TL neuro

An Open Experiment in Open Experimenting

Please Note: All of the following was originally presented as work in progress. The work on this page has not been peer reviewed and thus the data are not considered to be a citable finding in the traditional sense. Any manuscripts that were finally accepted for publication are peer-reviewed and citable- those versions should be used for authoritative reference. The text and images on this pages are copyright protected. If you would like to use this material in some way please contact mtaffe AT scripps DOT edu.

UPDATE Aug 2015: The revised manuscript describing the male rat data was returned with a decision of accepted pending minor revisions. One reviewer expressed satisfaction and another had minor issues with some of the references we added in response to the previous review. Specific requests boiled down to asking us to add/reinforce reference to the work of one research team and delete reference to the work of two other teams. Gee, I wonder who this reviewer was? We made a few changes, and returned it for re-review. Two days later the manuscript was accepted for publication.

UPDATE May-Jul 2015: The manuscript was returned with a decision of major revisions. The biggest issue to deal with was a statement from the handling editor that it was totally inappropriate to include any comparison with the females in this paper, given that the female study had been published. I disagreed and dashed off a request for clarification, including my arguments for why it was appropriate. (One reviewer had asked for assurance that the males and females had been treated identically but otherwise there was no demand from the reviewers to remove the female data.) It took me weeks to get a response and the editor did not address my arguments, merely reiterated the original request. However this time the editor added a suggestion that it might be acceptable to put it in the Supplemental Materials. Since we’d already pushed part of the male-female comparison off into a Supplemental Materialsfile and the comments were otherwise pretty light, I decided to take all the sex comparisons out of the main paper and push all of it into the Supplemental file. This is not ideal, given the poor visibility, but such is life.

UPDATE 24 Mar 2015: The manuscript containing the male rat data along with a comparison with the female groups already published has now been submitted for review.

UPDATE 9 Jan 2015: The paper has been accepted for publication! (The speed of turn-around indicates that the editor who was handling the submission felt it unnecessary to send it back out for re-review.)

UPDATE Jan 2015: The journal has returned the manuscript with an opportunity for revisions. The reviewers found interest in the study but, naturally, had a list of criticisms. We felt we could respond to the comments adequately so we have prepared a revision and resubmitted it for additional review. One of the reviewers went pretty lightly on us, and mostly seemed to take issue with the breadth of context we presented as well as disagreement over the discussion of relevant literature. Another reviewer had fairly fundamental problems with our design and I wouldn’t be surprised if that one recommended rejection. So it will be interesting to see how this goes in re-review.

UPDATE Nov 2014: The study has been submitted for consideration for publication. The submitted manuscript includes the FR and PR dose-substitution for the Methylone-trained animals which I didn’t include here (as yet); they looked very similar to the MDMA-trained group. It also included a median split analysis. This is a division of the acquisition data into the upper and lower halves of the distributions based on the number of infusions the individuals obtained. This is relevant because 40-50 percent of rats have been reported not to meet acquisition criterion for MDMA self-administration in some prior studies (see Oakly et al, 2014; Schenk et al, 2007), thus individual differences may be a critical aspect of the self-administration of these drugs.

UPDATE Dec 2014: The initial review decision is an invitation to resubmit a revised version of the manuscript for further consideration. One area of criticism is mostly directed towards our interpretive comments with respect to related literature. These remarks are of the type that can easily be handled by adding to the Discussion. There is another area of complaining about the design being too complicated. While our repeated dose-substitutions in the same groups of animals are perhaps slightly more complex than if it were all between-groups, we have strong reasons for doing things this way. First and foremost is that the interpretation of effects are always strongest within-subject. Second, this is consistent with reduction and refinement goals that are endemic to the ethical use of animals in research. Finally, a study being complex is not in and of itself a reason not to publish it. So we’re going to have to do what we can to clarify the description. Then there was a “Gee, it would be better to see the direct sex-comparison” comment. Not quite sure which way we’re going to jump on this one. We already have a paper-sized manuscript. Adding the sex-comparison would mean more complexity (which one reviewer already doesn’t like), more length and possibly having to drop some of the analysis.


The experiment is, roughly speaking, in Open Science. I have a line of work in the laboratory that lends itself to an experiment in semi-public presentation of data as we move along, rather than waiting for final publication. Publication of findings can take years from the initial finding, thus slowing the progress of science since other laboratories cannot springboard forward from what we have found. The idea for this experiment is that I will post the work as we progress and invite commentary on the meaning, next steps we might take, potential limitations, etc. Consider it an educational exercise for all of us.


StructureFig-MDMA-Methylone-MephedroneBackground Cathinones, aka “bathsalts”
There are a number of synthetic cathinone stimulants that are in reasonably substantial and continued use in the US, as well as elsewhere worldwide. Cathinone, the core molecule differs from amphetamine in the addition of a ketone in the beta position. In the figure, 3,4-methylenedioxymethamphetamine (MDMA or “Ecstasy”) can be contrasted with its cathinone cousing Methylone, which might otherwise be called 3,4-methylenedioxymethcathinone. If you see Methylone referred to as “bk-MDMA”, as it sometimes is with users, you will now be able to recognize what “beta-keto-MDMA” means. Mephedrone more or less led the emergence of substituted cathinones with one death noted in Sweden in 2008 and a major increase in prevalence in the UK throughout 2009 and 2010. To my view there has never been a major place in the recreational pharmacopeia for 4-methylmethamphetamine, the amphetamine cousin of mephedrone.

Background 2: How addictive is it?
One of the main layperson and public policy questions we need to address with novel and emerging drugs is “How addictive is it?”. This is not a simple question and opinions vary about how to best answer this in animal models. Without going into the many pages it would take, for this we are interested in relative drug liking in non-addicted animals. The assessment is pretty simple: Rats are prepared with intravenous catheters and allowed to press a lever to obtain small infusions of that drug. By comparing behavioral responses under various conditions we can come to some determination of how pleasurable that drug is with respect to other drugs. The available data on the intravenous self-administration (IVSA) of mephedrone and methylone are not extensive. Data on mephedrone IVSA are in Hadlock et al (blog post), Aarde et al, 2013b and Motbey et al, 2013. The only available data on the self-administration of methylone (as of Sep 2014) are in Watterson et al, 2012 (blog post). There is more rationale on why we are interested in comparing these drugs with MDMA in The Mephedrone Conundrum blog post.

Background 3: Female animals
The NIH has recently issue a policy position which reinforces the critical importance of conducting sex-difference comparisons across biomedical domains (Clayton and Collins 2014). It has been shown that female rats will self-administer more cocaine (Roth and Carroll 2004b; Smith et al. 2011) and more methamphetamine (Reichel et al. 2012; Roth and Carroll 2004a) than males; these sex differences can be more pronounced under long-access escalation and/or Progressive Ratio procedures. Little is known about any possible sex differences in the self-administration of atypical stimulants like MDMA or the recently emerging synthetic cathinones. Thus, the present study was conducted in female rats to expand understanding of the comparative reinforcing properties of these compounds. For now the comparison with males will be indirect although we may decide to run male groups in a similar way for comparison.

Purpose:
To compare the self-administration efficacy and potency of mephedrone and methylone with that of MDMA in female rats.

Methods
The methods follow the same techniques we’ve published in Aarde et al, 2013a and Aarde et al, 2013b for the most part.

This experiment was conducted in Female Wistar rats, with separate groups trained initially to self-administer either MDMA (N=15), Mephedrone (N=16), or Methylone (N=16). The animals had no prior lever training, were not food restricted and drug infusions were delivered on a fixed-ratio 1 contingency (FR1; each press of the drug-associated lever resulted in an infusion). These were two hour sessions and there was a 20 sec timeout after each drug infusion during which no additional infusions could be obtained.

Results
N.b.: The results may be continuously updated and changed. There are a number of reasons for that, including the exclusion of subjects for various reasons that were not applicable when specific graphs were created. Potentially because we run more subjects and have judged them worthy of inclusion with a prior group. Or possibly animals may be excluded in one analysis and further consideration suggests this was the wrong approach.

Acquisition:
IVSA-acquistionV2

At this point it appears that female rats self-administer all three compounds quite readily. MDMA and Methylone lead to similar intakes at the end of acquisition whereas more Mephedrone infusions are obtained. There are only marginal differences in the lever discrimination but if anything, Mephedrone leads to higher active-lever discrimination.

MDMA-methylone-FR-DRv1Fixed-Ratio Dose Substitution:
In this part of the study, the available dose of drug that was available in each infusion was varied from day to day. The point of doing so is that the shape of the curves can give you more information about the relative potency (per-infusion dose at which a given effect occurs, such as the number of lever presses) and efficacy (maximum effect on lever pressing, in essence) of the drugs. If you just evaluate a single dose, you may misinterpret dose effects as general properties of the drug in question. So for these graphs, the dose order was balanced across the training group. The first dose-response series was conducted in the Mephedrone (4MMC) and MDMA trained groups with their respective training drug. The next series permitted both groups to respond for Methylone. The statistical analysis confirmed that the groups differed in responding for saline and the lowest dose of their training drugs in the upper panel. So this would tend to be interpreted as a potential difference in efficacy, not too surprising given where the two groups ended up at the end of acquisition. However there is a potential problem here in that we may not have tested a low enough dose!
In the Methylone series, however, there were no longer any differences confirmed between the two training groups. This was interesting for two reasons. First because it shows that the MDMA-trained animals weren’t simply less interested in any of these drugs than the 4MMC trained group. Rather, the difference during acquisition and the training-drug dose substitution probably was related to MDMA being less effective as a reinforcer and 4MMC being more-effective. When the two groups were being rewarded with the same drug, Methylone, the curves were indistinguishable.
XX-MMC-PRbreakpt-quick [Interim Note: The FR studies continued with the groups being examined on first each other’s training drug and then being returned to their original drug for a final FR dose-substitution. In this, the MDMA-trained animals looked more like the Mephedrone/4MMC rats. This is setup for the PR finding…]

Progressive-Ratio Dose Substitution:
A Progressive-Ratio (PR) test differs from the FR in a key way. For each successive infusion of drug within a session, the rat has to press more times than she did previously. For example, it might take 1 lever press for the first infusion, 2 for the next, then 4, then 8, then 16, etc. The idea is that you let animals work until they quit- which is inferred as them telling you it is not worth it to work that hard for that particular reward anymore. The “breakpoint” is the final ratio that was completed (i.e. that resulted in a drug infusion).
In this figure the Mephedrone/4MMC trained group was tested on two different per-infusion doses (0.125, 1.0 mg/kg/inf) of MDMA, Mephedrone/4MMC or Methylone in a randomized order. The statistical analysis found an effect of the available drug dose, but not of the drug identity on this test. Significantly higher breakpoints were achieved for the higher dose of drug in each case, indicating that they would work harder for it. No differences between the drugs were found, thus they appear to be about equivalently reinforcing and potent on this test.
XX-MDMA-PRbreakpt-quickIn comparison, while the MDMA-trained group shows the same lack of distinction between the drugs, notice that they are less willing to work for these compounds, at either dose, than are the Mephedrone/4MMC trained rats. This didn’t amount to a statistically reliable difference in the three-way analysis but the analysis within this group did not confirm a group effect.

UPDATE: Dec 16, 2014
We’ve been working on making this a better sex-comparison study by running three male rat groups in a similar study to the one outlined above for the females. Additional self-administration studies were conducted in male Wistar rats with separate groups trained initially to self-administer either MDMA (N=17), Mephedrone (N=15), or Methylone (N=14). The male rats had no prior lever training, were not food restricted and drug infusions were delivered on a fixed-ratio 1 contingency (FR1; each press of the drug-associated lever resulted in an infusion). These were two hour sessions and there was a 20 sec timeout after each drug infusion during which no additional infusions could be obtained.

MDMA-Methylone-AcqThe initial acquisition data were similar to the outcome for the females, depicted above, in that the curves for MDMA and Methylone were similar and shallower than the acquisition for Mephedrone. In this figure we show the acquisition data for the males and the original females who were trained on MDMA (0.5 mg/kg/inf). As you can see there is a modest difference (supported by a main effect of sex, p<0.05, on the ANOVA) with females self-administering more MDMA. There was no sex difference confirmed for Methylone or for Mephedrone (not shown).
At this interim point we have evidence for a sex-difference in the self-administration of MDMA but not for Methylone or Mephedrone. This is somewhat surprising giving the studies that we reviewed at the top of this page showing that female rats tend to self-administer more cocaine and methamphetamine. The dose substitution experiments are under way and will eventually support additional sex-comparisons so it may be the case that sex-differences may still be found.

As mentioned in the Update at the top of the page, we performed a median-split analysis on the female acquisition data. This was in large part due to reports that high percentages of male rats in MDMA IVSA studies fail to meet (arbitrary) acquisition criteria. Although we will probably end up with insufficient statistical power to confirm the data, an initial comparison of the median splits for the male and female groups trained on MDMA suggests the major difference may be in the lower half of the male distribution. These male rats exhibited negligible discrimination between active and inactive levers and averaged about half as many infusions per session as the lower half of the female distribution.

There are two studies of human users that report increased odds of dependence in women compared with men, see Bruno et al 2009 and Uosukainen et al, 2014. Another study failed to find any sex-difference in dependence rates (de Almeida et al, 2009). Our data suggest that one measure of interest in humans would be the aversion rate. That is, how many people try MDMA and really do not like it? This human outcome might be consistent with the lowest-preferring rats in our study and it is there that our studies would predict the sex-differences to be largest.

Funding: This work is supported under a research project funded by the US National Institutes of Health (NIDA grant DA024105). The NIH and NIDA have no further role in the conduct of this project, analysis of the data, the decisions to publish data, etc.

Oversight: The animal studies are conducted under oversight of the Institutional Care and Use Committee of The Scripps Research Institute.

Literature cited
Aarde SM, Huang PK, Creehan KM, Dickerson TJ, Taffe MA. The novel recreational drug 3,4-methylenedioxypyrovalerone (MDPV) is a potent psychomotor stimulant: self-administration and locomotor activity in rats. Neuropharmacology. 2013 Aug;71:130-40. doi: 10.1016/j.neuropharm.2013.04.003. Epub 2013 Apr 15. [PMC (free) Link]

Aarde SM, Angrish D, Barlow DJ, Wright MJ Jr, Vandewater SA, Creehan KM, Houseknecht KL, Dickerson TJ, Taffe MA. Mephedrone (4-methylmethcathinone) supports intravenous self-administration in Sprague-Dawley and Wistar rats. Addict Biol. 2013 Sep;18(5):786-99. doi: 10.1111/adb.12038. Epub 2013 Jan 30.[PMC (free) Link]

Clayton JA, Collins FS (2014) Policy: NIH to balance sex in cell and animal studies. Nature 509: 282-3

Hadlock GC, Webb KM, McFadden LM, Chu PW, Ellis JD, Allen SC, Andrenyak DM, Vieira-Brock PL, German CL, Conrad KM, Hoonakker AJ, Gibb JW, Wilkins DG, Hanson GR, & Fleckenstein AE (2011). 4-Methylmethcathinone (mephedrone): neuropharmacological effects of a designer stimulant of abuse. The Journal of pharmacology and experimental therapeutics, 339 (2), 530-6 PMID: 21810934

Motbey CP1, Clemens KJ, Apetz N, Winstock AR, Ramsey J, Li KM, Wyatt N, Callaghan PD, Bowen MT, Cornish JL, McGregor IS.High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: neural consequences and comparison with methamphetamine.J Psychopharmacol. 2013 Sep;27(9):823-36. doi: 10.1177/0269881113490325. Epub 2013 Jun 5.

Roth ME, Carroll ME (2004a) Sex differences in the acquisition of IV methamphetamine self-administration and subsequent maintenance under a progressive ratio schedule in rats. Psychopharmacology 172: 443-449

Roth ME, Carroll ME (2004b) Sex differences in the escalation of intravenous cocaine intake following long- or short-access to cocaine self-administration. Pharmacol Biochem Behav 78: 199-207

Watterson LR, Hood L, Sewalia K, Tomek SE, Yahn S, Johnson CT, Wegner S, Blough BE, Marusich JA, Olive MF (2012) The Reinforcing and Rewarding Effects of Methylone, a Synthetic Cathinone Commonly Found in “Bath Salts”. J Addict Res Ther S9:002: 1-8
____

© Michael A Taffe and TL Neuro, A laboratory blog, 2014. Unauthorized use and/or duplication of this material without express and written permission from this blog’s author and/or owner is strictly prohibited. Excerpts and links may be used only with explicit permission and provided that full and clear credit is given to Michael A Taffe and TL Neuro, A laboratory blog, with appropriate and specific direction to the original content.

Advertisements

1 Comment »

  1. […] An Open Experiment in Open Experimenting […]

    Pingback by Mephedrone is more reinforcing than methylone or MDMA in female rats | TL neuro — January 9, 2015 @ 9:27 am


RSS feed for comments on this post. TrackBack URI

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

Create a free website or blog at WordPress.com.

%d bloggers like this: