05; environment challenge interaction, F(1, 55)=22.71, p<0.0001;>0.05), indicating that environmental effects on repeated cocaine-induced behaviors and neuroadaptations are due to reduced effects of each acute cocaine administration. Motor impairment could not account for the reduced reactivity to cocaine of EE mice, as EE mice perform actually better than SE on a rotarod paradigm (data not shown).
Figure 2.
Behavioral sensitization to cocaine in mice reared in standard (SE) or enriched (EE) environments. (a) Development of behavioral sensitization to cocaine (15 mg/kg i.p.). Both SE and EE mice develop behavioral sensitization i.e. cocaine-induce locomotor activity increases over time, but EE consistently show reduced locomotor response to cocaine. (b) Expression of behavioral sensitization to cocaine (10 mg/kg i.p.) after 30 days of withdrawal. Both SE and EE mice show a sensitized response to cocaine (coc-coc vs sal-coc groups) but EE show reduced locomotor response to cocaine compared to SE mice. Post-hoc Student–Newman–Keuls's test: * and **p<0.05 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f2.html#figure-title" target="_blank">Full figure and legend (28K)
Cocaine-induced Elevation in Dopamine Levels in the NAc do not Differ Between EE and SE Mice
Dopamine basal levels did not differ between EE and SE mice neither in the NAc (SE=46.594.85; EE=47.906.71 fmol/10 l) nor in the Caudate Putamen (SE=51.554.87; EE=52.986.03 fmol/10 l). Saline injections did not alter extracellular levels of dopamine, whereas cocaine produced dose-dependent and time-dependent increases in dopamine levels both in the NAc and in the Caudate Putamen (three-way ANOVA for repeated measures, NAc: dose effect, F(1, 40)=21.28, p<0.0001;>0.05) (Figure 3). Increases in the NAc were more pronounced than those in the Caudate Putamen at both 10 mg/kg (300 vs 200% of basal levels) and 20 mg/kg (450 vs 300% of basal levels) of cocaine. Importantly, cocaine-induced elevations in extracellular levels of dopamine did not differ in EE and SE mice (three-way ANOVA for repeated measures, NAc: environment effect, F(1, 40)=0.34, p>0.05; environment dose interaction, F(5, 40)=0.002, p>0.05; environment time interaction, F(5, 40)=0.35, p>0.05; environment dose time interaction, F(5, 40)=1.68, p>0.05; CPu: environment effect, F(1, 40)=0.01, p>0.05; environment dose interaction, F(5, 40)=0.01, p>0.05; environment time interaction, F(5, 40)=0.48, p>0.05; environment dose time interaction, F(5, 40)=0.59, p>0.05).
Figure 3.
Cocaine-induced elevations of dopamine levels in mice reared in standard (SE) or enriched (EE) environments. Injections of 10 and 20 mg/kg of cocaine increase the extracellular levels of dopamine (a) in the nucleus accumbens and (b) in the caudate putamen to similar extent in SE and EE mice. Arrows indicate the time of injections. Results represent meansSEM from five to six mice.Full figure and legend (33K)
Cocaine Increases the Expression of the Immediate Early Gene zif-268 in the Striatum of SE but not of EE Mice
As the differences in behavioral responses to cocaine could not be explained by different effects of cocaine at the presynaptic levels, we monitored the expression of Zif-268 (Beckmann and Wilce, 1997) by in situ hybridization in the striatum as a measure of reactivity to cocaine at the postsynaptic levels. Basal levels of zif-268 did not differ between SE and EE in any region examined. In addition, zif-268 mRNA levels did not differ between mice chronically pretreated with cocaine (15 mg/kg i.p) and saline pretreated controls (groups coc–sal vs sal–sal). Cocaine challenge (10 mg/kg) increased zif-268 mRNA levels (about 25%) in the NAc shell and the core (but not in the caudate putamen) of SE mice similarly in chronically cocaine- (coc–coc) and saline-treated (sal–coc) mice, indicating that these effects of cocaine do not undergo sensitization. In contrast, in EE mice, cocaine (10 mg/kg i.p.) did not increase expression of zif-268 in any condition (three-way ANOVA: Shell, environment effect, F(1, 37)=7.72, p<0.01;>0.05; challenge effect, F(1, 37)=5.66, p<0.05;>0.05; environment challenge, F(1, 37)=6.94, p<0.05;>0.05; environment treatment challenge, F(1, 37)=0.10, p>0.05; Core; challenge effect, F(1, 37)=16.70, p<0.01; href="http://www.nature.com/npp/journal/v34/n5/full/npp200851a.html#fig4" target="_blank">Figure 4).
Figure 4.
Expression of zif-268 in mice reared in standard (SE) or enriched (EE) environments sensitized to cocaine. For induction of behavioral sensitization mice were injected with cocaine (15 mg/kg) or saline as in Figure 2 and injected with cocaine (10 mg/kg) or saline 30 days after the last cocaine or saline injection. Challenge injections of cocaine increase zif-268 expression in the shell (a) and core (b) of the nucleus accumbens in SE but not in EE mice. Importantly, zif-268 expression was not enhanced by sensitization. No significant change was found in the caudate putamen in SE or EE mice (c). Post-hoc Student–Newman–Keuls's test: *p<0.05 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f4.html#figure-title" target="_blank">Full figure and legend (118K)
EE and SE Have Opposite Effects on the Levels of Delta-Fos B Following Cocaine Administration
By western blot, we measured Delta-Fos B levels in the striatum of SE and EE mice sensitized to cocaine (15 mg/kg) and in saline controls. As previously reported (Hiroi et al, 1997), repeated administration of cocaine produced a significant increase in Delta-Fos B levels in the striatum of SE mice (about 40%). Interestingly, levels of Delta-Fos B in the striatum of saline-treated EE mice were elevated compared to saline-treated SE mice (about 50%) and were comparable to cocaine-treated SE mice. Surprisingly, in EE mice cocaine did not produce an increase but rather a decrease in Delta-Fos B levels (two-way ANOVA, environment effect, F(1, 44)=2.15, p>0.05; treatment effect, F(1, 44)=0.28, p>0.05; environment treatment effect, F(1, 44)=21.97, p<0.0001) href="http://www.nature.com/npp/journal/v34/n5/full/npp200851a.html#fig5" target="_blank">Figure 5).
Figure 5.
Levels of Delta-Fos B protein in the striatum of mice reared in standard (SE) or enriched (EE) environments sensitized to cocaine. Mice underwent the same protocol for the development of cocaine sensitization as in Figure 2a and were killed 18 h after the last cocaine or saline injection. EE mice show higher basal levels of Delta-Fos B than SE mice. Importantly, repeated administration of cocaine induced an increase of Delta-Fos B levels in SE and a decrease in EE mice. (a) Graphical representation of group means and (b) representative immunoblots. Post-hoc Student–Newman–Keuls's test: **p<0.01 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f5.html#figure-title" target="_blank">Full figure and legend (47K)
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DISCUSSION
In this study, we found that EE mice are protected against the effects of cocaine. EE mice develop less conditioned place preferences for compartments associated with cocaine injections and are less sensitive to the activating effects of cocaine in a sensitization paradigm than SE mice. In addition, cocaine-induced increases in dopamine levels in the striatum do not differ between EE and SE mice, whereas ventral striatal neurons are activated by cocaine in SE but not in EE mice, as measured by expression of the immediate early gene zif-268. Finally, we found that saline-treated EE mice have higher striatal levels of Delta-Fos B than saline-treated SE mice and that, whereas repeated administration of cocaine increases Delta-Fos B levels in the striatum of SE mice, it decreases Delta-Fos B levels in the striatum of EE mice. These results suggest that exposure to positive environmental conditions during early stages of life can greatly influence brain functioning and can provide protection against the effects of addictive drugs.
In a previous study, we found that the activating effects of an acute administration of cocaine were reduced by exposure to EE (Bezard et al, 2003). Here, we show that the rewarding effects of cocaine are also diminished in EE mice. In addition, EE reduce the activating effects of repeated administrations of cocaine. Indeed, in a behavioral sensitization paradigm, EE mice show reduced locomotion both during the development and the expression of behavioral sensitization. Although the validity of behavioral sensitization models to study drug addiction in humans is debated (Lecca et al, 2007), behavioral sensitization is considered an important preclinical tool to study the increases in the effects of drugs and the neuroadaptations that take place in the brain upon repeated administration of cocaine (Vanderschuren and Kalivas, 2000; Berridge, 2007; Bradberry, 2007). In our study, the rate of sensitization was similar between SE and EE mice with cocaine-treated mice having an average increase of 100% in locomotor counts as compared to the first day of cocaine exposure, whereas the sustained reduction of the effects of cocaine indicates that EE mice are protected against the effects of this drug. In a previous study, it was shown that rats reared in EE are less sensitive to the discriminative effects of cocaine (Fowler et al, 1993). In addition, in rats, EE decrease the rewarding effects of amphetamine in a self-administration paradigm (but not in a conditioned place preference paradigm) (Bowling and Bardo, 1994; Bardo et al, 2001; Green et al, 2002) and the activating effects of amphetamine (Bowling et al, 1993; Bardo et al, 1995) or nicotine (Green et al, 2003). However, in those studies, control groups for the effects of environmental enrichment were animals reared and housed in impoverished/isolated conditions. Whereas such controls may have helped magnifying the effects of EE, in our opinion, they do not represent a proper control to extrapolate the effects of environmental enrichment because of the known important behavioral, neurochemical, and molecular alterations produced by social isolation (Lu et al, 2003; Van den Buuse et al, 2003). Thus, our model, which compares enriched to standard housing conditions, has the advantage to provide direct and uncontroversial information of the influences of enrichment on the effects of cocaine. A recent study comparing mice reared in EE to mice reared in SE has found that EE mice show less activation and less place preference to morphine than SE mice (Xu et al, 2007).
Cocaine elevates dopamine levels by binding to the dopamine membrane transporter (DAT) and preventing dopamine reuptake by presynaptic terminals (Jaber et al, 1997) and these dopamine elevations in the NAc are considered central in the mediation of the reinforcing effects of psychostimulants (Di Chiara and Bassareo, 2007). Given that previous studies have shown reduced DAT levels in mice reared in EE (Bezard et al, 2003), we expected that the reduced effects of cocaine were related to a deficit in its ability to elevate dopamine in the NAc. In contrast, we found that the levels of dopamine under basal conditions and after cocaine administration did not differ between EE and SE. It is possible that, although significant, reductions of about 20% in DAT levels (Bezard et al, 2003) were not sufficient to alter cocaine-induced effects. It is also possible that given the chronic nature of environmental enrichment, neuronal adaptations occur in EE mice that compensate the decrease in DAT. For example, it has been shown that in DAT knockout mice, cocaine induces normal increases in dopamine accumbal levels through an action on noradrenaline transporter (Carboni et al, 2001).
We have previously shown that acute cocaine (20 mg/kg) increases c-fos levels in dorsolateral striatum in EE mice and in the ventromedial striatum in SE mice (Bezard et al, 2003). Here, in the same cocaine sensitization paradigm used for our behavioral experiments, we have measured mRNA levels of zif-268, another immediate early gene, whose expression is tightly correlated with cellular activation and synaptic plasticity (Beckmann and Wilce, 1997). Acute administration of cocaine increases the expression of zif-268 in the ventral and dorsal striatum in rats (Hope et al, 1992; Moratalla et al, 1992; Thiriet et al, 2002) and in mice (Brami-Cherrier et al, 2005). In addition, in mice genetically lacking zif-268 behavioral sensitization and conditioned place preferences are dramatically reduced (Valjent et al, 2006). Finally, injections of zif-268 antisense oligodeoxynucleotides in the basolateral amygdala disrupt the reconsolidation of drug-related memories and reduces cocaine-seeking behavior (Lee et al, 2005). Consistent with those studies, we found that zif-268 expression in the shell and core of the NAc was induced by cocaine administration in SE mice to the same extent in sensitized and nonsensitized mice. In contrast, in EE mice cocaine did not induce zif-268 expression in the NAc. Thus, the reduced expression of zif-268 in response to cocaine found in EE mice may participate to the reduced behavioral effects of cocaine in these mice. Several mechanisms could explain how similar levels of extracellular dopamine induced differential reactivity to cocaine in striatal neurons. For example, changes in dopamine receptors availability or functionality might account for the reduced response to cocaine. However, previous studies have failed to show a significant effect of environmental enrichment on D1 and D2 binding (Bardo and Hammer, 1991) and mRNA expression (Bezard et al, 2003). Future studies are needed to investigate whether other systems such as the glutamate system (Beckmann and Wilce, 1997), at the presynaptic or postsynaptic level, are involved in the protective effects of environmental enrichment.
Delta-Fos B is a member of the Fos family of transcription factors. In contrast to the other Fos proteins, Delta-Fos B is induced to only a small degree in response to acute drug administration but because of its unique stability, after repeated drug administration, Delta-Fos B gradually accumulates in the striatum and stays elevated for weeks or months after discontinuation of drug exposure (Hope et al, 1994; Chen et al, 1995; Moratalla et al, 1996; Hiroi et al, 1997). Thus, it has been hypothesized that Delta-Fos B functions as a sustained molecular switch that mediates some of the more persistent adaptations of the brain that underlie addiction (McClung et al, 2004). Importantly, striatal Delta-Fos B has been shown to be increased not only by drugs of addiction but also by nonaddictive drugs, such as antipsychotics as well as environmental manipulations such as exposure to stress (McClung et al, 2004). In our study, environmental stimulation per se increased striatal levels of Delta-Fos B in saline-treated mice. This increase is likely due to the constant activation of striatal neurons produced by environmental enrichment. A previous study has shown that wheel running increases striatal levels of Delta-Fos B in rats (Werme et al, 2002). Thus, it is possible that wheel running, which is a constant part of our EE, was, at least in part, responsible for increases in Delta-Fos B. Interestingly, a recent study has found that Delta-Fos B activation parallels and may be responsible for the increases in spine density subsequent to chronic cocaine administration (Lee et al, 2006). Given that EE increase striatal dendritic arborization (Kolb et al, 2003), Delta-Fos B may be involved in these structural modifications.
Elevations in Delta-Fos B levels are generally believed to be one of the mechanisms responsible to the sensitized reactions to cocaine (McClung and Nestler, 2003), whereas in our study, EE mice that had higher basal levels of Delta-Fos B showed reduced responses to cocaine. Although somewhat unexpected, these results are in agreement with the work of Hiroi et al. (1997) who found that the constitutive Fos B knock-out mice are more sensitive to the effects of cocaine than wild-type control. This supports the view that the influences of Delta-Fos B on subsequent behavioral, neurochemical, and molecular effects of addictive drugs depend on several factors, such as the duration of the increase in Delta-Fos B levels (McClung and Nestler, 2003) and the striatal type neurons where Delta-Fos B is actually increased (McClung et al, 2004).
Surprising findings of this study were that when cocaine was administered to EE mice, it decreased, instead of increasing, Delta-Fos B striatal levels indicating that EE induces a form of neuronal plasticity that dramatically alters the effects of subsequent cocaine administration. To our knowledge, this is the first study showing that in vivo manipulations can induce decreases in striatal levels of Delta-Fos B. Interestingly, in vitro experiments have recently shown that the exceptional resistance of Delta-Fos B to metabolic degradation appears to depend on the phosphorylation of a serine residue (Ulery et al, 2006). Although we could not test this hypothesis, it is intriguing to speculate that in EE mice repeated administration of cocaine alters the activity of kinases or phosphatases and results in the dephosphorylation of Delta-Fos B.
In conclusion, in this study we demonstrate that environmental stimulation during early stages of life provides protection against the abuse-related effects of cocaine. This protection appears to be mediated by a reduced activation of striatal neurons by cocaine and by dramatic changes in the neuronal adaptations usually observed after repeated cocaine administration. These results support the hypothesis that positive life experiences during critical periods, such as the adolescence decrease the sensitivity to drugs of abuse and the vulnerability to addiction.
Notes
DISCLOSURE/CONFLICT OF INTEREST
The authors declare that except for finance received from their primary employer no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interests.
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Acknowledgments
We thank Dr M Iadarola (NIDCR, NIH, USA) for generously providing Fos antibody. We thank B Merceron for technical assistance and JP Poindessault for help with figures. This study was supported by CNRS, University of Poitiers, Fondation pour la Recherche Médicale (FRM, 2003), Mission Interministérielle de la Lutte contre les Drogues et la Toxicomanie (MILDT-INSERM, 2006–2007) and Région Poitou Charentes (2003). R El Rawas is a recipient of a CNRS PhD fellowship (BDI-PED, 2005–2008).
Supplementary Information accompanies the paper on the Neuropsychopharmacology website (http://www.nature.com/npp)
Environmental Enrichment During Early Stages of Life Reduces the Behavioral, Neurochemical, and Molecular Effects of Cocaine
Marcello Solinas1, Nathalie Thiriet1, Rana El Rawas1, Virginie Lardeux1 and Mohamed Jaber1
1Institut de Physiologie et Biologie Cellulaires, University of Poitiers, CNRS, Poitiers, France
Correspondence: Professor M Jaber, Institut de Physiologie et Biologie Cellulaires, University of Poitiers, CNRS-6187, 40 avenue du recteur Pineau, Poitiers 86022, France. Tel: +33 (0)5 49 453985; Fax: +33 (0)5 49 454014; E-mail: mohamed.jaber@univ-poitiers.fr
Received 25 January 2008; Revised 4 March 2008; Accepted 5 March 2008; Published online 7 May 2008.
Abstract
It is known that negative environmental conditions increase vulnerability to drugs, whereas little is known on whether positive environmental conditions such as enriched environments (EE) have protective effects against addiction. We have previously found that EE consisting of bigger cages containing several toys that were changed once per week reduce cocaine-induced increases in locomotor activity. Here, we also show that the rewarding effects of cocaine are blunted in mice reared from weaning to adulthood in EE compared to mice reared in standard environments (SE). In addition, although both EE and SE mice develop behavioral sensitization to cocaine, EE mice show less activation in response to repeated administration of cocaine injections and reduced responses to cocaine challenges. In vivo microdialysis experiments demonstrate that the protective effects of EE do not depend on reduced cocaine-induced increases in the dopamine levels in the ventral or dorsal striatum. On the other hand, they were associated with reduced cocaine-induced expression of the immediate early gene zif-268 in the nucleus accumbens (shell and core) of EE mice. Finally, basal levels of Delta-Fos B, a transcription factor known to be increased by sustained activation of striatal neurons, are higher in the striatum of EE compared to SE mice and repeated administration of cocaine increases Delta-Fos B levels in SE mice but decreases them in EE mice. Altogether our results demonstrate that exposure to complex environments during early stages of life produce dramatic changes in the striatum that result in reduced reactivity to drugs of abuse.
Keywords:
addiction, enrichment, behavior, dopamine, nucleus accumbens, molecular biology
INTRODUCTION
Of the many people who experience psychoactive drugs, only a small percentage develops drug addiction. It is believed that environmental factors play a central role in determining sensitivity to the rewarding effects of drugs and vulnerability to develop addiction (Sinha, 2001; Goeders, 2002; Marinelli and Piazza, 2002; Kreek et al, 2005). For example, several studies have demonstrated that environmental stressors increase the unconditioned, conditioned, and reinforcing effects of drugs such as amphetamine, cocaine, and heroin (Sinha, 2001; Goeders, 2002; Marinelli and Piazza, 2002; Kreek et al, 2005). Preclinical studies have also provided important information on the mechanisms underlying this enhanced response to the effects of drugs and have shown that the dopamine system is implicated in this excessive reaction to drugs (Rouge-Pont et al, 1995; Goeders, 2002; Marinelli and Piazza, 2002).
The investigation of negative factors exacerbating addiction has been extensive, whereas considerably less attention has been dedicated to environmental manipulations that may mimic positive life experiences such as enrichment environments (EE) and may provide protection against drugs' effects. EE have been shown to increase learning and memory (van Praag et al, 2000), to facilitate recovery from brain injuries (Will et al, 2004), to reduce the insurgence of neurodegenerative diseases (Nithianantharajah and Hannan, 2006) and some authors have suggested that EE represent more natural and healthy environment than current standard housing conditions (Wurbel, 2001). In addition, some studies have shown that the reinforcing effects of amphetamine and nicotine are reduced in rats reared in EE compared to rats reared in social isolation (Bowling et al, 1993; Bardo et al, 2001; Green et al, 2002, 2003).
In a previous study, we have shown that mice reared in EE show less locomotor activity and a different pattern of c-fos activation in the striatum in response to an acute injection of cocaine compared to mice reared in a standard environments (SE) (Bezard et al, 2003). We also found that EE mice had less dopaminergic neurons in the substantia nigra pars compacta, lower levels of the dopamine transporter (DAT), the molecular target of cocaine, and higher levels of neurotrophin BDNF in the striatum (Bezard et al, 2003). These findings provided an initial demonstration that EE may be crucial in determining resistance to drugs of abuse such as cocaine. However, little information is available on the effects of EE on cocaine's rewarding properties, which are considered directly responsible for the development of drug addiction and on the behavioral and molecular consequences of repeated administration of cocaine.
In this study, we housed mice either in EE or SE from weaning to adulthood and we used (1) conditioned place preferences (Cunningham et al, 2006) to assess the rewarding effects of cocaine; (2) behavioral sensitization, an animal model for the intensification of drug craving and relapse believed to underlie addiction in humans (Vanderschuren and Kalivas, 2000; Robinson and Berridge, 2003; Bradberry, 2007), to assess the consequences of repeated exposure to cocaine; (3) in vivo microdialysis techniques to assess the response to cocaine of the dopamine mesolimbic system at the presynaptic level (Di Chiara and Bassareo, 2007); (4) in situ hybridization techniques to assess the reactivity to cocaine of the dopamine mesolimbic system at the postsynaptic level by measuring expression of immediate early genes (Hope, 1998; Berke and Hyman, 2000; Gerfen, 2000) and (5) western blot techniques to assess whether the differences observed in the behavioral effects of cocaine could be due to changes in the levels of Delta-Fos B, a transcription factor that is implicated in long-term neuroadaptations associated with drug addiction (McClung et al, 2004).
MATERIALS AND METHODS
Subjects
Male C57Bl/6J mice (Janvier, France) were housed in a temperature-controlled environment on a 12-h light/dark cycle with the lights on from 0700 to 1900 hours and had ad libitum access to food and water. All experimentation was conducted during the light period. Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) for the care of laboratory animals.
Housing Environmental Conditions
After weaning (3 weeks of age), mice were randomly divided in two different housing environmental conditions: SE or EE. SE consisted of common cage housing (25 20 15 cm). The EE consisted of larger (60 38 20 cm) cages containing a constantly running wheel and a small house and four-five toys that were changed once a week with new toys of different shape and color. For both SE and EE conditions, mice were housed in groups of four (each mouse from a different sibling) for at least 2 months and maximum 3 months before the start of the experiments. All the experiments were conducted at adulthood.
Conditioned Place Preference
Conditioned place preference experiments were performed in four identical boxes (IMETRONIC, Pessac, France) formed by two lateral chambers (15 15 20 cm) connected by a central alley (5 15 20 cm). Two sliding doors separated the alley from the chambers. In each chamber, two Plexiglas prisms with triangular bases (5 7 19 cm) were arranged to form different patterns (always covering the same surface of the chamber) and were used as conditioned stimuli. Two different metallic grids, one with large (1 cm) squares and the other with small (0.5 cm) circles were also used as conditioned stimuli. Behavioral data were collected by an Imetronic interface connected to a PC. Two infrared photocells were present in each compartment and detected the presence and movements of mice. Conditioned place preference procedure consisted of three phases: preconditioning, conditioning, and test. For each manipulation, mice were brought to the experimental room 60 min before the start of the experiment to allow for habituation and to reduce stress. Preconditioning was performed on day 1. Mice were placed in the central compartment with the doors closed. After 15 s, the doors were opened and mice were free to explore the entire two-compartment apparatus for 20 min. The time spent in each compartment was recorded and was considered a measure of spontaneous preference. Conditioning sessions were performed on days 2, 3, and 4, twice per day with morning and afternoon sessions separated by at least 6 h. During these sessions, mice were injected with either saline or cocaine (10 and 20 mg/kg) and immediately confined to one of the pairing compartments for 30 min. The order of treatments (saline or cocaine), the time of cocaine injection (morning or afternoon), and the compartment (right or left) were counterbalanced in a semirandom order in both SE and EE mice. Control mice received saline on both sides of the cage. A total of three cocaine- and three saline-conditioned sessions were performed. Test sessions for conditioned place preference were conducted on day 6 and were similar to preconditioning sessions with animals placed in the central compartment for 15 s and then left free to choose a compartment for 20 min. The time spent in each compartment was measured and compared to the time spent in the same compartment during the preconditioning session. Preference scores, which served as a measure of rewarding effects of cocaine, were calculated by subtracting the time in seconds spent during the pretest from the time spent during the test day in the compartment paired to cocaine injections. For saline–saline groups, given that there was not a natural preference for either compartment, we arbitrarily chose to calculate preference score in the left compartment. We also verified that results would not have been different if we had chosen to calculate preference scores for the right compartment.
Locomotor Activity and Behavioral Sensitization Procedure
Motor activity was measured in Plexiglas cages (19 11 14 cm) placed in frames mounted with computer-monitored photocell beams (IMETRONIC). Horizontal locomotion was measured by the number of cage crossings. Behavioral data were collected by an Imetronic interface connected to a PC. Mice were first habituated to locomotor chambers for 60 min. Then, they were injected intraperitoneally with drug or saline and immediately placed back into the chamber.
Behavioral sensitization consisted of two phases: development and expression. For development of behavioral sensitization six injections of cocaine (15 mg/kg intraperitoneally (i.p.)) or saline were administered every second day. Expression of behavioral sensitization was measured 30 days after the last injection (withdrawal day 30). A total of eight groups were obtained (N=8 per group): SE sal–sal; sal–coc; coc–sal and coc–coc and EE sal–sal; sal–coc; coc–sal and coc–coc. Sal–sal mice received saline both during development and expression of behavioral sensitization; sal–coc mice received saline during development and cocaine for the expression of behavioral sensitization; coc–sal mice received cocaine during development and saline for the expression of behavioral sensitization; coc–coc mice received cocaine both during development and expression of behavioral sensitization. Locomotor activity was measured for 90 min for cocaine during the development of behavioral sensitization. On withdrawal day 30, locomotor activity was measured for only 45 min to allow the killing of mice and for obtaining brains to perform in situ hybridization studies.
In Vivo Microdialysis
Two groups of SE and two groups of EE mice (N=5–6 per group) were used for in vivo microdialysis experiments. Concentric dialysis probes design was similar to the one previously described for rats (Solinas et al, 2006), but sizes were adapted for mice. The exposed dialyzing surface of the probes was limited to the lowest 1.0 mm portion of the probes. Probe diameter was 0.250 mm. Mice were deeply anaesthetized with Avertin (0.4–0.75 mg/g) and placed in a stereotaxic apparatus (Stoelting Co., Illinois, USA). Coordinates for the nucleus accumbens (NAc) (A=1.5; L=0.6; V=5.0) and caudate putamen (A=0.0; L=1.8; V=4.0) were in accordance with the mouse brain atlas (Paxinos and Franklin, 2001). Each mouse was implanted with a single probe in the left hemisphere. About 24 h after probe implant, experiments were performed in freely moving mice. Ringer's solution (147.0 mM NaCl, 2.2 mM CaCl2, and 4.0 mM KCl) was delivered through the dialysis probes at a constant flow rate of 1 l/min. Dialysate samples (20 l) were collected every 20 min and 10 l were immediately injected for analysis by high-performance liquid chromatography (5200a Coulochem III, ESA, Chelmsford, MA, USA). The mobile phase contained 100 mM NaH2PO4, 0.1 mM Na2EDTA, 0.5 mM n-octyl sulfate, and 18% (v/v) methanol (pH adjusted to 5.5 with Na2HPO4). After stable dopamine level values (less than 10% variability) were obtained for at least three consecutive samples (typically after about 3 h), treatments started. Mice were first injected with saline and dopamine levels monitored for 60 min, then with 10 mg/kg of cocaine and dopamine levels monitored for 120 min and, finally, with 20 mg/kg of cocaine and dopamine levels monitored for 120 min. Assay sensitivity for dopamine was 2 fmoles per sample. At the end of the microdialysis experiments, mice were euthanized by dislocation and brains were removed and left to fix in 4% formaldehyde in saline solution. Brains were then frozen and cut on a cryostat in serial coronal slices. Slices were colored with cresyl violet to identify the location of the probes (see Supplementary Figures S1 and S2). Only data from mice with correct probe location were considered for statistical analysis. Dopamine levels are expressed as a percentage of basal dopamine values. Basal dopamine values were calculated as the mean of three consecutive samples (differing no more than 10%) immediately preceding the first drug or vehicle injection.
In Situ Hybridization
In situ hybridization experiments were performed on the brain of mice sensitized as described above. The same eight experimental groups were analyzed: SE sal–sal; sal–coc; coc–sal and coc–coc and EE sal–sal; sal–coc; coc–sal and coc–coc (N=6 per group). Mice were killed 45 min after the last injection of cocaine at day 30 of withdrawal. This postmortem time was chosen because levels of immediate early genes peak within an hour after cocaine injections (Yano and Steiner, 2005). Brains were removed, frozen in isopentane at -40°C, and then stored at -80°C. Coronal tissue sections (14 m thick) were thaw mounted onto SUPERFROST slides (VWR International, Fontenay sous Bois, France) and stored at -80°C.
In situ hybridization was performed with [35S] (UTP)-labeled RNA for zif-268 (Thiriet et al, 2002). Sections were delipidated, acetylated (0.25% acetic anhydride in 0.1 M triethanolamine (pH 8)), prehybridized for 10 min at 60°C in 50% formamide/SSC 1 (150 mM NaC1 and 15 mM sodium citrate, pH 7), dehydrated and air-dried. Thirty microliters of the labeled probes diluted to 25 000 cpm/l with hybridization buffer (50% formamide, 10% dextran sulfate, SSC 4 , 10 mM dithiothreitol) was placed on tissue sections and covered with coverslips and left overnight at 52°C. Post-hybridization was then carried out as previously described (Thiriet et al, 2002). The sections were exposed to X-ray film (Kodak, biomax-MR) for 4 days, then 7 days to Storm phosphorimager system for quantitative analysis using Image Quant software. Optical densities for each area (see Supplementary Figure S3) were calculated by subtracting to all areas the background level determined between sections of each slide. A total of two slides per animal (four sections per slide) were used for each hybridization experiment. All experiments were duplicated.
Western Blot
To measure Delta-Fos B (35–37 kDa) protein levels in the striatum, mice were exposed to the same regimen for the development of cocaine sensitization used for behavioral and in situ hybridization experiments. A total of four groups were used: SE sal, SE coc, EE sal and EE coc (N=12 per group). Sal groups received six injections of saline, whereas coc groups received six injections of 15 mg/kg of cocaine every second day. Mice were killed by decapitation 18 h after the last injection because the levels of Delta-Fos B are maximal, whereas other Fos proteins are absent at this time point (McClung et al, 2004). Brains were immediately removed and striata (NAc and caudate putamen) were immediately dissected. Proteins were extracted using a slightly modified version of the protocol described by Brenhouse and Stellar (2006). Briefly, brain tissue was homogenized in boiling lysis buffer (350 l for 25 mg) containing 50 mM Tris–HCl pH 6.8, 100 mM DTT, 2% SDS, with 0.05U aprotinin A, 1 M pepstatin, 1 M leupeptin, 0.1 mM benzamidine, 10 nM chloroquin, 10 nM Soybean Trypsin inhibitor, 0.1 mM N-Tosyl-L-lysine chloromethyl ketone hydrochloride, 100 g/ml N-p-Tosyl-L-arginine methyl ester hydrochloride, and 0.1 mM PMSF protease inhibitors. After homogenization using ultra-turrax for 15 s (IMLAB Laboratories technology, France), samples were then boiled for 3 min and centrifuged at full speed for 10 min. Supernatant were collected and then stored at -20°C until analysis. Protein samples (about 30 g) were subjected to SDS–polyacrylamide gel electrophoresis (10% acrylamide/0.27% N,N'-methylenebisacrylamide resolving gel) and proteins were transferred to PVDF membrane. Blots were blocked for 1 h in 5% nonfat dry milk in TBS-Tween 0.1%. Primary antibody incubation was then performed in blocking buffer for 120 min at room temperature, using rabbit Fos-related antigen (FRA, dilution 1:500), which recognizes the Delta-Fos B isoforms (kindly provided by Dr Iadarola, National Institute of Dental and Craniofacial Research, National Institute of Health, USA). The blots were then washed 3 10 min in blocking buffer and were subsequently incubated for 60 min in a 1 : 1000 dilution of goat anti-rabbit antibody conjugated to horseradish peroxidase (Bio-Rad Laboratories, France) in blocking buffer. The blots were then washed again 2 10 min in blocking buffer and then for 10 min in TBS, and developed with an enhanced chemiluminescence system (Pierce, France) before being exposed to Kodak MS Film (VWR, France) for 1 min. The blots were subsequently incubated with an anti--tubulin antibody (1/2000, Sigma-Aldrich, France). Levels of band density were quantified by densitometry with the VisioLab 2000 image analyser (Biocom, Paris, FRANCE), and for each sample the results are expressed as level of delta-Fos B over level of -tubulin. The experiment was repeated twice (each time with N=6 for each group). Care was taken to assure that quantification was performed within a linear range (Jaber et al, 1999). As relative values among experimental groups did not vary, data were pooled together by normalizing data to the mean value of each SE saline-treated group.
Drugs
Cocaine HCl (15 mg/kg, 10 mg/kg) was obtained from the Research Triangle Institute (Research Triangle Park, NC, USA). It was dissolved in sterile saline solutions (0.9%) and administered i.p. in a volume of 1 ml/100 g.
Statistics
All results are presented as group meansSEM. Differences in behavioral scores, in the levels of extracellular dopamine, mRNA and protein expression between groups were assessed by one, two or three way analysis of variance ANOVA. Results showing significant overall changes were subjected to Student–Newman–Keuls post-hoc test. Differences were considered significant when p<0.05.
RESULTS
EE Mice are Less Sensitive than SE Mice to the Reinforcing Effects of Cocaine
Neither SE nor EE mice showed significant natural preferences for either conditioned place preference compartments during pretests or developed preferences for one or the other compartment when saline was given in both compartments (Figure 1a and b). On the other hand, SE mice developed significant place preferences to cocaine at the dose of 10 and 20 mg/kg, whereas EE mice did not develop significant preferences at either doses (Figure 1b) (two-way ANOVA, environment effect, F(1, 67)=5.93, p<0.05;>0.05).
Figure 1.
Rewarding effects of cocaine in mice reared in standard (SE) or enriched (EE) environments. EE and SE mice were tested in the conditioned place preference paradigm after conditioning to cocaine (10 and 20 mg/kg i.p.) or saline. EE showed less conditioned place preference to cocaine than SE mice. (a) Shows time in sec spent in the cocaine-paired compartments before and after conditioning, whereas (b) shows preference scores. Preference score was calculated by the time spent in the cocaine-paired compartment during the test session minus the time spent in the same compartment during the pretest session. Post-hoc Student–Newman–Keuls's test: **p<0.01 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f1.html#figure-title" target="_blank">Full figure and legend (43K)
EE Mice are Less Sensitive than SE Mice to the Stimulant Effects of Repeated Administration of Cocaine
As previously reported (Bezard et al, 2003), the first injection of cocaine (15 mg/kg i.p.) produces a higher activation in SE mice as compared to EE mice (Figure 2a and b). With repeated administrations, both groups developed sensitization, ie, for both groups the effects of cocaine on day 6 were higher than on day 1 (Figure 2a). However, the effects of cocaine were consistently lower (about 50%) in EE compared to SE mice (Figure 2a) (three-way ANOVA, environment effect, F(1, 59)=3772, p<0.0001;>0.05; treatment day interaction, F(5, 354)=4.04, p<0.01;>0.05). In addition, sensitization was maintained in the two groups after 30 days of withdrawal from cocaine as demonstrated by the sensitized response to 10 mg/kg of cocaine (Figure 2b). However, once again, the amplitude of cocaine's effects was higher in SE than in EE mice (Figure 2b) (three-way ANOVA, environment effect, F(1, 55)=25.55, p<0.0001;>0.05; environment challenge interaction, F(1, 55)=22.71, p<0.0001;>0.05), indicating that environmental effects on repeated cocaine-induced behaviors and neuroadaptations are due to reduced effects of each acute cocaine administration. Motor impairment could not account for the reduced reactivity to cocaine of EE mice, as EE mice perform actually better than SE on a rotarod paradigm (data not shown).
Figure 2.
Behavioral sensitization to cocaine in mice reared in standard (SE) or enriched (EE) environments. (a) Development of behavioral sensitization to cocaine (15 mg/kg i.p.). Both SE and EE mice develop behavioral sensitization i.e. cocaine-induce locomotor activity increases over time, but EE consistently show reduced locomotor response to cocaine. (b) Expression of behavioral sensitization to cocaine (10 mg/kg i.p.) after 30 days of withdrawal. Both SE and EE mice show a sensitized response to cocaine (coc-coc vs sal-coc groups) but EE show reduced locomotor response to cocaine compared to SE mice. Post-hoc Student–Newman–Keuls's test: * and **p<0.05 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f2.html#figure-title" target="_blank">Full figure and legend (28K)
Cocaine-induced Elevation in Dopamine Levels in the NAc do not Differ Between EE and SE Mice
Dopamine basal levels did not differ between EE and SE mice neither in the NAc (SE=46.594.85; EE=47.906.71 fmol/10 l) nor in the Caudate Putamen (SE=51.554.87; EE=52.986.03 fmol/10 l). Saline injections did not alter extracellular levels of dopamine, whereas cocaine produced dose-dependent and time-dependent increases in dopamine levels both in the NAc and in the Caudate Putamen (three-way ANOVA for repeated measures, NAc: dose effect, F(1, 40)=21.28, p<0.0001;>0.05) (Figure 3). Increases in the NAc were more pronounced than those in the Caudate Putamen at both 10 mg/kg (300 vs 200% of basal levels) and 20 mg/kg (450 vs 300% of basal levels) of cocaine. Importantly, cocaine-induced elevations in extracellular levels of dopamine did not differ in EE and SE mice (three-way ANOVA for repeated measures, NAc: environment effect, F(1, 40)=0.34, p>0.05; environment dose interaction, F(5, 40)=0.002, p>0.05; environment time interaction, F(5, 40)=0.35, p>0.05; environment dose time interaction, F(5, 40)=1.68, p>0.05; CPu: environment effect, F(1, 40)=0.01, p>0.05; environment dose interaction, F(5, 40)=0.01, p>0.05; environment time interaction, F(5, 40)=0.48, p>0.05; environment dose time interaction, F(5, 40)=0.59, p>0.05).
Figure 3.
Cocaine-induced elevations of dopamine levels in mice reared in standard (SE) or enriched (EE) environments. Injections of 10 and 20 mg/kg of cocaine increase the extracellular levels of dopamine (a) in the nucleus accumbens and (b) in the caudate putamen to similar extent in SE and EE mice. Arrows indicate the time of injections. Results represent meansSEM from five to six mice.Full figure and legend (33K)
Cocaine Increases the Expression of the Immediate Early Gene zif-268 in the Striatum of SE but not of EE Mice
As the differences in behavioral responses to cocaine could not be explained by different effects of cocaine at the presynaptic levels, we monitored the expression of Zif-268 (Beckmann and Wilce, 1997) by in situ hybridization in the striatum as a measure of reactivity to cocaine at the postsynaptic levels. Basal levels of zif-268 did not differ between SE and EE in any region examined. In addition, zif-268 mRNA levels did not differ between mice chronically pretreated with cocaine (15 mg/kg i.p) and saline pretreated controls (groups coc–sal vs sal–sal). Cocaine challenge (10 mg/kg) increased zif-268 mRNA levels (about 25%) in the NAc shell and the core (but not in the caudate putamen) of SE mice similarly in chronically cocaine- (coc–coc) and saline-treated (sal–coc) mice, indicating that these effects of cocaine do not undergo sensitization. In contrast, in EE mice, cocaine (10 mg/kg i.p.) did not increase expression of zif-268 in any condition (three-way ANOVA: Shell, environment effect, F(1, 37)=7.72, p<0.01;>0.05; challenge effect, F(1, 37)=5.66, p<0.05;>0.05; environment challenge, F(1, 37)=6.94, p<0.05;>0.05; environment treatment challenge, F(1, 37)=0.10, p>0.05; Core; challenge effect, F(1, 37)=16.70, p<0.01; href="http://www.nature.com/npp/journal/v34/n5/full/npp200851a.html#fig4" target="_blank">Figure 4).
Figure 4.
Expression of zif-268 in mice reared in standard (SE) or enriched (EE) environments sensitized to cocaine. For induction of behavioral sensitization mice were injected with cocaine (15 mg/kg) or saline as in Figure 2 and injected with cocaine (10 mg/kg) or saline 30 days after the last cocaine or saline injection. Challenge injections of cocaine increase zif-268 expression in the shell (a) and core (b) of the nucleus accumbens in SE but not in EE mice. Importantly, zif-268 expression was not enhanced by sensitization. No significant change was found in the caudate putamen in SE or EE mice (c). Post-hoc Student–Newman–Keuls's test: *p<0.05 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f4.html#figure-title" target="_blank">Full figure and legend (118K)
EE and SE Have Opposite Effects on the Levels of Delta-Fos B Following Cocaine Administration
By western blot, we measured Delta-Fos B levels in the striatum of SE and EE mice sensitized to cocaine (15 mg/kg) and in saline controls. As previously reported (Hiroi et al, 1997), repeated administration of cocaine produced a significant increase in Delta-Fos B levels in the striatum of SE mice (about 40%). Interestingly, levels of Delta-Fos B in the striatum of saline-treated EE mice were elevated compared to saline-treated SE mice (about 50%) and were comparable to cocaine-treated SE mice. Surprisingly, in EE mice cocaine did not produce an increase but rather a decrease in Delta-Fos B levels (two-way ANOVA, environment effect, F(1, 44)=2.15, p>0.05; treatment effect, F(1, 44)=0.28, p>0.05; environment treatment effect, F(1, 44)=21.97, p<0.0001) href="http://www.nature.com/npp/journal/v34/n5/full/npp200851a.html#fig5" target="_blank">Figure 5).
Figure 5.
Levels of Delta-Fos B protein in the striatum of mice reared in standard (SE) or enriched (EE) environments sensitized to cocaine. Mice underwent the same protocol for the development of cocaine sensitization as in Figure 2a and were killed 18 h after the last cocaine or saline injection. EE mice show higher basal levels of Delta-Fos B than SE mice. Importantly, repeated administration of cocaine induced an increase of Delta-Fos B levels in SE and a decrease in EE mice. (a) Graphical representation of group means and (b) representative immunoblots. Post-hoc Student–Newman–Keuls's test: **p<0.01 href="http://www.nature.com/npp/journal/v34/n5/fig_tab/npp200851f5.html#figure-title" target="_blank">Full figure and legend (47K)
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DISCUSSION
In this study, we found that EE mice are protected against the effects of cocaine. EE mice develop less conditioned place preferences for compartments associated with cocaine injections and are less sensitive to the activating effects of cocaine in a sensitization paradigm than SE mice. In addition, cocaine-induced increases in dopamine levels in the striatum do not differ between EE and SE mice, whereas ventral striatal neurons are activated by cocaine in SE but not in EE mice, as measured by expression of the immediate early gene zif-268. Finally, we found that saline-treated EE mice have higher striatal levels of Delta-Fos B than saline-treated SE mice and that, whereas repeated administration of cocaine increases Delta-Fos B levels in the striatum of SE mice, it decreases Delta-Fos B levels in the striatum of EE mice. These results suggest that exposure to positive environmental conditions during early stages of life can greatly influence brain functioning and can provide protection against the effects of addictive drugs.
In a previous study, we found that the activating effects of an acute administration of cocaine were reduced by exposure to EE (Bezard et al, 2003). Here, we show that the rewarding effects of cocaine are also diminished in EE mice. In addition, EE reduce the activating effects of repeated administrations of cocaine. Indeed, in a behavioral sensitization paradigm, EE mice show reduced locomotion both during the development and the expression of behavioral sensitization. Although the validity of behavioral sensitization models to study drug addiction in humans is debated (Lecca et al, 2007), behavioral sensitization is considered an important preclinical tool to study the increases in the effects of drugs and the neuroadaptations that take place in the brain upon repeated administration of cocaine (Vanderschuren and Kalivas, 2000; Berridge, 2007; Bradberry, 2007). In our study, the rate of sensitization was similar between SE and EE mice with cocaine-treated mice having an average increase of 100% in locomotor counts as compared to the first day of cocaine exposure, whereas the sustained reduction of the effects of cocaine indicates that EE mice are protected against the effects of this drug. In a previous study, it was shown that rats reared in EE are less sensitive to the discriminative effects of cocaine (Fowler et al, 1993). In addition, in rats, EE decrease the rewarding effects of amphetamine in a self-administration paradigm (but not in a conditioned place preference paradigm) (Bowling and Bardo, 1994; Bardo et al, 2001; Green et al, 2002) and the activating effects of amphetamine (Bowling et al, 1993; Bardo et al, 1995) or nicotine (Green et al, 2003). However, in those studies, control groups for the effects of environmental enrichment were animals reared and housed in impoverished/isolated conditions. Whereas such controls may have helped magnifying the effects of EE, in our opinion, they do not represent a proper control to extrapolate the effects of environmental enrichment because of the known important behavioral, neurochemical, and molecular alterations produced by social isolation (Lu et al, 2003; Van den Buuse et al, 2003). Thus, our model, which compares enriched to standard housing conditions, has the advantage to provide direct and uncontroversial information of the influences of enrichment on the effects of cocaine. A recent study comparing mice reared in EE to mice reared in SE has found that EE mice show less activation and less place preference to morphine than SE mice (Xu et al, 2007).
Cocaine elevates dopamine levels by binding to the dopamine membrane transporter (DAT) and preventing dopamine reuptake by presynaptic terminals (Jaber et al, 1997) and these dopamine elevations in the NAc are considered central in the mediation of the reinforcing effects of psychostimulants (Di Chiara and Bassareo, 2007). Given that previous studies have shown reduced DAT levels in mice reared in EE (Bezard et al, 2003), we expected that the reduced effects of cocaine were related to a deficit in its ability to elevate dopamine in the NAc. In contrast, we found that the levels of dopamine under basal conditions and after cocaine administration did not differ between EE and SE. It is possible that, although significant, reductions of about 20% in DAT levels (Bezard et al, 2003) were not sufficient to alter cocaine-induced effects. It is also possible that given the chronic nature of environmental enrichment, neuronal adaptations occur in EE mice that compensate the decrease in DAT. For example, it has been shown that in DAT knockout mice, cocaine induces normal increases in dopamine accumbal levels through an action on noradrenaline transporter (Carboni et al, 2001).
We have previously shown that acute cocaine (20 mg/kg) increases c-fos levels in dorsolateral striatum in EE mice and in the ventromedial striatum in SE mice (Bezard et al, 2003). Here, in the same cocaine sensitization paradigm used for our behavioral experiments, we have measured mRNA levels of zif-268, another immediate early gene, whose expression is tightly correlated with cellular activation and synaptic plasticity (Beckmann and Wilce, 1997). Acute administration of cocaine increases the expression of zif-268 in the ventral and dorsal striatum in rats (Hope et al, 1992; Moratalla et al, 1992; Thiriet et al, 2002) and in mice (Brami-Cherrier et al, 2005). In addition, in mice genetically lacking zif-268 behavioral sensitization and conditioned place preferences are dramatically reduced (Valjent et al, 2006). Finally, injections of zif-268 antisense oligodeoxynucleotides in the basolateral amygdala disrupt the reconsolidation of drug-related memories and reduces cocaine-seeking behavior (Lee et al, 2005). Consistent with those studies, we found that zif-268 expression in the shell and core of the NAc was induced by cocaine administration in SE mice to the same extent in sensitized and nonsensitized mice. In contrast, in EE mice cocaine did not induce zif-268 expression in the NAc. Thus, the reduced expression of zif-268 in response to cocaine found in EE mice may participate to the reduced behavioral effects of cocaine in these mice. Several mechanisms could explain how similar levels of extracellular dopamine induced differential reactivity to cocaine in striatal neurons. For example, changes in dopamine receptors availability or functionality might account for the reduced response to cocaine. However, previous studies have failed to show a significant effect of environmental enrichment on D1 and D2 binding (Bardo and Hammer, 1991) and mRNA expression (Bezard et al, 2003). Future studies are needed to investigate whether other systems such as the glutamate system (Beckmann and Wilce, 1997), at the presynaptic or postsynaptic level, are involved in the protective effects of environmental enrichment.
Delta-Fos B is a member of the Fos family of transcription factors. In contrast to the other Fos proteins, Delta-Fos B is induced to only a small degree in response to acute drug administration but because of its unique stability, after repeated drug administration, Delta-Fos B gradually accumulates in the striatum and stays elevated for weeks or months after discontinuation of drug exposure (Hope et al, 1994; Chen et al, 1995; Moratalla et al, 1996; Hiroi et al, 1997). Thus, it has been hypothesized that Delta-Fos B functions as a sustained molecular switch that mediates some of the more persistent adaptations of the brain that underlie addiction (McClung et al, 2004). Importantly, striatal Delta-Fos B has been shown to be increased not only by drugs of addiction but also by nonaddictive drugs, such as antipsychotics as well as environmental manipulations such as exposure to stress (McClung et al, 2004). In our study, environmental stimulation per se increased striatal levels of Delta-Fos B in saline-treated mice. This increase is likely due to the constant activation of striatal neurons produced by environmental enrichment. A previous study has shown that wheel running increases striatal levels of Delta-Fos B in rats (Werme et al, 2002). Thus, it is possible that wheel running, which is a constant part of our EE, was, at least in part, responsible for increases in Delta-Fos B. Interestingly, a recent study has found that Delta-Fos B activation parallels and may be responsible for the increases in spine density subsequent to chronic cocaine administration (Lee et al, 2006). Given that EE increase striatal dendritic arborization (Kolb et al, 2003), Delta-Fos B may be involved in these structural modifications.
Elevations in Delta-Fos B levels are generally believed to be one of the mechanisms responsible to the sensitized reactions to cocaine (McClung and Nestler, 2003), whereas in our study, EE mice that had higher basal levels of Delta-Fos B showed reduced responses to cocaine. Although somewhat unexpected, these results are in agreement with the work of Hiroi et al. (1997) who found that the constitutive Fos B knock-out mice are more sensitive to the effects of cocaine than wild-type control. This supports the view that the influences of Delta-Fos B on subsequent behavioral, neurochemical, and molecular effects of addictive drugs depend on several factors, such as the duration of the increase in Delta-Fos B levels (McClung and Nestler, 2003) and the striatal type neurons where Delta-Fos B is actually increased (McClung et al, 2004).
Surprising findings of this study were that when cocaine was administered to EE mice, it decreased, instead of increasing, Delta-Fos B striatal levels indicating that EE induces a form of neuronal plasticity that dramatically alters the effects of subsequent cocaine administration. To our knowledge, this is the first study showing that in vivo manipulations can induce decreases in striatal levels of Delta-Fos B. Interestingly, in vitro experiments have recently shown that the exceptional resistance of Delta-Fos B to metabolic degradation appears to depend on the phosphorylation of a serine residue (Ulery et al, 2006). Although we could not test this hypothesis, it is intriguing to speculate that in EE mice repeated administration of cocaine alters the activity of kinases or phosphatases and results in the dephosphorylation of Delta-Fos B.
In conclusion, in this study we demonstrate that environmental stimulation during early stages of life provides protection against the abuse-related effects of cocaine. This protection appears to be mediated by a reduced activation of striatal neurons by cocaine and by dramatic changes in the neuronal adaptations usually observed after repeated cocaine administration. These results support the hypothesis that positive life experiences during critical periods, such as the adolescence decrease the sensitivity to drugs of abuse and the vulnerability to addiction.
Notes
DISCLOSURE/CONFLICT OF INTEREST
The authors declare that except for finance received from their primary employer no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interests.
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Acknowledgments
We thank Dr M Iadarola (NIDCR, NIH, USA) for generously providing Fos antibody. We thank B Merceron for technical assistance and JP Poindessault for help with figures. This study was supported by CNRS, University of Poitiers, Fondation pour la Recherche Médicale (FRM, 2003), Mission Interministérielle de la Lutte contre les Drogues et la Toxicomanie (MILDT-INSERM, 2006–2007) and Région Poitou Charentes (2003). R El Rawas is a recipient of a CNRS PhD fellowship (BDI-PED, 2005–2008).
Supplementary Information accompanies the paper on the Neuropsychopharmacology website (http://www.nature.com/npp)
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