Results reported for methylenedioxypyrovalerone (MDPV) and methylone were obtained from previous publications (Watterson et al., 2012, 2014). There were two scenarios in which neurotransmitter data were excluded from graphs and analyses. First, there were a few cases in which amygdala samples had very elevated levels of DA, DOPAC, and HVA. Because of the closeness of amygdala and striatum in the brain, it is likely that a small amount of striatum was inadvertently included as part of the amygdala sample.

All statistically significant effects of condition (LgA vs naïve) on neurotransmitters, and interactions between condition and sex are shown in Table 1 and Fig. Self-administration of α-PVP during LgA conditions had no effect on 5-HT in any brain region but increased 5-HIAA in all brain regions compared to naïve. In contrast, self-administration of 4MMC during LgA conditions decreased 5-HT in all brain regions except thalamus and decreased 5-HIAA in most brain regions compared to naïve (Table 1 and Fig. 3). Small, but significant changes in DA levels for LgA groups compared to naïve were found in select brain regions where α-PVP decreased DA levels in thalamus, whereas 4MMC increased DA levels in hypothalamus and thalamus. The most striking effects of condition were large elevations of DOPAC and HVA levels in striatum that were noted following LgA self-administration of both α-PVP and 4MMC. DOPAC level was also elevated in hypothalamus for LgA groups compared to naïve (Table 1 and Fig. 3).

Flakka was added to the Schedule I list in 2014 by the Drug Enforcement Administration (DEA). This was part of an effort to curb the abuse of synthetic drugs, which were becoming increasingly popular and causing significant health issues. Exposure of Hep G2 cells to PVP, PV8, and PV9 for 15 min significantly lowered the fluorescence anisotropy of the DPH probe, which is negatively related to the fluidity of the inner part of the cell membrane. Statistically significant increases in membrane fluidity were observed in the concentration range from 50 to 300 μM for PVP and 25 to 300 μM for PV8 and PV9, while the most pronounced effect was observed after treatment with PV8 (300 μM) (Fig. 8). However, neither PVP, PV8, or PV9 lowered the fluorescence anisotropy of the TMA-DPH probe, which reflects the fluidity of the polar head-group portion of the cell membrane (Fig. 8). Hep G2 and H9c2(2-1) cells were cultivated in DMEM, SH-SY5Y in DMEM/F12, and RPMI 2650 in MEM with Earle’s salts and 1× Non-Essential Amino Acids Solution media, supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/ml)-streptomycin (100 μg/ml) at 37 °C in a humidified atmosphere enriched with 5% CO2.

Effects of Flakka abuse

Pyrovalerone derivatives (α-pyrrolidinophenones) constitute a branch of synthetic cathinones, a second most prominent group of novel psychoactive substances (NPS). Since 2008, each year has seen the introduction of a number of novel synthetic cathinone derivatives into the dynamic, clandestine NPS market, in an attempt to circumvent legal restrictions (EMCDDA 2017; Majchrzak et al. 2018; Zawilska and Wojcieszak 2017). The most remarkable examples of α-pyrrolidinophenones include 3,4-methylenedioxypyrovalerone (3,4-MDPV) and α-pyrrolidinopentiophenone (α-PVP). Frozen tissues were weighed, sonically disrupted in 100 µl of 0.3 N HClO4 and centrifuged for 10 minutes at 4ºC to remove cellular debris. A 100 µl aliquot of the supernatant was placed in an WPS-3000TBSL autosampler maintained at 10ºC, and 10 µl was injected onto a Thermo Scientific (Waltham, MA) Hypersil BDS C18 column (35ºC) with Thermo Scientific Dionex Test Phase running at a flow rate of 0.5 mL/min. Coulometric detection was accomplished with a Thermo Scientific Dionex 6011RS electrode cell, and the signal analyzed on a Thermo Scientific Dionex Chromeleon CDS processing platform.

• Another limitation was that only one self-administration dose of α-PVP and 4MMC was included.
• Drug users take flakka to get a feeling of euphoria, a heightened sense of awareness, stimulation, and energy.
• ED50 values represent the mean dose leading to 50% maximal response with upper 95% confidence limits (UL) and lower 95% confidence limits (LL).
• In contrast, self-administration of 4MMC during LgA conditions decreased 5-HT in all brain regions except thalamus and decreased 5-HIAA in most brain regions compared to naïve (Table 1 and Fig. 3).
• All graphical data presentations were created using GraphPad Prism 7.0 (GraphPad Software Inc.; La Jolla, CA).

What are the complications of Flakka use?

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.

Comedown Effects

Second, an unknown peak interfered with the integration of the DOPAC or NE peak in select samples of PFC and hypothalamus. Additionally, in some samples of thalamus, DOPAC levels were below the level of quantitation. 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. For self-administration alpha-pyrrolidinopentiophenone function data, autoshaping data were analyzed with separate mixed factors ANOVAs (day x lever x sex) to compare active and inactive lever presses, with day and lever as within-subjects factors, and sex as a between-subjects factor. The additional 21 days of self-administration were also analyzed with separate mixed factors ANOVAs (day x lever x sex), which compared responses on the active and inactive levers. If the assumption of circularity was violated, the Geisser-Greenhouse Adjustment was employed.

Cedrick Maceo Daphney

Brain tissue samples were weighed and placed in cryovials containing stainless steel grinding balls. Supernatant was then transferred to a 96 well plate for analysis by electrochemical detection (ECD). A Thermo Scientific CoulArray Multi-Channel ECD Array system (model 5600A; Thermo Scientific, Waltham, MA, USA) was used to analyze neurotransmitter concentrations. The array detector contained 16 coulometric electrochemical cells that provided quantitation of multiple neurotransmitters and metabolites simultaneously.

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.

LgA self-administration of α-PVP increased 5-HIAA levels in all brain regions, compared to control. In contrast, both LgA and ShA 4MMC self-administration decreased 5-HT and 5-HIAA levels in most brain regions. LgA exposure to both synthetic cathinones increased DOPAC levels in hypothalamus and striatum, and increased HVA levels in striatum compared to control.

Links to NCBI Databases

A 10-μl aliquot was injected onto a Luna Omega 2.1 x 150 mm column (Phenomenex, Torrance, CA) coupled to a LPG-3400RS pump, WPS-3000TBRS autosampler, and a CoulArray electrochemical detector. The mobile phase consisted of 50 mM sodium phosphate, 47 mM citric acid, 0.14 mM EDTA, 0.64 mM octanesulfonic acid, and 5% methanol, with a flow rate of 0.4 ml/min. The detector was set to sequentially deliver potentials of −150 mV, 150 mV, 400 mV, and 600 mV.

• After extending incubation time to 72 h, PV9 caused almost complete loss of viable SH-SY5Y (max. reduction by 94%), Hep G2 (max. reduction by 95%), and RPMI 2650 cells (max. reduction by 99%) when applied at 200 and 300 μM, and a 91% reduction of H9c2(2-1) when applied at 300 μM.
• 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.
• In contrast, female ShA rats had lower DA levels than males in PFC, higher DOPAC levels than males in amygdala, hypothalamus, and striatum, and higher HVA levels than males in amygdala and striatum.
• The detector was set to sequentially deliver potentials of −150 mV, 150 mV, 400 mV, and 600 mV.
• In contrast, other studies measured neurochemical effects several days after the last drug exposure (Briand et al., 2008; Hadlock et al., 2011; Schwendt et al., 2009), which may be during withdrawal.
• For example, previous studies have shown that depletion of serotonin through provision of a tryptophan-deficient diet to rats results in impaired spontaneous alternations (González-Burgos et al. 1995).

Substituted analogs differ from native PV8 as they affect H9c2(2-1) cell viability even after 24 h. 4-F-PV8 applied for 24 h markedly reduced the viability of SH-SY5Y (100–300 μM), Hep G2 (50–300 μM), RPMI 2650 (10–300 μM), and H9c2(2-1) (200 and 300 μM) cell lines, with the greatest reduction by 42% (SH-SY5Y), 77% (Hep G2), 79% (RPMI 2650), and 72% (H9c2(2-1)) (Fig. 4b). Cell viability was significantly decreased in SH-SY5Y (25–300 μM; maximal reduction by 83%), Hep G2 (50–300 μM; maximal reduction by 97%), RPMI 2650 (10–300 μM; maximal reduction by 97%), and H9c2(2-1) cells (10–300 μM; maximal reduction by 79%) (Fig. 4b).

Flakka has emerged as a significant substance of abuse, yet research on its addictive properties is still limited due to its relatively recent appearance on the drug scene. Despite this, there is a strong consensus among experts that Flakka has a high potential for abuse, dependence, and addiction. This assessment is based on the drug’s potent effects, its ability to alter brain chemistry, and comparisons with other synthetic drugs like bath salts.