Interestingly, ShA drug self-administration also led to escalation of intake, particularly for 4MMC. This finding suggests that dysregulated drug intake is not limited to LgA conditions for α-PVP and 4MMC, similar to a past study showing that LgA and ShA cocaine self-administration both led to escalation of intake (Beckmann et al., 2012). To our knowledge, this is the first study to statistically compare male and female rodents for 4MMC and α-PVP self-administration, and the resulting neurochemical changes.
Figure 1.
- Long incubation times were applied in order to show whether the cytotoxicity of studied compounds increase with time, which is relevant since the common abuse pattern of synthetic cathinones includes long sessions during which multiple doses are administered (Zawilska and Wojcieszak 2013).
- Likewise, adolescent rats exposed to methylone exhibited persistent depletions of serotonin and deficits in reference memory (Lopez-Arnau et al. 2014).
- The longer side-chain α-pyrrolidinophenones and their fluoro- and methoxy-analogs produce more pronounced maximal cytotoxicity, with regard to mitochondrial activity and cell membrane integrity, than the five-carbon α-PVP and its substituted derivatives.
- Significant reduction of cell viability was observed in RPMI 2650 cells at concentrations of 10 to 300 μM, in SH-SY5Y cells between 50 and 300 μM, in Hep G2 cells between 100 and 300 μM, and in H9c2(2-1) cells at 200 and 300 μM after 24-h incubation.
- However, mephedrone and methylone have also been reported to not persistently deplete brain monoamine content (Baumann et al. 2012).
- Instead of omitting these values from graphs and analyses, we substituted a value of 0.005 ng/mg, which is halfway between zero and the level of quantitation of 0.01 ng/mg.
Each animal had a familiarization (training) session with a pair of identical objects (~ 5 cm long x 5 cm wide x 10 cm high) placed 5 cm away from the walls but adjacent in the open field. 6 hours after familiarization (Leger et al. 2013), each mouse was tested in the same box with a novel object replacing one of the familiar objects.Familiarization and testing sessions lasted 10 minutes, were video recorded, and were assessed offline. Exploration was defined as sniffing the object or orienting the head towards the object while the subject was within 1 cm of distance from the object. The percentage of the total exploration time spent exploring the novel object was calculated to reflect recognition memory.
Figure 1:
- Treatment with PV9 for 24 h caused a significant decrease in the survival of Hep G2 and RPMI 2650 (10–300 μM), SH-SY5Y (100–300 μM), and H9c2(2-1) (200 and 300 μM) cells.
- The identified studies that used more than 21 sessions of self-administration (Ahmed and Koob, 1999; Belin et al., 2009; Greenwell et al., 2009a; Greenwell et al., 2009b) provided very little information on the neurochemical changes that occur beyond 21 sessions.
- Each animal had a familiarization (training) session with a pair of identical objects (~ 5 cm long x 5 cm wide x 10 cm high) placed 5 cm away from the walls but adjacent in the open field.
- Substituted analogs differ from native PV8 as they affect H9c2(2-1) cell viability even after 24 h.
- Sex differences for α-PVP during autoshaping (Fig. S2) did not lead to sex differences in α-PVP self-administration (Fig. 2).
- Despite this, there is a strong consensus among experts that Flakka has a high potential for abuse, dependence, and addiction.
Pyrrolidinophenones are a class of stimulant recreational designer drugs including many substituted cathinones. Brains were sliced into 1 mm thick coronal sections, and these slices were placed flat on a cold plate over ice. Using a 1.5 mm diameter tissue biopsy-punch, regions of interest were taken from individual slices, as we have described previously (Murnane et al. 2012). Synthetic cathinones (“bath salts”) are β-ketone analogs of amphetamines, yet few studies have examined their potential neurotoxic effects. Rats were given 7 days to recover from surgery before commencement of ICSS procedures, during which they received daily injections of 2.5mg/mL meloxicam (0.15mL volume) to minimize postsurgical discomfort.
Flakka Vs. Bath Salts
Spontaneous alternation in the Y-maze has been proposed to measure hippocampus-dependent spatial working memory (Walker and Gold 1994). Performance in the Y-maze was assessed by both automated quantitation (Maze Engineers; Cambridge, MA) and manual observation. Spontaneous alternations were calculated as the percentage of the total arm entries minus two composed of triads containing entries into all arms. For α-PVP, 4-MEC, and METH, higher doses (5, 100, and 3mg/kg, respectively) produced elevations in ICSS threshold values, with only METH producing significant elevations (mean±95% CI; α-PVP, 19.83±38.64; 4-MEC, 28.00±31.72; METH, 84.39±69.48%).
What are the side effects of Flakka?
Although spontaneous alternation behavior has been closely linked to brain cholinergic systems (Lalonde 2002), previous studies have documented a role for catecholamines, as dopamine depletions in the striatum or septum result in impaired spontaneous alternations (Taghzouti et al. 1985). Likewise, norepinephrine levels in the hippocampus are elevated during spontaneous alternations (Men et al. 1999) and depletion of norepinephrine from forebrain projections results in impaired spontaneous alternation (Pisa and Fibiger 1983). Relating specific neurochemical depletions to specific behavior impairments presents rich research opportunities. In this study, we document persistent effects of the second-generation pyrrolidine synthetic cathinone α-PPP on behavior and neurochemistry. We document that, consistent with previous studies of amphetamine derivatives (Murnane et al. 2012), α-PPP acutely decreases body weight over the course of a standard 6-hour dosing regimen.
Alpha-PVP is known for producing intense stimulation alpha-pyrrolidinopentiophenone function and can lead to extreme behavioral changes, including agitation and violent outbursts. Both substances can induce euphoria, heightened sex drive, increased sociability, and hallucinations. When used in excessive amounts, they can lead to severe negative effects, including high blood pressure, elevated heart rate, paranoia, aggression, panic attacks, and psychosis. Both drugs can be consumed in various ways—such as smoking, injecting, or snorting—and carry a significant risk of overdose and death. The initial effects of taking Flakka are similar to those of bath salts and methamphetamines.
Standards for GLU analysis were prepared by weighing approximately 25 mg of GLU (Alfa Aesar, Ward Hill, MA). The standard was transferred to a volumetric flask and diluted to volume with mobile phase A (5 mM ammonium acetate and 0.1% formic acid, aqueous) and labeled as stock solution. The stock solution containing approximately 2.5 mg/ml of GLU was then diluted to encompass a concentration range from 2500 to 10 ng/ml. Standards for ECD were prepared by weighing approximately 1 mg of analytes DA (Sigma-Aldrich, Buchs, Switzerland), DOPAC (Sigma-Aldrich, Buchs, Switzerland), HVA (Sigma-Aldrich, Buchs, Switzerland), 5-HT (Sigma-Aldrich, St. Louis, MO), 5-HIAA (Sigma-Aldrich, St. Louis, MO), and NE (Sigma-Aldrich, St. Louis, MO).
We report that α-PPP persistently depletes dopamine, DOPAC, serotonin, and norepinephrine levels in the striatum, as well as norepinephrine levels in the prefrontal cortex, in male mice five days after treatment, using an 80mg/kg unit dose of α-PPP. Our findings suggest that the striatum may be a brain region that is particularly sensitive to the untoward effects of α-PPP, and possibly other synthetic cathinones. Moreover, at least for dopamine, these changes do not appear to be related to changes in neurotransmitter production or metabolism, as we did not detect any changes in dopamine turnover, suggesting that they are related to poisoning of the dopamine system rather than temporary changes in dopamine metabolism. Notwithstanding these findings, much work remains to be completed to elucidate the role of sex, age, species, strain, dose, drug class,structural modification, and pharmacological mechanism in the putative neurotoxicity effects of cathinones, including the second-generation pyrrolidines. Stimulant-induced neurochemical changes may occur at different times for different brain regions or neurotransmitter systems.
Self-administration of α-PVP and 4MMC induced plasticity in neural circuitry that may be driving compulsive drug taking or producing a deficit state for normal reward, thereby increasing motivation to continue self-administration (Koob and Le Moal, 2005). Furthermore, the increased GLU levels observed for LgA groups may be enhancing drug seeking (Koob, 2010), although drug seeking was not directly measured in the present study. Cocaine and amphetamine, which have similar mechanisms of action as α-PVP and 4MMC, respectively, induce synaptic plasticity within the DA system and DA receptive neurons.
Adverse effects
When heated up, it gives off a foul-smelling smoke characterized as smelling like dirty socks. In H9c2(2-1) cells, the viability was reduced by 76–78% at 200 μM and by approximately 96% at 300 μM, irrespective of the incubation time. In the concentration range of 10–300 μM, significant cytotoxic effects were observed in SH-SY5Y, Hep G2, and RPMI 2650 cells after 24-h incubation, and in SH-SY5Y and RPMI 2650 cells after 72-h incubation. However, at 25–300 μM, the viability of Hep G2 cells was affected only after 72-h incubation. In H9c2(2-1) cells, significant effects were observed at 100–300 μM, irrespective of the incubation time (Fig. 6c).