TL neuro

June 3, 2016

Inhalation delivery of psychostimulants to rats using e-vape technology

Filed under: 4-MMC/Mephedrone, Cathinones, E-cigarettes, MDPV, Methamphetamine — mtaffe @ 4:03 pm

Although inhaled exposure of drugs is a prevalent route of administration for human substance abusers, animal models of inhaled exposure to psychomotor stimulants (cocaine, methamphetamine, synthetic cathinones, etc) are not commonly available. Inhaled use of methamphetamine is more common than other routes of administration in habitual and dependent users (Das-Douglas et al. 2008; Heinzerling et al. 2010; Wood et al. 2008) and the SAMHSA/TEDS treatment admission database for 2012 shows 4.7% of treatment seekers in the USA were admitted for smoked cocaine vs 2.2% for other routes of cocaine administration. There is limited evidence that people are using e-cigarettes for inhalation of methamphetamine (Evans 2014; Rass et al. 2015), “bath salts” (Johnson and Johnson 2014; Rass et al. 2015) and “flakka” (presumptively α-pyrrolidinopentiophenone; alpha-PVP) as reported (Anderson 2015).

We have therefore developed a method for the delivery of psychostimulant drugs to rats and evaluated the impact of methamphetamine (MA), 3,4-methylenedioxypyrovalerone (MDPV; “bath salts”) and 4-methylmethcathinone (4-MMC; mephedrone). The following paper describing our initial studies has been recently accepted for publication in Neuropsychopharmacology:

Locomotor stimulant and rewarding effects of inhaling methamphetamine, MDPV and mephedrone via electronic cigarette-type technology. Jacques D. Nguyen1, Shawn M. Aarde1, Maury Cole2, Sophia A. Vandewater1, Yanabel Grant1 and Michael A. Taffe1
1Committee on the Neurobiology of Addictive Disorders; The Scripps Research Institute; La Jolla, CA, USA
2La Jolla Alcohol Research, Inc, La Jolla, CA, USA

Schematic of the inhalation chamber

Schematic of the inhalation chamber

Our exposure model for this study involved a standard sized rat housing chamber with a sealed lid- these are commercially available for a variety of purposes. The chamber was plumbed for regulated airflow and incorporated the ability to deliver and exhaust the vapor from an e-cigarette type device. The overall approach for delivery to rodents is under patent to La Jolla Alcohol Research, Inc which has been instrumental in developing the equipment for our studies. This collaboration has resulted in a number of studies so far, this one is the second one to be published. The first paper described the effects of THC inhalation (blogpost). The company has also recently been awarded an SBIR Phase II Grant (R44 DA041967) to further develop and enhance commercialization of the device.

Control of the dose administered to the rat in this system is a key initial topic of investigation. We determined in this paper whether the dose can be altered with the manipulation of a number of variables. The concentration off the drug may be altered in the propylene glycol (PG) vehicle (aka “e-juice”)- our standard condition for this study was 100 mg/mL but effects from 12.5-200 mg/mL were also explored for different drugs. For the most part this study found concentration-dependent effects only across drugs (4-MMC was much less potent than MA or MDPV) when the puffing and inhalation duration was held constant. The puffing regimen and duration of inhalation exposure can be altered as well. In most of our studies we delivered 10-s vapor puffs with 2-s intervals between them every 5 minutes for durations of 10-40 min (approximately 0.125 ml was used in a 40 min exposure session). Varying the total duration from 10 to 30 min resulted in dose dependent effects of inhaling MA (12.5 mg/mL) or MA (12.5 mg/mL).

Vape decreases ICSS thresholds

A decrease in ICSS threshold was produced by inhalation exposure to 4MMC (200 mg/mL), MA (100 mg/mL) and MDPV (100 mg/mL). Similar effects were produced by i.p. administration of 4MMC (1.0mg/kg), MA (0.5 mg/kg) or MDPV (0.5 mg/kg). Significant differences from the respective Vehicle condition are indicated by *.

We present data on the intracranial self-stimulation reward paradigm in this paper. This is a model in which electrodes are implanted into the medial forebrain bundle of the rat and it is trained to respond for small deliveries of electrical current which has a rewarding or reinforcing effect. The procedure used for this study ramps the stimulation up and down during a session until the threshold necessary for the individual to experience a reinforcing effect is determined. Once the animals are trained to generate stable thresholds, they can be tested by administering drugs before the session. If a drug has a rewarding or reinforcing effect, it tends to lower the threshold below the baseline level. Here we show that all three drugs decrease reward thresholds in male rats. The reduction in the reward threshold was of a similar magnitude when drug was administered by injection or by vapor inhalation. This is a key indication that this procedure can generate reinforcing or rewarding levels of drug in the rats.

Activity rates after inhalation of  3,4-methylenedioxypyrovalerone (MDPV; 25,50,100mg/mL) or 4-methylmethcathinone (4MMC/mephedrone; 100, 200mg/mL).

Mean (N=13; + SEM) activity rates after inhalation of 3,4-methylenedioxypyrovalerone (MDPV; 25,50,100mg/mL) or 4-methylmethcathinone (4MMC/mephedrone; 100, 200mg/mL). Gray shaded symbols indicate a significant difference from PG vehicle at the corresponding time point. Base = pre-inhalation baseline.

Locomotor activity was measured after vapor inhalation using a radiotelemetry system that generates activity rates as counts per minute. In this figure we show the activity before and after inhalation of the PG vehicle and then three concentrations of MDPV and two concentrations of 4-methylmethcathinone (4-MMC, mephedrone) for 40 min. Locomotor activity was increased for 2-3 h after the initation of vapor for all three MDPV concentrations and for the 200 mg/mL concentration of 4-MMC. Similar effects were observed for MA and we went on to show that the dopamine D1-like receptor antagonist SCH23390 (10 ug/kg, i.p., prior to inhalation) blocked locomotor increases caused by inhalation of each drug. This is as would be expected, similar to the effect of SCH23390 on locomotor stimulant effects of these drugs when injected in rodents.

Wheel Activity after MDPV or MA

Mean (N=7; ± SEM) wheel activity of male rats after inhalation of methamphetamine (100 mg/mL in PG), MDPV (100 mg/mL in PG) or the PG alone for 40 minutes. Gray shaded symbols indicate a significant difference from PG. A significant difference from the 30 min time point within an inhalation condition is indicated with *, and a difference from MA with #, for corresponding time points.

Another study in the paper investigated effects of vapor inhalation of the MA and MDPV on wheel activity. Under vehicle treatment, rats run more on the wheel in the first 30 minutes and then run significantly less for the subsequent 90 min of a 2 h session. When allowed to use the wheel after vapor exposure to MA or MDPV, activity is initially suppressed and then rebounds as the session continues. Presumably, the initial suppression of wheel activity is related to the increase in chamber locomotor activity found in the radiotelemetry study- if the rats were running around the cage they might be unlikely to enter the wheels. In the study depicted, MDPV caused significantly more activity than vehicle inhalation 60-90 min after finishing the vapor inhalation. MA in this experiment did not increase activity compared with vehicle, however activity was significantly higher in the last thirty minutes than the first 30 min after MA inhalation. Additional data found no significant effects of 40 min of inhalation of either MDPV or MA at a 25 mg/mL concentration and a third study found elevations of wheel activity 90-120 min after a 20 min inhalation of MA (100 mg/mL). In total, the wheel activity data confirm dose-dependent effects on a second measure of locomotion.

Overall, this study is the first to demonstrate behavioral effects of e-cigarette type inhalation delivery of psychostimulants to rats. This further validates our model and encourages additional study of the risks of e-cigarette delivery of psychoactive substances in laboratory animal models.
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J. D. Nguyen, S. M. Aarde, M. Cole, S. A. Vandewater, Y. Grant and M. A. Taffe. Locomotor stimulant and rewarding effects of inhaling methamphetamine, MDPV and mephedrone via electronic cigarette-type technology, 2016, accepted article preview 9 June 2016; doi: 10.1038/npp.2016.88 [ PublisherSite ][ PubMed ]

Funding and Disclosures for this paper: This work was funded by support from the United States Public Health Service National Institutes of Health (R01 DA024105, R01 DA024705, R01 DA035281 and R44 DA041967) which had no direct input on the design, conduct, analysis or publication of the findings. Subsets of these data were first presented at the Experimental Biology meeting in 2015 and the Annual Meeting of the Society for Neuroscience 2015. Development of the apparatus was supported by La Jolla Alcohol Research, Inc and MC is inventor on a patent for this device. SAV consults for La Jolla Alcohol Research, Inc.

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May 31, 2016

Inhalation model for evaluation of e-cigarette based delivery of THC

Filed under: Cannabis, E-cigarettes, Vape inhalation — mtaffe @ 11:13 am

Our interest in developing inhalation techniques for delivering cannabinoids, most especially the primary active constituent Δ9-tetrahydrocannabinol (THC), to rats arose from the realization that increasing numbers of people were using non-combusted methods for inhalation. When we started this project there were no studies using a Volcano type or e-cigarette type of system to deliver THC to rodents. Of course the majority of cannabis consumption has always been via smoke inhalation and there have been a few prior studies in laboratory models, primarily from the Lichtman laboratory. Our focus was therefore on the non-combustible techniques stemming from the evidence of personal acquaintance reports, a plethora of Web sites advertising methods, an emerging literature showing human practices (Giroud et al, 2015, Morean et al, 2015) and from suggestions that e-cigarette delivery may offer a safer alternative for medical cannabis consumers (Varlet et al, 2016).

The following has been recently accepted for publication in Neuropharmacology.
Inhaled delivery of Δ9-tetrahydrocannabinol (THC) to rats by e-cigarette vapor technology. Jacques D. Nguyen1, Shawn M. Aarde1, Sophia A. Vandewater1, Yanabel Grant1, David G. Stouffer1, Loren H. Parsons1, Maury Cole2 and Michael A. Taffe1
1Committee on the Neurobiology of Addictive Disorders; The Scripps Research Institute; La Jolla, CA, USA
2La Jolla Alcohol Research, Inc; La Jolla CA, USA

Schematic of the inhalation chamber

Schematic of the inhalation chamber


Our exposure model for this study involved a standard sized rat housing chamber with a sealed lid- these are commercially available for a variety of purposes. The chamber was plumbed for regulated airflow and incorporated the ability to deliver and exhaust the vapor from an e-cigarette type device. We tried a number of commercial tanks in this study, one specific example is the Protank 3 Atomizer by Kanger Tech. The overall approach for delivery to rodents is under patent to La Jolla Alcohol Research, Inc which has been instrumental in developing the equipment for our studies. This collaboration has resulted in a number of studies so far, this one is the first to be published. The company has also recently been awarded an SBIR Phase II Grant (R44 DA041967) to further develop and enhance commercialization of the device.

Dosing control was managed in this system with the manipulation of a number of variables. One of the major goals of this study was to determine how the dose delivered to the animal might be regulated by altering these vaping parameters. The concentration off the drug (in this case Δ9-tetrahydrocannabinol (THC) may be altered in the propylene glycol (PG) vehicle (aka “e-juice”)- our standard condition for this study was 200 mg/mL but effects from 25-100 mg/mL were also explored and showed a concentration-dependent effect when the puffing and inhalation duration was held constant. The puffing regimen and duration of inhalation exposure can be altered as well. In most of our studies we delivered 10-s vapor puffs with 2-s intervals between them every 5 minutes for durations of 10-40 min (approximately 0.125 ml was used in a 40 min exposure session). This study established that for a given THC concentration in the vehicle, the duration over which animals were exposed could produce graded effects consistent with a dose-dependent pattern.

Mean (N=8; ±SEM) temperature response to THC inhalation for 10, 20 or 30 min in 5 min intervals. A significant difference from both the baseline and the other exposure conditions is indicated by the open symbols and from the 10 min condition by the shaded symbols.

Mean (N=8; ±SEM) temperature response to THC vapor inhalation for 10, 20 or 30 min in 5 min intervals. A significant difference from both the baseline and the other exposure conditions is indicated by the open symbols and from the 10 min condition by shaded symbols.


This first figure depicts THC-induced reductions in body temperature produced by THC inhalation for 10-30 minutes in male rats, using a radiotelemetry system for reporting temperature every 5 minutes. The figure depicting this experiment in the paper depicts 30 min averages but I really like this version so I’m including it here. [For those concerned with statistics, see below.] The points to the left indicate a pre-inhalation baseline interval in the telemetry recording chambers. There is a break in the series because we didn’t record them during vapor inhalation (see our SFN 2014 poster presentation for a pilot study recording during inhalation). The main point here is that 10 min of inhalation doesn’t change body temperature, 30 min has a major hypothermic effect and 20 min produces an intermediate effect. Thus, this system is able to produce dose-dependent effects that are so helpful for interpretation of behavioral pharmacology studies. We show in the paper that i.p. injection of 10-20 mg/kg THC produces a temperature nadir similar to that produced by 20-30 min of inhalation (see a blog post on our 2015 paper on temperature responses to injected THC for comparison). Our telemetry measure of locomotion did not show any suppression in this experiment but we do show a suppression of activity in both males and females in Figure 2 of the paper. There was some evidence that female rats are more sensitive to the hypothermia induced by, e.g., THC 50 mg/mL for 30 min in this study, likely because of their lower bodyweight compared with the male rats.

Mean tail-flick latency measured following 20 min of exposure with pre-treatment with SR141716 (SR; 4 mg/kg, i.p.) or Vehicle (N=8). Significant differences compared with respective vehicle condition are indicated by *, differences from SR+THC vapor by #.

Mean tail-flick latency measured following 20 min of exposure with pre-treatment with SR141716 (SR; 4 mg/kg, i.p.) or Vehicle (N=8). Significant differences compared with respective vehicle condition are indicated by *, differences from SR+THC vapor by #.


One of the major tests of cannabinoid activity in a rodent is a decrease in nociception. The ability to sense a noxious stimulus was tested by placing the tail in a 52°C water bath and timing the latency for it to flick it out. The experiment in the figure depicts a study in which the animals were exposed to PG or 200 mg/mL THC for 20 min with and without prior treatment with the cannabinoid 1 receptor antagonist SR141716 (Rimonabant; 4 mg/kg, i.p.). This shows that THC inhalation extends the time for the animal to flick its tail out of the warm water and that this effect is blocked with the antagonist pre-treatment. Although not shown here, the magnitude of the latency change caused by vapor inhalation of THC was the same as that produced by a 10 mg/kg THC i.p. injection. This comparability of the effect of inhaled versus injected THC was also highly consistent with data we generated showing that blood concentrations of THC were very similar when observed 30 min after THC (200 mg/mL) inhalation or 30 min after 10 mg/kg, i.p. injection.

In summary, we’ve created a new model for evaluating inhaled delivery of THC to rats via an e-cigarette type of method. We’ve found significant effects on three of the four traditional measures (Tetrad Test) of cannabinoid activity in a rodent- hypothermia, hypolocomotion and antinociception (the fourth, catalepsy, was not assessed). Effects were of comparable magnitude to those produced by intraperitoneal injection, allowing these data to be placed in context with prior studies using injection delivery of THC. There are several advantages of this model, most pertinently the more rapid timecourse of effects compared to what is produced with an i.p. injection.
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Jacques D. Nguyen, Shawn M. Aarde, Sophia A. Vandewater, Yanabel Grant, David G. Stouffer, Loren H. Parsons, Maury Cole and Michael A. Taffe. Inhaled delivery of Δ9-tetrahydrocannabinol (THC) to rats by e-cigarette vapor technology, 2016, Neuropharmacology, in press. DOI: 10.1016/j.neuropharm.2016.05.021 [PubMed]

Funding and Disclosures for this paper: This work was funded by support from the United States Public Health Service National Institutes of Health (R01 DA024105, R01 DA035281 and R44 DA041967) which had no direct input on the design, conduct, analysis or publication of the findings. Development of the apparatus was supported by La Jolla Alcohol Research, Inc and MC is inventor on a patent for this device. SAV consults for La Jolla Alcohol Research, Inc.

[Stats for body temperature figure: The ANOVA of the five minute temperature intervals (including three baseline samples, -15 to -5, and 40-180 min following initiation of vapor) confirmed main effects of Time post-initiation [F (32, 224) = 38.36; P < 0.0001], Duration of vapor exposure [F (2, 14) = 38.66; P < 0.0001] and the interaction of factors [F (64, 448) = 16.64; P < 0.0001].
The Tukey post-hoc test confirmed significant temperature reductions after 20 (40-70 min post-vapor initiation) or 30 min (40-155 min post-vapor initiation) of vapor exposure to THC compared with each of three baseline samples. Furthermore, the post-hoc test confirmed that temperature after all three exposure durations differed significantly from each other from 40-150 and 160-165 min following vapor initiation. Significant differences in temperature between 10 and 30 min vapor exposures were confirmed for the entire post-vapor duration.]

April 10, 2016

Caffeine and Cathinones

Filed under: Cathinones, MDPV — mtaffe @ 3:06 pm

An interesting presentation at the recent 2016 annual meeting of ASPET (part of Experimental Biology) from Gregory Collins reported on the interactive effects of caffeine with other stimulant drugs, including MDPV. It appears that Dr. Collins has recently been funded by the NIH to work on just such interactions of caffeine with MDPV and methylone.

I was interested about the premise here, due to to a longstanding interest in MDMA and its effects on people. In a short summary, the street “Ecstasy” supply has been notoriously contaminated with all sorts of psychoactive compounds other than MDMA. The ecstasydata.org testing site was set up in part as a warning/surveillance system. At present, if you go to advanced search and identify materials they have tested that return MDMA and nothing else you find 1281 (105 in 2016 to date) entries. If you search for items they have found positive for MDMA and at least one other psychoactive constituent you find 659 (17 in 2016 to date) items. This is 34% of the total number of MDMA-positive samples. Undoubtedly this has ebbed and flowed over the years but my recollection in doing similar searches now and again is that it has generally been the case that at least half of the tested items have been pure MDMA. [As always, do note that there is a selection factor for who bothers to send samples in to ecstasydata for testing. Likely to be non-random in terms of users (I would expect repeat submissions from afficionados or other highly interested and aware parties) and in terms of chances of non-MDMA constituents (I am making an assumption here that items of suspicious subjective effects/experiences are more likely to be submitted.).]

Returning to the caffeine story, I note that ecstasydata returns 347 items containing both MDMA and caffeine, representing 18% of the total MDMA-positive population or 53% of the contaminated subset.

Moving on, we can searched for MDPV only (9 items), MDPV plus some other psychoactive (28 items, 76% of MDPV -containing) and MDPV plus caffeine (17 items, 46% of all MDPV -containing, 61% of MDPV +other). Wondering if this was a function of MDPV really not being very Ecstasy-like and therefore being unlikely to turn up by itself in the population who are sending samples to ecstasydata, I looked at alpha-PVP (14 pure, 3 alpha-PVP+other and 0 with caffeine). Hmmm.

I also searched for methylone only (64 items), methylone plus some other psychoactive (30 items, 32% of methylone-containing) and methylone plus caffeine (7 items, 7.4% of all methylone-containing, 23% of methylone+other).

So the rationale for looking at caffeine interactions for methylone (and MDMA for that matter) is pretty good, even if we must recognize that the majority (going by this particular measure of epidemiology) of the street Ecstasy / Molly / methylone is probably pure MDMA or pure methylone.

The MDPV supply looks highly contaminated with caffeine (46%) but there is, to my view, a slightly bigger problem with assuming ecstasydata.org submissions reflect the drug that is available on the street. Going by media reports, MDPV (and alpha-PVP) seem to be very common in people that fit the profile of (or are reported to be) those who use prototypical stimulant drugs such as methamphetamine and cocaine. They seem less similar to the clubbing/raving Ecstasy using consumer. On average. This would, potentially, mean that ecstasydata.org is getting submissions from a much less representative part of those people exposed to MDPV versus methylone.

This is, of course, barely better than speculation and it will require information from other sources, such as DEA legal seizure activities, to further explore this issue.

Still, I think we can conclude that caffeine interaction with cathiones are of interest, even if they are not perhaps the first order of business (i.e., the effects of each drug by itself).

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It was in the session in the afternoon of Mon the 4th of April. It was organized by Li and Gerak and titled “Division for Behavioral Pharmacology Symposium: Quantitative Pharmacological Analysis of In Vivo Data and Its Implications in CNS Drug Discovery”.

Predicting Additivity: Abuse—Related Effects of “Bath-Salt” Mixtures
Gregory Collins—South Texas Veterans Hlth. Care Syst.—Audie L. Murphy VA Hosp.

March 2, 2016

Escalation of mephedrone IVSA under long-access conditions

Filed under: 4-MMC/Mephedrone, Cathinones, Methylone — mtaffe @ 10:41 am

StructureFig-MDMA-Methylone-MephedroneWe continue to be interested in assessing the relative abuse liability of new synthetic cathinone stimulants that pop up in recreational users. The most established entities such as mephedrone (4-methylmethcathinone; 4-MMC) and methylone (3,4-methylenedioxymethcathinone) are of particular interest to our research because they share some pharmacological properties with MDMA (Ecstasy), constituting a class of stimulants sometimes called entactogens. As you can see from the structures at the left, methylone is the direct cathinone cousin of MDMA– the ketone group on the beta carbon is the element that differentiates a cathinone from an amphetamine.

The 2013 and 2014 NFLIS showed that methylone may be more common than MDMA in the US and mephedrone continues to be popular in the UK. Our recent papers (Vandewater et al, 2015 and Creehan et al, 2015) compared the intravenous self-administration (IVSA) of methylone, mephedrone and MDMA within relatively short (2 h) daily training sessions in male and female rats, respectively. We found that rats will IVSA greater amounts of mephedrone compared with MDMA with methylone falling in between the other two. One prior study had found that rats will IVSA methylone at very high rates, more like a traditional stimulant than like MDMA, thus we were curious to further examine potential differences.

It has been shown that relatively long (6 h) daily sessions of access to cocaine (Ahmed and Koob, 1998; Larson et al., 2007) or methamphetamine (Kitamura et al., 2006; Schwendt et al., 2009) IVSA results in both higher daily drug intake and a progressive increase across sessions (termed “escalation”) relative to animals trained only in 1-2 h sessions. This has been conceptualize as a better rat model of the state of human stimulant addiction, as opposed to the interpretation of mere drug liking. In contrast, a prior study found no difference in total session intake of the entactogen class stimulant MDMA between long (6 h) and short (2 h) access groups over the first 11 sessions (Schenk et al., 2003). This seemed a little unusual to us and we showed in Vandewater et al (2015) that when run in the dark cycle (the rats’ active period of the day), male rat IVSA of MDMA
Fig1-LgA-ShA-MethyloneMMC-Revunder 6 h daily access conditions is higher than under 2 h access conditions. So we conducted a new study to determine how the rat IVSA of the two entactogen (MDMA-like) cathinones would fare under 6 h access conditions. The following has been recently accepted for publication:

Nguyen, J.D., Grant, Y., Creehan, K.M., Vandewater, S.A. and Taffe, M.A. Escalation of intravenous self-administration of methylone and mephedrone under extended access conditions., Addict Biol, 2016, in press. [ Publisher Site ][ PubMed ]

This study was conducted in male rats, trained to intravenously self-administer methylone or mephedrone in Short Access (ShA; 2 h) or Long Access (LgA; 6 h) sessions. The training dose was 0.5 mg/kg per infusion for each drug. The mean (SEM) number of infusions obtained by the four different groups is depicted in the first figure from the paper, reproduced here. There are two takeaway messages. First, the total daily intake is higher for the LgA groups for both drugs. Secondly the mephedrone LgA group obtained more infusions than did the methylone LgA group. [Significant differences from the first three sessions within group are indicated by shaded symbols. Significant differences between Access groups within a drug are indicated by * and differences between drugs, within Access condition, by †.] This further confirms, as did our MDMA LgA study, that there is nothing weird about entactogen IVSA under LgA vs ShA conditions- rats take more drug in 6 h than in 2 h. It also emphasizes that rats will take more mephedrone than methylone.

First 2 h intake of LgA groups

First 2 h intake of LgA groups Significant differences from the first three sessions within group are indicated by shaded symbols. Significant differences from MDMA are indicated by * and from methylone by †.

In some senses that is a trivial observation and one of the key measures of rats having achieved a state more similar to the addicted human is whether the LgA animals gradually take more drug in the time interval commensurate with the ShA animals- in our case the initial two hours of their 6 h session. This graph depicts the first 2 h infusions for the mephedrone (4-MMC) and methylone trained animals from this new study as well as the similar data for the MDMA 6 h animals from* Vandewater et al (2015). As you can see in this graph, the three drugs are clearly distinguished from each other on this key measure of “escalated” drug seeking behavior. First 2 h intake of MDMA is relatively stable across this training interval, first 2 h methylone intake increases across sessions and first 2 h mephedrone intake increases even more. The conclusion we reach from this is that both methylone and mephedrone have enhanced abuse liability compared with MDMA and they are more likely to lead to patterns of relatively uncontrolled or compulsive drug use in humans.

We also took this new study one step farther by asking how hard the four groups would work for a given magnitude of drug infusion. We do this by using a Progressive Ratio procedure. In the normal training the animals have a Fixed Ratio (as it is called) of lever presses to infusions. In this study, it was FR1 meaning they had only to make one press on the drug-associated lever to get an infusion of drug. In the PR procedure, the number of responses required for each successive drug infusion is progressively increased throughout the session (e.g., 1, 2, 4, 8, 16….). Eventually the rats will stop obtaining drug infusions. The last ratio they completed for a drug infusion is called the “breakpoint”, indicating how many lever presses they made for that final infusion. We also varied the available drug dose per infusion in a random order across session. Thus, we obtain an estimate of how hard each group will work for a given dose of drug. In order to directly compare liability for stimulant drug seeking across the groups we used the same two test drugs, methamphetamine (MA) and mephedrone/4-MMC.

The top panels contrast breakpoints during methamphetamine (MA) substitution in A) ShA and B) LgA groups. The bottom panels contrast breakpoints reached during mephedrone (4-MMC) dose substitution in C) ShA and D) LgA groups. Significant differences from vehicle control within-group are indicated by *, from the 0.125 dose by # and from all other dose conditions by %. Significant differences from all other groups, within a dose condition, are indicated by †.

The top panels contrast breakpoints during methamphetamine (MA) substitution in A) ShA and B) LgA groups. The bottom panels contrast breakpoints reached during mephedrone (4-MMC) dose substitution in C) ShA and D) LgA groups. Significant differences from vehicle control within-group are indicated by *, from the 0.125 dose by # and from all other dose conditions by %. Significant differences from all other groups, within a dose condition, are indicated by †.

This direct comparison study found that the rats trained to IVSA mephedrone under LgA conditions worked harder for either their training drug mephedrone or MA than did any other the other groups. There was no similar LgA/ShA difference for methylone-trained rats. This further emphasizes the substantial abuse liability of mephedrone/4-MMC. This drug appears to be quite similar to classical stimulants like methamphetamine and cocaine in this respect.

It continues, therefore, to be a mystery why a drug which releases serotonin in the nucleus accumbens to a greater degree than it releases dopamine would be such an effective reinforcer in the rat IVSA assay. There is considerable evidence, beyond just the fact that rats are pretty reluctant to IVSA MDMA compared with methamphetamine, that increasing serotonergic over dopaminergic effects of drugs is going to decrease the effectiveness as a reinforcer. And therefore decrease the liability for repeated use patterns. One of the scientific benefits of looking into the rewarding properties of some of these new cathinone stimulants is precisely this. It can suggest places where the existing dogma, based on the amphetamines in large part, may need some reconsideration.

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*We originally submitted this paper including a comparison with the prior MDMA group, cited and referenced so that there was no confusion as to where the data came from. First, a reviewer mentioned that this may be inappropriate. Second, the handling Editor noted that this was against journal policy. After a bit of back and forth with the Editor over the reasons for making this comparison we had to cave and remove the direct (i.e. including statistical comparisons) contrast with those prior data.

December 17, 2015

Daily Marijuana Use In Adolescents

Filed under: Cannabis — mtaffe @ 9:38 am

The Monitoring the Future Study of longitudinal drug trends releases the latest updates in December each year. The website has links to the updated tables and a few selected Figures.
2015-DailyPot-MtFThis graph depicts the percentage of 8th, 10th and 12th grade students in the US who indicate that they have used marijuana at least 25 days out of the past 30 (their definition of “Daily” in the survey). For those who want precision, the 2015 numbers are 1.1% for 8th graders, 3.0% for 10th graders and 6% for 12th graders. You may be inclined to view single digit percentages as no big deal. It seems like a small number. One percent? Hardly worth talking about, right?

Except if, as I do, you have children in one or more of these age ranges. And you go so far as to become acquainted with some of your children’s friends and schoolmates. And acquainted with some of their parents. What you quickly realize is that you know at least 50 kids within your child’s circle at least a little bit. Enough to know their name and something about them. Maybe 100. And your kid probably knows at least 200 fairly well.

So look around? Which 1 of these kids is already smoking pot every day in 8th grade? Which 6 are at the end of high school?

EVERY day. Smoking pot. And the odds are very good that this kid is smoking multiple times a day. Staying high for extended periods.

Remember this when you think dismissively to yourself that “that 7th grader looks stoned, hahaha” as I once did before catching myself. I should know better. And even I don’t really think specific kids are the ones smoking pot every day. Until I think about it.

But the stats say they are. Some of them.

 

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It is also the case that 6.5% of 8th graders, 15% of 10th graders and 21% of 12th graders have used marijuana at least once in the past month. 35% of 12th graders in the past year. This means the daily use rate is 17% of 12th graders who have tried marijuana at least once in the past year.

September 21, 2015

MDMA purity in Ecstasy 2013 to 2015

Filed under: MDMA — mtaffe @ 12:21 pm

I was just looking at the ecstasydata.org data on the content of pills submitted for analysis to see what new cathinones were turning up. While there, I did a little search on pills that were found to contain MDMA. There is a common thought that Ecstasy pills are notoriously contaminated with non-MDMA psychoactive drugs, supported by the data places such as ecstasydata.org have generated. My dabbling in the database in the past has my memory thinking that it was never any worse than about 50% of submitted pills that were positive for MDMA containing other drugs.

[Note, as always that this is not in any way a representative sample. It depends on voluntary submission of pill samples and it can, obviously, only ever cover a tiny fraction of what is being sold on the clandestine market at any time. There are arguments to be made that severe selection biases might creep in- from the type of people who bother to submit pills, or know about the service, to a possibility that those pills reputed to be suspicious in subjective outcome might be more likely to be submitted. So take this with caution.]

what was interesting is that when I limited the search parameters to 2013-2015, there were 285 entities with MDMA-only and 387 with MDMA+something else. Thus, 74% of the submitted items that contained MDMA were pure. It’s 85% in 2015, to date.

2000-2002: 80%
2003-2005: 53%
2006-2008: 31%
2009-2011: 46%
2012: 60%

So the purity of MDMA-containing street drugs appears to have rebounded across this 15 year interval.

I wonder how this is related to the MDMA-drought that heralded the onset of popularity of mephedrone and methylone and the “Molly” marketing situation by which consumers think they are getting the pure molecule, i.e., MDMA.

September 9, 2015

MDPV conditions a place preference in rats; but what about this CTA?

Filed under: Animal Models, Behavior, Cathinones, MDPV — mtaffe @ 10:44 am

A new paper presents what appear to be conflicting results about the rewarding properties of MDPV (“bathsalts”) in rats:

King HE, Wakeford A, Taylor W, Wetzell B, Rice KC, Riley AL. Sex differences in 3,4-methylenedioxypyrovalerone (MDPV)-induced taste avoidance and place preferences. Pharmacol Biochem Behav. 2015 Jul 26;137:16-22 [ PubMed ][ Publisher Link ]

King15-MDPV-CPPKing and colleagues have conducted a conditioning experiment in male and female Sprague-Dawley rats. In outline, place conditioning involves a two-compartment apparatus in which the two sides can be distinguished by tactile, visual and/or odor cues. Typically, a baseline session will be conducted in which the rat is allowed to roam around the entire apparatus (in this experiment for 15 min). The baseline amount of time spent on each side is then recorded. During the conditioning training, animals are injected with a drug (in this study 0.0, 1.8 or 3.2 mg/kg of MDPV, intraperitoneally) and confined in their non-preferred side of the apparatus for 30 min. On alternate days, all animals are injected with saline and confined to their initially-preferred side of the apparatus for 30 min. The key 15 min test, depicted in this graph, comes after 4 repetitions of each training session; the data are represented by the proportion of time spent on the drug-paired side. As you can see, the group of animals that received only saline injections on all eight days changed from the slightly less-preferring baseline test (~39% on the drug-paired side) to no-preference (~50% time spent on each side). In contrast, the groups that got active MDPV doses during active drug conditioning sessions increased the time spent on the drug-paired side (* depicts a significant difference from the 0 dose group on the post-conditioning test). These data are collapsed across the rat sex and the paper indicates that no sex differences were confirmed in the statistical analysis.

This is interpreted as a rewarding effect of MDPV at these doses.

So far so good.

There is a slightly less-intuitive finding in this paper as well.

King15-MDPV-CTAThe authors also conducted a taste-aversion study in the same animals. In a classic Conditioned Tasted Aversion study, rats are allowed to consume some food or fluid that they find palatable, such as saccharin-sweetened water. If the rats are given some treatment that makes them feel bad (such as an injection of lithium chloride) after consuming the highly-palatable food/fluid, they tend to avoid it on a subsequent re-test. In this King study, the investigators allowed the rats to consume saccharin solutions for 20 min just prior to receiving their MDPV injections for the above-described place preference assay. Pretty efficient design!

What you can see from this graph is that the male and female rats that only received saline injections after drinking saccharin (M0 and F0 groups) gradually consumed more saccharin prior to each of the three remaining active-drug conditioning sessions. In contrast, the groups that received MDPV consumed less saccharin solution. The stats symbols are a little complicated on this one so from the paper: [Panel A (males): *M0 significantly greater than M1.8 and M3.2; ^M0 significantly greater than all drug-treated groups; %M1.0 and M1.8 significantly greater than M3.2; #M3.2 significant decrease from Trial 1; αM3.2 significant decrease from Trial 2. Panel B (females); *F0 significantly greater than F1.8 and F3.2; ^F0 and F1.0 significantly greater than F3.2; #F1.0 significant increase from Trial 1.].

Wait, huh? This implies that the animals are expressing feeling bad because of MDPV (hence the Taste Aversion) and feeling good because of MDPV (the Place Preference).

At the same time and from the exact same doses, thanks to this efficient experimental design.

The answer to this conundrum lies in an older paper by Linda Parker.

Parker LA. Rewarding drugs produce taste avoidance, but not taste aversion. Neurosci Biobehav Rev. 1995 Spring;19(1):143-57. [ PubMed ]

Parker95-CTA-AversiveTasteReactionThis paper contrasted changes in sucrose preference induced by lithium chloride versus a number of abused drugs that are are reinforcing in self-administration and place preference assays such as amphetamine, cocaine, nicotine, morphine, etc. There were low/medium dose and high doses tested, I’m including only the high-dose graph here for simplicity but the takeaway message isn’t changed. As you can tell from this high-dose graph, all test drugs except morphine produced a significant reduction in sucrose drinking compared with the injection of saline (CTA). The key part of this study was that the rats were video taped for behavioral scoring by a reviewer blinded to drug treatment condition. Importantly, the rats were scored for Aversive Taste Reactions (ARs) to the drinking spouts / fluid taste. This included gaping, chin rubbing, paw pushing and letting fluid drip from the spout without consuming it. Lithium Chloride and nicotine were the only drugs that resulted in drug-injected rats expressing more ARs to the sucrose on the test day compared with the saline-injected rats. This implies, as the author underlined in discussion comments, that the mechanisms by which saccharin preference is reduced differs. Sometimes when a CTA is produced in a rat, it really is a reflection of the conditioning process making the rat find the saccharin taste unpleasant. Other times, the conditioning process doesn’t make the taste unpleasant, but rats certainly find it less pleasurable, reinforcing or rewarding.

In slightly different language, we might interpret this as meaning that the experience of the type of pleasurable experience caused by drugs of abuse can diminish the value of other stimuli or experiences that rats would otherwise find to be pleasurable.

This interpretation dovetails nicely with our prior paper in which rats were permitted to both self-administer MDPV intravenously and to run on an activity wheel [see blog post]. What we found was that as the rats initiate the consistent self-administration of MDPV, they decrease the amount of wheel activity that they engage in. That study was consistent with an earlier one we published showing that self-administration of methamphetamine also gradually decreases the amount rats will use the activity wheel [ see blog post ].

We interpret this phenomenon to reflect the drug stimulus devaluing the degree to to which a rat finds wheel activity rewarding.

As such, this gives us an animal model to further explore concepts that may explain why some individuals who use drugs regularly become addicted whereas some other individuals can cease drug use. The comparative value of other sources of reinforcement (social, family, vocational, recreational, etc) versus the drug experience may be a critical determinant of who will spiral into a compulsive drug use problem.

___
Additional reading: Hunt and Amit, 1987.

September 1, 2015

MDPV and Methylone self-administration in rats

Filed under: Cathinones, Methylone — mtaffe @ 1:19 pm

A new paper from the Designer Drugs Research Unit within the NIDA IRP compares the self-administration of MDPV and methylone in rats. Methylone (3,4-methylenedioxymethcathinone) appears to have replaced MDMA in the NFLIS forensic laboratory database 2013-2014. This is only the third group to publish self-administration data with methylone so this is a very welcome tie-breaker. Watterson and colleagues (2012) reported fairly robust self-administration of methylone in male Sprague-Dawley rats and we reported marginal to weak self-administration in both female and male Wistar rats.

Schindler CW1, Thorndike EB, Goldberg SR, Lehner KR, Cozzi NV, Brandt SD, Baumann MH. Reinforcing and neurochemical effects of the “bath salts” constituents 3,4-methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxy-N-methylcathinone (methylone) in male rats. Psychopharmacology (Berl). 2015 Aug 29. [Epub ahead of print] [ PubMed ]

Schindler15-Fig1IVSAIn this new study the authors trained male Sprague-Dawley rats to self-administer MDPV (0.03 mg/kg/infusion) or methylone (0.3 mg/kg/infusion) in two hour sessions during the dark (active) cycle. This figure to the right depicts nose-poke responses for the drug-associated (active) and non-associated (inactive) holes. Sessions indicated by A indicate Acquisition and E1-E6 indicate extinction sessions in which no drug resulted from a nose-poke response. Shaded symbols indicate when active and inactive responding was significantly different. The comparison makes it clear that while basic self-administration criteria were established for both drugs (active and inactive responding differed) the MDPV group made more responses than the methylone-trained group. They also exhibited a clear patter of self-administration from the second day of training compared with the seventh session for methylone. A followup study (Fig 2 of the paper) trained a group on a slightly higher 0.5 mg/kg/inf methylone dose (identical to the one we used and the highest, most effective dose used in the Watterson et al (2012) study). This group was given 17 acquisition sessions and the trajectory of behavior matched that of the 0.3 mg/kg group.

Schindler15-DoseResponse After acquisition, and before the extinction sessions reported in the first figure, the authors conducted a dose-substitution procedure in which the MDPV and 0.3 mg/kg/inf methylone [I didn’t mention the cocaine-trained group in the above discussion, but they were included in the study.] animals were given varying doses of drug in each infusion on different days. Each dose was provided on three consecutive days and the intakes on days 2-3 are reported here. As you can see, the methylone trained animals were insensitive to dose-substitution, particularly in comparison with MDPV (or cocaine) trained rats. This is very similar to our finding, particularly if you consider that the Schindler paper didn’t use a dose of methylone higher than 0.5 mg/kg/infusion in their procedure. In Vandewater et al (2015) and in Creehan et al (2015) the methylone-trained male and female rats really only expressed differences in intake between 0.125 and 2.5 mg/kg/inf doses. The functions were pretty linear (and descending, like the cocaine one shown above) but they were very shallow.

The take-away message is pretty similar to our studies and discordant with the findings of Watterson, et al 2012, 2014. Methylone supports low levels of self-administration in rats, but to a much lower extent than does MDPV in a direct comparison here. A similar indirect comparison of our studies (Vandewater et al 2015; Creehan et al 2015 vs Aarde et al, 2013, Aarde et al, 2015) comes to the same conclusion.

August 19, 2015

Mephedrone is also more reinforcing than MDMA or Methylone in male rats

Filed under: 4-MMC/Mephedrone, Cathinones, MDMA, Methylone — mtaffe @ 2:30 pm

StructureFig-MDMA-Methylone-MephedroneMethylone has now surpassed MDMA in Forensic Laboratory samples in the US. Mephedrone is less popular in the US but maintains a high degree of popularity in the UK. We recently published a paper [Creehan et al, 2015; PubMed; blogpost] showing that the drug mephedrone was a more effective reinforcer than either MDMA or methylone in female rats.

Our followup study which compared the self-administration of these three drugs in male rats has been accepted for publication.

Vandewater, S.A., Creehan, K.M. and Taffe, M.A. Intravenous self-administration of entactogen-class stimulants in male rats. Neuropharmacology, 2015, 99:538-545. [ PubMed ][ Publisher Site ]

We are interested in the reinforcing potential of mephedrone and methylone due in large part to their neuropharmacological similarity to MDMA. Specifically, these three drugs have a relatively greater enhancement of serotonin in the nucleus accumbens of the rat brain compared with the enhancement of dopamine. The latter effect is associated with pleasurable/rewarding effects of traditional stimulant drugs like methamphetamine whereas the preferential serotonin release/accumulation is associated with reduced reward potential. In short, rats have shown much less intravenous self-administration of MDMA versus methamphetamine.

Fig1-XY-3drg-Acq The fact that Mephedrone and Methylone share the MDMA-like neurochemical profile groups them all together as atypical or entactogen-class stimulant drugs and predicts less compulsive use compared with traditional stimulants like methamphetamine. Yet several self-administration studies with mephedrone have already shown a greater abuse potential compared with MDMA. The one available study with methylone (Watterson et al, 2012; blogpost) prior to our work implied the same. Our study in female rats was the first to directly compare these three drugs and we confirmed that mephedrone is a much more effective reinforcer, thus predicting higher liability for compulsive use. MDMA and methylone, however, appeared to be quite similar.

Our new study in the male rats used essentially the same procedures as the study in females and had the same conclusion. In this figure we show mean (±SEM) daily infusions of drug (upper panel) and the proportion of responses on the drug-associated lever versus the inactive lever (lower panel) obtained for groups of male rats trained to self-administer MDMA (N=17), Methylone (N=14) or Mephedrone (N=15) in 2 hour sessions. [Significant differences from the first session within group are indicated by *, differences between mephedrone and both other groups by #, differences from methylone by ‡ and differences from MDMA by †.] The MDMA and mephedrone groups differed from each other and the methylone-trained group was intermediate.

The results from the female animals in the previous study differed slightly in that the methylone and MDMA intakes were nearly identical and were both significantly different from the mephedrone self-administration.

MDMA-Methylone-AcqWe did some follow up comparisons between males and females for the new paper and those ended up in the Supplemental Materials file. The only significant sex-difference was for the MDMA-trained groups where there was a significant main effect of rat sex confirmed in the analysis. The methylone intakes appeared to be nearly identical between males and females, as is depicted and the mephedrone intakes did not differ either (not shown).We did some further sub-groups analysis and found that this difference in MDMA self-administration was mostly in the less-preferring half of the male group.

At this point we have a direct confirmation of the enhanced liability of mephedrone for compulsive use over that of MDMA. This is a clear rejection of the suggestion based on intra-cranial self-stimulation reward data that it has reduced liability- clearly something is off about the way the results were interpreted by Bonano et al (2014). Methylone appears to be much more similar to MDMA although it might be a slightly more effective reinforcer in male rats. Nevertheless we still cannot reconcile our results with methylone self-administration with the apparently robust self-administration reported by Watterson et al (2012). As there are only now three published studies of the self-administration of methylone, we must await further studies to better understand the reasons for these different outcomes.

July 29, 2015

Modeling the preference for peers that you get high with

Filed under: Animal Models, Behavior, Cocaine, Neuroscience — mtaffe @ 1:02 pm

A new paper from Mark Smith and colleagues addresses whether drug exposure can differentially condition a preference for certain peers in a rat model.

Smith MA, Strickland JC, Bills SE, Lacy RT. The effects of a shared history of drug exposure on social choice. Behav Pharmacol. 2015 Apr 28. [Epub ahead of print]

The study focused on “choice” rat groups, one of which (N=16) was to receive cocaine injections and one of which (N=16) was to receive saline injections. The choice rats were then destined to have social interactions with a social peer rat that had received cocaine or a social rat that had received saline.

Smith15-RatPrefThe social choice apparatus is depicted in this figure from the paper. The choice rat is permitted to roam about the apparatus and choose proximity to one of two partner rats. A pre-conditioning test established the amount of time a given choice rat spent in proximity to each of the saline- or cocaine-treated partner rats.

The choice rats then underwent a total of 10 conditioning sessions in normal home cages. For these sessions, the choice rats would receive their cocaine or saline injections and then interact with a single partner rat for 30 min. On five of those sessions the partner rat was as cocaine treated animal and on the other five the partner was saline treated.

The critical post-conditioning preference test was then conducted.

A change in preference was expressed as the amount of time spent on the side of the apparatus containing each partner rat divided by the time spent with that partner during the pre-conditioning test. Two analyses were conducted, one just scoring time in each half of the apparatus and a second analysis scoring time spent in ~the front half of each side, i.e., in closer proximity to the partner. This made no difference in the results, including the fact that there was no change in the amount of time spent in this “neutral” or non-social zone after conditioning.

The takeaway message was that there was a significant increase in the amount of time spent with the similarly-treated partner when all choice rats were considered. However when the group analysis was conducted, only the cocaine-treated choice rats exhibited increased preference for the cocaine-treated partner. Saline-treated choice rats had no partner preference.

The takeaway message is that cocaine-treated rats prefer to hang out with other cocaine-treated rats. It wasn’t a general social-conditioning effect, since there was no differential effect on time spent in the non-social part of the apparatus.

There is one major caveat. The size of the effect was about an 8% increase in the time cocaine-treated choice rats spent with the cocaine-treated partner during the choice test. This amounted to about 49 seconds.

This is a limited initial finding but it obviously has promise for investigating social factors that enhance or diminish drug preferences, drug reward and the power of drug-related cues to shape behavior.

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