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. Author manuscript; available in PMC: 2023 Dec 1.
Published in final edited form as: Psychopharmacology (Berl). 2022 Oct 21;239(12):3833–3846. doi: 10.1007/s00213-022-06252-z

Methamphetamine-induced Lethal Toxicity in Zebrafish Larvae

Yu Chen 1,‡, Alexander Wisner 1, Isaac Schiefer 2,3, Frederick E Williams 1, F Scott Hall 1,*
PMCID: PMC10593407  NIHMSID: NIHMS1936771  PMID: 36269378

Abstract

Rationale.

The use of novel psychoactive substances has been steadily increasing in recent years. Given the rapid emergence of new substances and their constantly changing chemical structure, it is necessary to develop an efficient and expeditious approach to examine the mechanisms underlying their pharmacological and toxicological effects. Zebrafish (Danio rerio) have become a popular experimental subject for drug screening due to their amenability to high-throughput approaches.

Objectives.

In this study, we used methamphetamine (METH) as an exemplary psychoactive substance to investigate its acute toxicity and possible underlying mechanisms in 5-day post-fertilization (5 dpf) zebrafish larvae.

Methods.

Lethality and toxicity of different concentrations of METH were examined in 5 dpf zebrafish larvae using a 96-well plate format.

Results.

METH induced lethality in zebrafish larvae in a dose-dependent manner, which was associated with initial sympathomimetic activation, followed by cardiotoxicity. This was evidenced by significant heart rate increases at low doses, followed by decreased cardiac function at high doses and later time-points. Levels of ammonia in the excreted water were increased but decreased internally. There was also evidence of seizures. Co-administration of the glutamate AMPA receptor antagonist GYKI-52466 and the dopamine D2 receptor antagonist raclopride significantly attenuated METH-induced lethality, suggesting that this lethality may be mediated synergistically or independently by glutamatergic and dopaminergic systems.

Conclusions.

These experiments provide a baseline for the study of the toxicity of related amphetamine compounds in 5 dpf zebrafish as well as a new high throughput approach for investigating the toxicities of rapidly emerging new psychoactive substances.

Keywords: Zebrafish, Methamphetamine, Seizure, Glutamate, Ammonia, Heart, Dopaminergic Receptors

Introduction

Although problems associated with the use of psychostimulant drugs have been overshadowed in recent years by the opioid crisis, overdoses resulting from abuse of psychostimulant drugs has also been increasing in recent years (Bose et al. 2018; United Nations Office on Drugs and Crime 2021), and psychostimulant overdoses or combined opioid/stimulant overdoses appear to reflect the next stage of the current opioid epidemic (Hall and Miczek 2019). Among psychostimulant drugs, methamphetamine (METH) is one of the most commonly abused substances. METH is known for its neurotoxicity for dopaminergic and serotonergic neurons, which is evidenced by the depletion of dopamine and serotonin and the oxidation of their synaptic terminals (Albers and Sonsalla 1995; Battaglia et al. 2002; Graham et al. 1978; Granado et al. 2010; Spina and Cohen 1989). Currently there is no approved treatment for METH dependence, nor are there treatments for the adverse effects of METH associated with chronic use (Soares and Pereira 2019). Treatments for acute overdose are limited to symptomatic treatments for tachycardia/high blood pressure (labetolol), myocardial ischemia/infarction (nitroprusside), platelet aggregation (aspirin), psychomotor agitation (haloperidol, lorazepam, and diphenhydramine), and seizures (benzodiazepines or barbiturates) (Richards et al. 2015; Center for Substance Abuse Treatment 1999). Better approaches to the treatment overdose from METH, and other stimulant drugs, are needed. It will be essential to base these treatments upon a deeper understanding of the mechanisms underlying acute stimulant overdose.

This situation is becoming even more pressing with the emergence of large numbers of new amphetamine-like stimulant drugs whose potential for toxicity and lethality are largely unknown (Riley et al. 2020), but some of which may be far more potent than METH. Essential to this effects is the development of an efficient and expeditious approach to assess the toxicity and potential mechanisms of these numerous emerging compounds. Zebrafish (Danio rerio) are a model organism that can bridge the gap between cell culture approaches and studies in mammals. Studies in larval zebrafish combine the high-throughput advantages of cell culture with the study of an organism with a complete and complex physiology that is highly homologous to mammals (Ali et al. 2011). Of critical relevance to the current report, larval zebrafish possess functional dopaminergic, serotonergic, and glutamatergic neurons (Chen et al. 2010a; Maximino and Herculano 2010). Additionally, at this age the heart is fully developed and functional. Studies have shown that METH produces cardiotoxicity and behavioral changes in zebrafish (Bugel and Tanguay 2018; Fang et al. 2016; Ickes 2015; Mi et al. 2016), but acute overdose and lethality have not yet been studied. In addition to the factors mentioned above, 5-day post-fertilization zebrafish larvae were chosen for study based upon correlations of LC50 measures at this age with LD50 measures in rodents (Ducharme et al. 2015).

In this study, we used METH as an exemplary psychoactive substance to investigate its acute toxicity in a high-throughput manner, both to increase understanding of the mechanisms underlying the lethal effects of METH overdose and to provide a framework for a broader examination of emerging psychoactive substances.

Additionally, experiments were conducted to determine the extent to which the mechanisms underlying METH-induced acute lethality overlap with those previously established for rodents. Some of these likely involve neural toxicity. Several mechanisms for explaining METH-induced neurotoxicity have been identified, including oxidative stress (Cubells et al. 1994), neuroinflammation (Du et al. 2017), excitotoxicity (Staszewski and Yamamoto 2006), mitochondrial dysfunction (Mashayekhi et al. 2014), hyperthermia (Bowyer et al. 1994), and leakage of the blood brain barrier (BBB) (Kiyatkin et al. 2007). An additional and related mechanism of METH toxicity connects peripheral toxicity to neural consequences of acute overdose. This mechanism involves elevation of plasma ammonia, resulting from METH-induced liver failure that subsequently contributes to the METH-induced elevations in brain glutamate levels contributing to neurotoxicity (Halpin and Yamamoto 2012). Indeed, the symptoms of acute METH overdose and hepatic encephalopathy share many similarities, likely because both result in elevated plasma ammonia levels that stimulate neurotoxicity via increased brain glutamate levels. Perhaps indicative of this glutamatergic activation, seizure is commonly observed in both patients with acute METH overdose and patients with hepatic encephalopathy (Oja et al. 2017; Romanelli and Smith 2006). Studies have shown that ammonia decreases the expression of the astrocytic glutamate transporter EAAT1 (Excitatory Amino Acid Transporter 1; also known as the Glutamate Aspartate Transporter 1, GLAST1; encoded by the SLC1A3 gene), which further increases extracellular glutamate and depolarization of glutamate receptors (Chan and Butterworth 1999). Elevated glutamate levels lead to overactivation of glutamate receptors, which in turn results in a massive influx of calcium ions that initiate apoptotic pathways leading to neuronal cell death (Yamamoto et al. 2010). Blockade of glutamate AMPA and NMDA receptors can attenuate METH-induced neurotoxicity (Namiki et al. 2005; Staszewski and Yamamoto 2006). Whether similar approaches may reduce other aspects of acute METH toxicity or lethality has not been widely examined, although studies have identified a role of dopamine systems in METH-induced lethality. Genetic deletion of the dopamine transporter, or the dopamine D1 or D2 receptors, increases the LD50 for METH (Ito et al. 2008; Numachi et al. 2007). To explore these mechanisms, the ability of glutamatergic and dopaminergic antagonists, as well as the indirect γ-amino-butyric acid (GABA) agonist, diazepam, and the ammonia scavenging agent lactulose, were also examined.

Methods:

Drugs

(+)-Methamphetamine hydrochloride, lactulose, GYKI 52466 hydrochloride, R(+)-SCH-23390 hydrochloride, and S(−)-Raclopride (+)-tartrate salt were purchased from Sigma-Aldrich. (+)-MK 801 maleate was purchased from Hello Bio. Diazepam was obtained from Spectrum Pharmaceutics.

Subjects

20 wild-type (AB) adult zebrafish (10 male and 10 female) were purchased from the Zebrafish International Research Center (Eugene, Oregon). Upon arrival in Toledo, zebrafish were acclimated to the laboratory for at least 14 days prior to breeding. Male and female fish were housed separately in two 5 L fish tanks. Adult fish were kept on a 14/10 hr light/dark circle, with lights on at 7 AM. Brine shrimp (Brine Shrimp Direct) were the primary food source and flake food (Daniolab, Zebrafish Research Diet, size 4) was provided for supplementary nutrition.

A ratio of 5 female and 3 male adult zebrafish were placed into a breeding tank to acclimate one day before breeding. After mating, zebrafish embryos were collected by siphoning at 1-hour post-fertilization (hpf). The embryos were raised in a petri dish with egg water (Instant Ocean®, 60mg/L) at 28.5 °C. The water was changed daily to ensure the optimal conditions for embryo development.

LC50 Determination

On the day of the experiment, 5-day post-fertilization (dpf) zebrafish larvae were transferred to a 96-well plate (EMD Millipore MultiScreen-Mesh Filter Plates). Each well of the 96-well plate contained one larval fish. A preliminary range-finding assay for METH determined the approximate lethal concentration of METH (data not shown). To more specifically determine the LC50 value, 5 dpf zebrafish larvae (N = 18 per concentration, total N = 162) were exposed to METH concentrations from 500 μM to 50 mM for 5 hours at 28.5 °C. Fish that were used as controls were treated with egg water (0 mM METH). In preliminary experiments water in wells on the outside of the 96-well plate tended to evaporate more quickly than in interior wells, which might lead to concentration differences across wells over time. To overcome this issue a humidity chamber was constructed based on the method described in Walzl et al. (2012). After the 5 hr exposure to METH, the lethality at each concentration was recorded. Death was defined by the absence of a heartbeat. After mortality was recorded, zebrafish larvae were transferred onto glass depression slides and embedded in 3% methyl cellulose. Images and videos were taken using a stereo microscope (Nikon, SMZ18) and a CCD camera (Ximea, XiD-MD028MU-SY) to identify any visually apparent morphological changes.

Cardiotoxicity

In the lethality studies, clear changes in cardiovascular function were observed. To quantify these changes and to determine whether the effect of METH on zebrafish heart rate is concentration-dependent, zebrafish larvae (N = 18 per concentration, total N = 72; final N, discounting subjects that died, was 65) were exposed to non-lethal concentrations of METH (500 μM, 1 mM, 5 mM, and 10 mM) or egg water (Control). Doses were chosen based on lethality assessments (MNLC 10 mM), and the heart rate was counted after 5 hr. Heart rate was determined by observation of the heart under a stereo microscope (ZEISS, Stemi 2000-C) for 10 seconds. Heart rate was expressed as beats/min. To assess the time-course of cardiac changes after METH exposure the 5 mM dose was chosen for study because it was the lowest dose that depressed heart rate after 5 hours. Over continuous exposure to 5 mM METH the heart rate of zebrafish larvae (N = 36) was determined by observation of the heart under a stereo microscope (ZEISS, Stemi 2000-C) for 10 seconds at 0.5, 1, 2, 3, 4, and 5 hrs of exposure. Heart rate was expressed as beats/min.

Ammonia Excretion and Internal Ammonia Measurement

In a preliminary study, a spike of ammonia in the water was found after the larval fish had died. Therefore, after determination of the LC50 curve for 5 hr METH exposure, sub-lethal concentrations of METH based on the LC50 curve were used to examine ammonia excretion. 5 dpf zebrafish larvae (N = 12 per concentration, total N = 24) were transferred to a 96-well plate containing 500 μM, 1 mM, 5 mM, or 10 mM METH for 5 hr. At the end of the experiment, a 100 μL sample of the water in each well was collected separately into 0.5 mL centrifuge tubes. The QuantiFluo™ Ammonia/Ammonium ammonia assay kit (BioAssay Systems) was used to determine the ammonia level for each sample. The procedure was performed according to the manufacturer’s instructions. Experiments using METH alone, but no fish, showed that only minimally detectable levels of ammonia were observed at all METH concentrations.

For internal ammonia measurements the procedure was based on methods described by Braun et al. (2009b) with a few modifications. Briefly, pools of zebrafish larvae (3 pools, with N = 6 per pool for each METH concentration; N = 6*3 total larvae for each concentration) were treated with 0 mM, 10 mM, or 30 mM METH for 5 hr before transfer to 1.5 mL centrifuge tubes. Zebrafish larvae were frozen in liquid nitrogen (−80 °C), and subsequently manually ground to a fine powder using a pellet pestle (KONTES® 749521–0500), deproteinized with 3 volumes of ice-cold 6% perchloric acid, and centrifuged at 16,000 g for 15 min (at 4 °C). The supernatant for each sample was transferred to a new 1.5 mL centrifuge tube, neutralized with ice-cold 2 mol/L K2CO3 and centrifuged again at 16,000g for 10min (4°C). The final supernatant was transferred to a new 1.5 mL centrifuge tube and the ammonia level was analyzed using the QuantiFluo™ Ammonia/Ammonium ammonia assay kit (BioAssay Systems) for each sample.

Antagonism of METH-induced lethality

A series of experiments examined the potential mechanisms underlying METH lethality by co-administration of the following drugs with METH: the glutamatergic receptor antagonists MK-801 (an NMDA receptor antagonist) and GYKI 52466 (an AMPA receptor antagonist), the indirect GABA agonist diazepam, the ammonia clearing agent lactulose, and the dopaminergic receptor antagonists SCH 23390 (a D1 receptor antagonist) and raclopride (a D2 receptor antagonist). Doses of the antagonists were chosen based upon previous experiments using larval zebrafish (Chen et al. 2010b; Irons et al. 2013) where possible. For other drugs, preliminary experiments examined doses from 30 to 200 μM, and the highest doses used in these experiments were those producing no observable adverse effects. For the primary experiment reported here, 5 dpf zebrafish larvae (N = 12 ~ 18 per condition, total N = 167) were transferred to a 96-well plate and pre-treated with 50 μM or 100 μM MK-801, GYKI 52446, diazepam, lactulose, SCH 23390, or raclopride for 1 hr. Zebrafish larvae were then transferred to another 96-well plate and were co-administered the same treatments (50 μM or 100 μM of MK-801, GYKI 52446, diazepam, lactulose, SCH 23390, or raclopride) with 50 mM METH for 5 hr. Lethality was recorded at every hour for 5 hr.

Statistical Analysis

All statistical analysis and LC50 curve-fitting analysis was performed using Prism Graphpad 7.0 (San Diego, California, USA). Data, including heart rate, ammonia excretion, and internal ammonia were analyzed by one-way ANOVA followed by Tukey’s post hoc comparisons. The survival rate for METH, with or without pretreatments, were analyzed by the Mantel-Cox Log-rank Test. The level of significance was set at p< 0.05 for all analyses.

Results

LC50 Determination

Acute METH-induced toxicity and lethality was first examined by determining the LC50 concentration of METH. Zebrafish larvae were placed in solutions containing different concentrations of METH for 5 hr to establish LC50 values (Fig. 1A). The LC50 value for 5 hr exposure to METH was determined to be 43.98 mM and the maximal non-lethal concentration (MNLC) was 10 mM. Concentrations of METH above 10 mM caused complete immobilization and a noticeably reduced and irregular heartbeat (~ one beat per 10 s), which was the only indicator of whether the zebrafish larvae were still alive. Although this did not meet our criterion for “death”, e.g. the absence of a heartbeat, it was clear from preliminary studies that larvae in this state would not survive through the next 24 hours; nonetheless, based on our stated criterion, these larvae that were “near death” (ND) were not counted as dead for the 5 hr LC50 calculations. METH induced clear morphological and behavioral changes in zebrafish larvae. Pronounced body curvature and distinct tail deflection was found at 30 mM METH concentrations and above indicating the occurrence of sustained muscle contractions typical of tonic seizures (Figs. 1B and 1C). Zebrafish larvae started disintegrating in the water at 5 hr when exposed to 50 mM METH, indicating that death had occurred at least 30 min prior to observation. Lower METH concentrations (>1 mM) produced periodic, rapid, and often asymmetrical, movements of pectoral fins of zebrafish larvae that may have been an indication of clonic seizure activity (see supplemental video S1).

Figure 1.

Figure 1.

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(A) Dose-response curve for METH-induced lethality after a 5 hr continuous exposure (N=18 per concentration). The calculated LC50 is 43.98 mM and the MNLC is 10 mM. (B) METH-induced morphological changes. (i) control, (ii) 30 mM METH (iii) 50 mM METH. Body curvature and tail deflection found in 30 mM METH-treated zebrafish (*). At 50 mM zebrafish larvae started disintegrating in water, indicating that death occurred at least 30 min prior to observation (#). (C) Dorsal view of METH-induced tail deflection (red circle) in zebrafish, an indicator of sustained muscle contraction. (D) Chemical structure of methamphetamine.

Cardiotoxicity

To examine the effect of METH on heart rate in zebrafish, zebrafish larvae were exposed to sub-lethal concentrations of METH for 5 hr. METH had a biphasic effect on heart rate (F(4,63)=50.5, p<0.0001; Fig. 2A), with low concentrations (500 μM and 1 mM) causing a significant increase in heart rate (p<0.05 versus Control), while high concentrations (5 mM and 10 mM) caused significant decreases in heart rate (p<0.05 vs. control). To further examine whether this effect was time-dependent, zebrafish larvae were treated with 5 mM METH for 5 hr and heart rate was recorded at 0.5, 1, 2, 3, 4 and 5 hr. The effect of METH on heart rate was also time-dependent (F(6,34)=31.60, p< 0.0001l Fig. 2B). METH caused a significant increase in heart rate at 0.5 hr (p<0.05 vs. control)) but significant decreases in heart rate at 4 and 5 hr (p<0.05 vs. Control). In addition, in the lethality experiment described in the previous section the heart of some zebrafish larvae treated with high METH doses showed an abnormal reddening, perhaps indicative of cardiac hemorrhage, or blood pooling in the heart due to a valvular impairment that impaired blood circulation. An example of this effect is shown at the 30 mM METH concentration in Fig. 2C.

Figure 2.

Figure 2.

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(A) The effect of METH on heart rate (beats per minute; bpm) in zebrafish larvae. 5 dpf zebrafish larvae were treated with non-lethal concentrations of METH (500 μM, 1 mM, 5 mM, or 10 mM; N=18 per concentration). (B) The heart rate (bpm) of 5 dpf zebrafish larvae at different time-points (0.5, 1, 2, 3, 4, and 5 hr) during continuous exposure to 5 mM METH. The data are expressed as mean ± SEM. * significant difference from control values (p<0.05). (C) Images of control zebrafish larvae (left), and zebrafish larvae treated with 30 mM METH (right), showing abnormal reddening of the heart (red arrow).

Ammonia Excretion and Internal Ammonia Measurement

To determine whether elevated ammonia levels, indicative of compromised liver function, might play a role in METH-induced lethality, the level of ammonia excretion and internal ammonia was measured after 5 hr of METH administration (Fig. 2A). Ammonia excretion was measured in the surrounding water after exposure 500 μM, 1 mM, 5 mM, or 10 mM METH for 5 hr. METH increased ammonia excretion as demonstrated by a significant effect of METH Dose in the ANOVA (F(4, 23)=3.259, p< 0.05), but this effect was only significant at the 5 mM METH concentration in means comparisons (p<0.05 vs. control values).

There was also a significant effect of METH on internal, whole-body ammonia concentrations (F(2, 21)=13.38, p<0.05; Fig. 3B). Internal ammonia levels were not elevated significantly at any concentration, but a significant decrease in internal ammonia was found at the 30 mM METH concentration (p<0.05 vs. control).

Figure 3.

Figure 3.

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(A) 5 dpf zebrafish larvae were treated with non-lethal concentrations of METH (500 μM, 1 mM, 5 mM, or 10 mM; N=12 per concentration) to measure ammonia excretion. A significant increase in ammonia excretion was observed at the 5 mM concentration. (B) 5 dpf zebrafish larvae were treated with a non-lethal (10 mM) and a lethal concentration (30 mM) of METH (3 pools per condition; N=6 larvae per pool). Data are expressed as mean ± SEM. * significant difference from control values (p<0.05).

Antagonism of METH-induced lethality

To investigate the mechanisms involved in METH-induced lethal toxicity, MK-801, GYKI 52466, lactulose, diazepam, SCH 23390, and raclopride were co-administered with 50 mM METH for 5 hr and lethality was recorded. Exposure for 5 hr to 50 mM METH caused 100% lethality in zebrafish, thus 50 mM of METH was used as positive control. The NMDA receptor antagonist MK-801 only partially prevented METH-induced lethality, and this effect did not quite reach statistical significance (p=0.06; Fig. 4A). The final survival rate for 50 μM and 100 μM MK-801 treated groups were 8.3% and 25%, respectively (Fig. 4B). The AMPA receptor antagonist GYKI-52466 significantly reduced METH-induced lethality (p< 0.05; Fig. 5A). The final survival rate for 50 μM and 100 μM GYKI-52466 was 56.25% and 43.45%, respectively (Fig. 5B). Other drugs including diazepam, lactulose and SCH 23390 did not have any effect on METH-induced lethality (Figs. 6A, 7A, and 8A respectively). The final survival rate was 0% at 5 hr exposure of METH for all of these treatments (Fig. 6B, 7B, and 8B, respectively). Raclopride did reduce METH-induced lethality (p< 0.05; Fig. 9A), with a final survival rate at 50 μM and 100 μM raclopride of 16.67% and 58.33%, respectively (Fig. 9B).

Figure 4.

Figure 4.

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(A) The effect of MK-801 on METH-induced lethality in zebrafish. 5 dpf zebrafish larvae were pre-treated with either 50 μM or 100 μM MK-801 for 1 hr, followed by co-administration of 50 μM or 100 μM MK-801 with 50 mM METH for 5 hr (N=12-18 per condition). Mortality was recorded every hour. (B) Final mortality was recorded at the 5 hr time point. Data are expressed as percent survival (%).

Figure 5.

Figure 5.

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(A) The effect of GYKI-52466 on METH-induced lethality in zebrafish. 5 dpf zebrafish larvae were pre-treated with either 50 μM or 100 μM MK-801 for 1 hr, followed by co-administration of 50 μM or 100 μM GYKI-52466 with 50 mM METH for 5 hr (N=12-18 per condition). Mortality was recorded every hour. (B) Final mortality was recorded at the 5 hr time point. Data are expressed as percent survival (%).

Figure 6.

Figure 6.

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(A) The effect of diazepam on METH-induced lethality in zebrafish. 5 dpf zebrafish larvae were pre-treated with either 50 μM or 100 μM diazepam for 1 hr, followed by co-administration of 50 μM or 100 μM diazepam with 50 mM METH for 5 hr (N=12-18 per condition). Mortality was recorded every hour. (B) Final mortality was recorded at the 5 hr time point. Data are expressed as percent survival (%).

Figure 7.

Figure 7.

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(A). The effect of lactulose on METH-induced lethality in zebrafish. 5 dpf zebrafish larvae were pre-treated with either 50 μM or 100 μM lactulose for 1 hr, followed by co-administration of 50 μM or 100 μM lactulose with 50 mM METH for 5 hr (N=12-18 per condition). Mortality was recorded every hour. (B) Final mortality was recorded at the 5 hr time point. Data are expressed as percent survival (%).

Figure 8.

Figure 8.

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(A) The effect of SCH-23390 on METH-induced lethality in zebrafish. 5 dpf zebrafish larvae were pre-treated with either 50 μM or 100 μM SCH-23390 for 1 hr, followed by co-administration of 50 μM or 100 μM SCH-23390 with 50 mM METH for 5 hr (N=12-18 per condition). Mortality was recorded every hour. (B) Final mortality was recorded at the 5 hr time point. Data are expressed as percent survival (%).

Figure 9.

Figure 9.

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(A) The effect of raclopride on METH-induced lethality in zebrafish. 5 dpf zebrafish larvae were pre-treated with either 50 μM or 100 μM raclopride for 1 hr, followed by co-administration of 50 μM or 100 μM raclopride with 50 mM METH for 5 hr (N=12-18 per condition). Mortality was recorded every hour. (B) Final mortality was recorded at the 5 hr time point. Data are expressed as percent survival (%).

Discussion

METH abuse is a long-standing public health problem in the United States and throughout the world. METH abuse is associated with both short-term and long-term health problems. Although much focus, in terms of public health debate and scientific research, has focused on opioid abuse and overdose in recent years, METH abuse is again growing and may represent part of the next stage of the ongoing drug overdose crisis in the United States (Fogger 2019; Hall and Miczek 2019). Most preclinical research examining METH toxicity has focused on neurotoxicity (Cadet and Krasnova 2009; Krasnova et al. 2016). By contrast, drug overdose and the mechanisms underlying acute lethal toxicity has received much less attention. Overdose patients are treated in a largely symptomatic manner (Richards et al. 2015; Center for Substance Abuse Treatment 1999). Those treatments focus on the primary toxicities known to be involved in acute METH overdose based on readily observable symptoms such as high blood pressure and hyperthermia. METH is well-known from clinical reports of METH overdose to produce neural, cardiac, renal, and hepatic toxicity (Isoardi et al. 2019; Richards et al. 1999; Willson 2019). Nonetheless, few preclinical studies have focused on the mechanisms underlying METH overdose or lethal toxicity, although in vitro studies have examined METH-induced hepatotoxicity (for review see Carvalho et al. (2012)). Consequently, it is not surprising that the lethal toxic mechanisms that primarily underlie METH-induced lethality are poorly understood.

One of the goals of the present studies was to develop a larval zebrafish model for the study of the toxicity and lethality of METH, which can subsequently be used to examine the wide range of emerging novel psychoactive substances, particularly the amphetamine-like synthetic cathinones. The present studies show that METH-induced lethality can be easily shown in 5 dpf zebrafish using a high throughput approach that can now be applied to the study the lethality/toxicity of other amphetamine-like drugs. The use of such a zebrafish model will also require translatability ultimately to humans, as well as to other animal models. This seems to be the case. The current results bear many similarities to studies in rodents, supporting the validity of this approach. To begin with, there was evidence for increased ammonia excretion into the surrounding water after METH treatment, consistent with observations that elevations in plasma ammonia resulting from liver impairment contribute to METH-induced toxicity in rodents (Halpin et al. 2013; Halpin et al. 2014). This mechanism may be important for other amphetamine derivatives as well, as similar increases in plasma ammonia are observed after treatment with methylenedioxymethamphetamine (MDMA) in mice (Chen et al. 2020). However, it is uncertain to what extent increased ammonia levels contribute to lethal METH toxicity in the present experiments since internal ammonia levels were reduced at high METH concentrations, and elevations in ammonia excretion (based on measurements in the surrounding water), although significant, were not large. Moreover, administration of lactulose did not affect METH lethality in zebrafish larvae. In rodent studies (Halpin et al. 2013; Halpin et al. 2014), lactulose treatment reduces plasma ammonia levels and subsequent neurotoxicity. However, those studies examined sublethal METH doses, finding a connection between METH-induced liver impairment, elevated plasma ammonia levels, elevated glutamate levels, and METH-induced neurotoxicity. The limited effects of METH on ammonia in the present study may reflect a limitation of the present approach, which used 5-dpf zebrafish larvae. At this point in larval development the gastrointestinal tract is not yet active as the larvae are just beginning to forage for food after the egg sac has been depleted. Studies in older larvae would address this issue and perhaps improve the comparison to rodent studies.

Another important component of the hypothesis connecting elevated ammonia levels to METH-induced neurotoxicity was the involvement of glutamate. Importantly, the present studies in larval zebrafish indicated a role for glutamate in METH-induced lethal toxicity. The AMPA antagonist GYKI-52466 substantially reduced METH-induced lethality in larval zebrafish. A previous study found that GYKI-52466 reduces METH-induced neurotoxicity (Halpin and Yamamoto 2012), without affecting METH-induced hyperthermia. The effects of brain ischemia, which also involve glutamate neurotoxicity, are also attenuated by GYKI-52466 (Gyertyán et al. 1999). Studies in rodents have also found that MK-801 is able to reduce METH-induced neurotoxicity (Ali et al. 1994; Namiki et al. 2005; Sonsalla et al. 1989). The current data suggest that MK-801 also reduces METH-induced lethality in larval zebrafish. The protective effects of MK-801 could involve CNS effects, but might also involve hypothermic effects (Buchan 1992; Corbett et al. 1990). This might be a reason for the limited effects in this zebrafish model, something that will require further study, including studies under high ambient (water) temperature conditions, and studies in rodents. Although the effects of METH on ammonia excretion in larval zebrafish were not as robust as expected, the data nonetheless provide some support for the ammonia-glutamate connection that has been shown to be involved in METH-induced neurotoxicity (Halpin et al. 2013; Halpin et al. 2014), suggesting that these same mechanisms might play a role in acute lethal toxicity. METH caused a transient increase in ammonia excretion at non-lethal concentrations, but a decrease in internal ammonia when exposed to lethal concentrations of METH. The decreases in internal ammonia levels may suggest that zebrafish larvae were coping with compromised liver function by increasing excretion. Unlike mammals, which convert ammonia to urea as a major mechanism of eliminating ammonia, zebrafish, especially larval zebrafish, excrete ammonia through their gills and skin as a detoxifying mechanism (Braun et al. 2009a; Shih et al. 2008). Studies in both zebrafish and mammals have found that the symptoms associated with elevated ammonia levels can be prevented by pre-treatment with MK-801 (Feldman et al. 2014; Kosenko et al. 2003). Pretreatment/co-treatment with lactulose did not have any effect on METH-induced lethality. Lactulose has been widely prescribed for symptoms of acute or chronic hyperammonemia to promote ammonia excretion (Riordan and Williams 1997). Larval zebrafish do not develop a fully functional gastrointestinal system by 5 dpf, having just exhausted the yolk sac. Therefore, ammonia cannot be excreted through the gastrointestinal tract which likely explains why the treatment with lactulose was ineffective.

Other mechanisms are also certainly involved in METH-induced lethality. Consistent with a primary role for dopamine in METH lethality (Ito et al. 2008; Numachi et al. 2007), raclopride also reduced METH lethality. Haloperidol is used in the treatment of METH overdose, but primarily for the purpose of controlling psychotic behavior and aggression (Richards et al. 2015). This data would suggest that there may be other benefits to dopamine antagonism in the case of METH overdose. The D1 receptor antagonist SCH 23390 did not reduce METH-induced lethality, suggesting that D2-like receptors may play a greater role than D1-like receptors in METH-induced lethality, at least in larval zebrafish. This contrasts with studies in rodents, where both D1 receptor and D2 receptor antagonists are effective in reducing METH-induced lethality (Bronstein and Hong 1995; Sabol et al. 2013; Sonsalla et al. 1986; Xu et al. 2005). Moreover, it appears that this protective effect of dopamine antagonists is independent of effects on METH-induced hyperthermia, at least in rodents (Numachi et al. 2007). The discrepancy between current findings in zebrafish and the findings in mammals may be due to several factors, including the differences in temperature physiology that have already been mentioned, as well as differences in the distribution of D1-like receptors and D2-like receptors in the brain or the peripheral nervous system (for review, see Maximino and Herculano (2010)), and perhaps also the early developmental stage of the animals studied here.

One of the other classes of drugs that are commonly used in cases of METH overdose are benzodiazepines. However, the indirect GABA agonist diazepam had no effect on METH-induced lethality in larval zebrafish. Although previous studies found that diazepam provides protection against METH-induced excitotoxicity and seizures (Ali et al. 1994; Gasior et al. 2000), it does not appear that these effects of diazepam extend to METH-induced lethality, at least in this model.

In rodent studies METH induces hyperlocomotion followed by reduced activity at higher doses, associated with the onset of stereotypical behavior (Baumann et al. 2008; Camp et al. 1994; Igari et al. 2015; Kitanaka et al. 2010; Singh et al. 2012; Spanos and Yamamoto 1989), while at even higher doses other forms of toxicity impair motor function in other ways. However, studies in zebrafish have observed primarily hypoactivity after METH treatment (concentrations of 10–1000 μM) (Bugel and Tanguay, 2018), including our own studies that showed locomotor reduction at the highest concentration (65 mg/L) (Tackie-Yarboi et al. 2020). This may have to do with differences in dosing or the circumstances in which activity is assessed in rodents and zebrafish. High METH concentrations caused complete immobilization of zebrafish larvae in the present studies. Immobility was associated with sustained muscle contraction characterized by body curvature and distinct tail deflection. This may be an indicator of seizure as a result of excessive glutamate release in the zebrafish brain. A previous characterization of seizure in larval zebrafish based on the effects of pentylenetetrazol (PTZ) divided the seizure activity into 3 stages, in which the last stage was identified by rapid circulatory swimming (Baraban et al. 2005), although these authors did describe complete immobility occurring at even higher doses. In the current study, these distinct behavioral changes described for PTZ seizures were not generally observed. In contrast, high concentrations of METH produced sustained muscle contractions which impaired the ability of the animals to move, and eventually to maintain their upright position. Although the PTZ pattern of changes were not observed here, low concentrations of METH did cause rapid, dis-coordinated movements of the pectoral fins, which may be evidence of clonic seizure activity. Given the different patterns of apparent seizure-like activity observed for PTZ and for METH in zebrafish, further investigations are needed to understand seizure behavior in zebrafish resulting from different types of treatments.

It has been widely reported in clinical studies that acute METH overdose can lead to cardiotoxicity. The common symptoms associated with METH-induced cardiotoxicity include cardiomyopathy, pulmonary edema, myocardial infarction, vasospasm, cardiac necrosis, tachycardia, and hypertension (Wijetunga et al. 2003). Consistent with these observations, METH elicited a biphasic effect on heart rate in larval zebrafish, with low concentrations of METH causing tachycardia, and high concentrations of METH causing bradycardia. Ickes (2015) reported that cardiac output was increased when METH was administered to embryonic (48 hpf) zebrafish at 3 mg/L (20 μM). The effect of METH on heart rate in the present study was also time dependent. METH initially elevated heart rate, but this was followed by a decrease in heart rate with prolonged exposure. Clinical reports of acute METH overdose show evidence of hypertension, vasospasm, and tachycardia, but this initial state is often followed by bradycardia and hypotension as the heart is damaged and cardiovascular function impaired. Fang et al. (2016) showed that prolonged METH exposure (500 mg/L) leads to a significant heartrate decline in 3 dpf zebrafish. METH can cause cardiac necrosis due to excessive release of catecholamines and direct cardiac stimulation (Ago et al. 2006; Kevil et al. 2019; Schurer et al. 2017), and perhaps other factors that may cause direct toxicity in cardiomyocytes (Won et al. 2013).

In the present study, cardiovascular impairments were further evidenced by what appear to be cardiac hemorrhage or pooling of blood in the heart. Hemorrhages are a likely outcome of elevated blood pressure, and we have previously observed hemorrhages in several organs after METH administration in mice (Muskiewicz et al. 2020). At least some of the observed cardiac effects may reflect blood pooling resulting from impaired valvular function, which we did observe in some video recordings. Valvulopathies have been associated with amphetamines acting via serotonergic mechanisms, including fenfluramine and MDMA (Ayme-Dietrich et al. 2019; Cosyns et al. 2013). These effects involve chronic drug exposure. The possibility of more acute effects of amphetamines on heart valve function, and a role in acute overdose, has not been explored. The present observations suggest the potential of such a role that is worthy of further investigation in zebrafish and mammalian models.

Here it is important to note differences in cardiac function between zebrafish and humans (for review see Genge et al. (2016)). Although fish have only two main heart chambers, the physiological basis of heart function is very similar to mammals, including action potential morphology and basic contractile dynamics (which, interestingly differ in important respects between humans and mice). The main difference between zebrafish and mammalian hearts is the lack of a pulmonary circulation in their two-chambered hearts. One consequence of this is different hemodynamic mechanisms in fish and mammals. Despite these differences zebrafish have been proven to be a tractable model for many human cardiovascular diseases, as well as pharmacological actions that impair or restore cardiac function, with a high degree of predictive validity (for review see Bowley et al. (2022).

In summary, the present studies of lethal METH toxicity in 5 dpf larval zebrafish identified many mechanisms that have been previously reported in preclinical studies in rodents, or clinical studies of METH overdose in humans. Specifically, in support of the validity of this model, these findings in larval zebrafish found: (1) METH is lethal to zebrafish larvae and this lethality involves glutamatergic and dopaminergic systems based on antagonism of lethality by the AMPA antagonist GYKI-52466, the NMDA antagonist MK-801, and the dopamine D2 receptor antagonist raclopride (Importantly, other drugs including diazepam, lactulose and SCH 23390 were without effect on lethality); (2) like humans and rodents, METH also induces cardiotoxicity in zebrafish larvae; (3) ammonia excretion is increased by METH, although its role in METH lethality is as yet uncertain. Thus, the larval zebrafish model used here to study the lethal toxicity of METH provides a higher throughput, less costly approach than rodent studies that can be used to study the large number of emerging amphetamine-like NPS.

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FUNDING

This work was supported by funding from the National Institute on Drug Abuse (NIDA), DA045350 (FSH) and DA045833 (ITS). We thank the Center for Drug Design and Development at The University of Toledo for use of the zebrafish core facilities.

Abbreviations:

BBB

blood brain barrier

dpf

day post-fertilization

hpf

hour post-fertilization

MNLC

maximal non-lethal concentration

METH

methamphetamine

ND

near death

NPS

novel psychoactive substances

Footnotes

CONFLICT OF INTEREST

The authors declare that there are no competing financial interests associated with this work.

References

  1. Ago M, Ago K, Hara K, Kashimura S, Ogata M (2006) Toxicological and histopathological analysis of a patient who died nine days after a single intravenous dose of methamphetamine: A case report. Legal Medicine 8: 235–239. [DOI] [PubMed] [Google Scholar]
  2. Albers DS, Sonsalla PK (1995) Methamphetamine-induced hyperthermia and dopaminergic neurotoxicity in mice: pharmacological profile of protective and nonprotective agents. Journal of Pharmacology and Experimental Therapeutics 275: 1104–1114. [PubMed] [Google Scholar]
  3. Ali S, Champagne DL, Spaink HP, Richardson MK (2011) Zebrafish embryos and larvae: A new generation of disease models and drug screens. Birth Defects Research Part C: Embryo Today: Reviews 93: 115–133. [DOI] [PubMed] [Google Scholar]
  4. Ali SF, Newport GD, Holson RR, Jr WS, Bowyer JF (1994) Low environmental temperatures or pharmacologic agents that produce hypothermia decrease methamphetamine neurotoxicity in mice. Brain Research 658. [DOI] [PubMed] [Google Scholar]
  5. Ayme-Dietrich E, Lawson R, Da-Silva S, Mazzucotelli JP, Monassier L (2019) Serotonin contribution to cardiac valve degeneration: new insights for novel therapies? Pharmacol Res 140: 33–42. [DOI] [PubMed] [Google Scholar]
  6. Baraban SC, Taylor MR, Castro PA, Baier H (2005) Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression. Neuroscience 131: 759–68. [DOI] [PubMed] [Google Scholar]
  7. Battaglia G, Fornai F, Busceti CL, Aloisi G, Cerrito F, De Blasi A, Melchiorri D, Nicoletti F (2002) Selective blockade of mGlu5 metabotropic glutamate receptors is protective against methamphetamine neurotoxicity. Journal of Neuroscience 22: 2135–2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Baumann MH, Clark RD, Rothman RB (2008) Locomotor stimulation produced by 3,4-methylenedioxymethamphetamine (MDMA) is correlated with dialysate levels of serotonin and dopamine in rat brain. Pharmacol Biochem Behav 90: 208–17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bose J, Hedden LS, Lipari NR, Park-Lee E (2018) Key Substance Use and Mental Health Indicators in the United States: Results from the 2017 National Survey on Drug Use and Health. Substance Abuse and Mental Health Services Administration. [Google Scholar]
  10. Bowley G, Kugler E, Wilkinson R, Lawrie A, van Eeden F, Chico TJA, Evans PC, Noël ES, Serbanovic-Canic J (2022) Zebrafish as a tractable model of human cardiovascular disease. British Journal of Pharmacology 179: 900–917. [DOI] [PubMed] [Google Scholar]
  11. Bowyer JF, Davies DL, Schmued L, Broening HW, Newport GD, Slikker W, Holson RR (1994) Further studies of the role of hyperthermia in methamphetamine neurotoxicity. Journal of Pharmacology and Experimental Therapeutics 268: 1571–1580. [PubMed] [Google Scholar]
  12. Braun MH, Steele SL, Ekker M, Perry SF (2009a) Nitrogen excretion in developing zebrafish (Danio rerio): a role for Rh proteins and urea transporters. American Journal of Physiology-Renal Physiology 296: F994–F1005. [DOI] [PubMed] [Google Scholar]
  13. Braun MH, Steele SL, Perry SF (2009b) The responses of zebrafish (Danio rerio) to high external ammonia and urea transporter inhibition: nitrogen excretion and expression of rhesus glycoproteins and urea transporter proteins. Journal of Experimental Biology 212: 3846–3856. [DOI] [PubMed] [Google Scholar]
  14. Bronstein DM, Hong J-S (1995) Effects of sulpiride and SCH 23390 on methamphetamine-induced changes in body temperature and lethality. Journal of Pharmacology and Experimental Therapeutics 274: 943–950. [PubMed] [Google Scholar]
  15. Buchan AM (1992) Do NMDA antagonists prevent neuronal injury? No. Archives of neurology 49: 420–421. [DOI] [PubMed] [Google Scholar]
  16. Bugel SM, Tanguay RL (2018) Multidimensional chemobehavior analysis of flavonoids and neuroactive compounds in zebrafish. Toxicol Appl Pharmacol 344: 23–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cadet JL, Krasnova IN (2009) Molecular bases of methamphetamine-induced neurodegeneration. Int Rev Neurobiol 88: 101–19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Camp DM, Browman KE, Robinson TE (1994) The effects of methamphetamine and cocaine on motor behavior and extracellular dopamine in the ventral striatum of Lewis versus Fischer 344 rats. Brain Res 668: 180–93. [DOI] [PubMed] [Google Scholar]
  19. Carvalho M, Carmo H, Costa VM, Capela JP, Pontes H, Remiao F, Carvalho F, Bastos Mde L (2012) Toxicity of amphetamines: an update. Arch Toxicol 86: 1167–231. [DOI] [PubMed] [Google Scholar]
  20. Chan H, Butterworth RF (1999) Evidence for an astrocytic glutamate transporter deficit in hepatic encephalopathy. Neurochemical research 24: 1397–1401. [DOI] [PubMed] [Google Scholar]
  21. Chen J, Patel R, Friedman TC, Jones KS (2010a) The behavioral and pharmacological actions of NMDA receptor antagonism are conserved in zebrafish larvae. International journal of comparative psychology/ISCP; sponsored by the International Society for Comparative Psychology and the University of Calabria 23: 82. [PMC free article] [PubMed] [Google Scholar]
  22. Chen J, Patel R, Friedman TC, Jones KS (2010b) The Behavioral and Pharmacological Actions of NMDA Receptor Antagonism are Conserved in Zebrafish Larvae. Int J Comp Psychol 23: 82–90. [PMC free article] [PubMed] [Google Scholar]
  23. Chen Y, Tran HTN, Saber YH, Hall FS (2020) High ambient temperature increases the toxicity and lethality of 3,4-methylenedioxymethamphetamine and methcathinone. Pharmacol Biochem Behav 192: 172912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Corbett D, Evans S, Thomas C, Wang D, Jonas RA (1990) MK-801 reduced cerebral ischemic injury by inducing hypothermia. Brain research 514: 300–304. [DOI] [PubMed] [Google Scholar]
  25. Cosyns B, Droogmans S, Rosenhek R, Lancellotti P (2013) Drug-induced valvular heart disease. Heart 99: 7–12. [DOI] [PubMed] [Google Scholar]
  26. Crime UNOoDa (2021) World drug report 2021. (United Nations publication, Sales No. E.21.XI.8). [Google Scholar]
  27. Cubells JF, Rayport S, Rajendran G, Sulzer D (1994) Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress. Journal of Neuroscience 14: 2260–2271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Du SH, Qiao DF, Chen CX, Chen S, Liu C, Lin Z, Wang H, Xie WB (2017) Toll-Like Receptor 4 Mediates Methamphetamine-Induced Neuroinflammation through Caspase-11 Signaling Pathway in Astrocytes. Front Mol Neurosci 10: 409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ducharme NA, Reif DM, Gustafsson JA, Bondesson M (2015) Comparison of toxicity values across zebrafish early life stages and mammalian studies: Implications for chemical testing. Reprod Toxicol 55: 3–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Fang J, Zhu D, Luo C (2016) An In Vivo Assessment: Cardiotoxicity Induced by Three Kinds of Addictive Drugs (Methamphetamine, Ketamine, and Methadone) in Zebrafish Embryos. International Journal of Public Health and Safety 1. [Google Scholar]
  31. Feldman B, Tuchman M, Caldovic L (2014) A zebrafish model of hyperammonemia. Molecular Genetics and Metabolism 113: 142–147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Fogger SA (2019) Methamphetamine Use: A New Wave in the Opioid Crisis? J Addict Nurs 30: 219–223. [DOI] [PubMed] [Google Scholar]
  33. Gasior M, Ungard JT, Beekman M, Carter RB, Witkin JM (2000) Acute and chronic effects of the synthetic neuroactive steroid, ganaxolone, against the convulsive and lethal effects of pentylenetetrazol in seizure-kindled mice: comparison with diazepam and valproate. Neuropharmacology 39: 1184–1196. [DOI] [PubMed] [Google Scholar]
  34. Genge CE, Lin E, Lee L, Sheng X, Rayani K, Gunawan M, Stevens CM, Li AY, Talab SS, Claydon TW, Hove-Madsen L, Tibbits GF (2016) The Zebrafish Heart as a Model of Mammalian Cardiac Function. Reviews of physiology, biochemistry and pharmacology 171: 99–136. [DOI] [PubMed] [Google Scholar]
  35. Graham DG, Tiffany SM, Bell WR, Gutknecht WF (1978) Autoxidation versus covalent binding of quinones as the mechanism of toxicity of dopamine, 6-hydroxydopamine, and related compounds toward C1300 neuroblastoma cells in vitro. Molecular pharmacology 14: 644–653. [PubMed] [Google Scholar]
  36. Granado N, Ares-Santos S, O’Shea E, Vicario-Abejón C, Colado MI, Moratalla R (2010) Selective vulnerability in striosomes and in the nigrostriatal dopaminergic pathway after methamphetamine administration. Neurotoxicity research 18: 48–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Gyertyán I, Gigler G, Simó A (1999) The neuroprotective and hypothermic effect of GYKI-52466, a non-competitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-antagonist on histological and behavioural variables in the gerbil global ischemia model. Brain research bulletin 50: 179–186. [DOI] [PubMed] [Google Scholar]
  38. Hall FS, Miczek KA (2019) Emerging threats in addiction: will novel psychoactive substances contribute to exacerbating the ongoing drug overdose epidemic? Psychopharmacology (Berl) 236: 839–843. [DOI] [PubMed] [Google Scholar]
  39. Halpin LE, Gunning WT, Yamamoto BK (2013) Methamphetamine causes acute hyperthermia-dependent liver damage. Pharmacol Res Perspect 1: e00008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Halpin LE, Northrop NA, Yamamoto BK (2014) Ammonia mediates methamphetamine-induced increases in glutamate and excitotoxicity. Neuropsychopharmacology 39: 1031–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Halpin LE, Yamamoto BK (2012) Peripheral ammonia as a mediator of methamphetamine neurotoxicity. J Neurosci 32: 13155–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ickes JR (2015) The Effects of Acute and Chronic Methamphetamine Exposure on Cardiovascular Development, Developmental Rate, and Aggression in Danio rerio. University of Akron [Google Scholar]
  43. Igari M, Shen HW, Hagino Y, Fukushima S, Kasahara Y, Lesch KP, Murphy DL, Hall FS, Uhl GR, Ikeda K, Yaegashi N, Sora I (2015) Attenuated methamphetamine-induced locomotor sensitization in serotonin transporter knockout mice is restored by serotonin 1B receptor antagonist treatment. Behav Pharmacol 26: 167–79. [DOI] [PubMed] [Google Scholar]
  44. Irons TD, Kelly PE, Hunter DL, Macphail RC, Padilla S (2013) Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish. Pharmacol Biochem Behav 103: 792–813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Isoardi KZ, Ayles SF, Harris K, Finch CJ, Page CB (2019) Methamphetamine presentations to an emergency department: Management and complications. Emerg Med Australas 31: 593–599. [DOI] [PubMed] [Google Scholar]
  46. Ito M, Numachi Y, Ohara A, Sora I (2008) Hyperthermic and lethal effects of methamphetamine: roles of dopamine D1 and D2 receptors. Neurosci Lett 438: 327–9. [DOI] [PubMed] [Google Scholar]
  47. Kevil CG, Goeders NE, Woolard MD, Bhuiyan MS, Dominic P, Kolluru GK, Arnold CL, Traylor JG, Orr AW (2019) Methamphetamine Use and Cardiovascular Disease: In Search of Answers. Arteriosclerosis, thrombosis, and vascular biology 39: 1739–1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Kitanaka J, Kitanaka N, Tatsuta T, Miyoshi A, Koumoto A, Tanaka K, Nishiyama N, Morita Y, Takemura M (2010) Pretreatment with l-histidine produces a shift from methamphetamine-induced stereotypical biting to persistent locomotion in mice. Pharmacol Biochem Behav 94: 464–70. [DOI] [PubMed] [Google Scholar]
  49. Kiyatkin EA, Brown PL, Sharma HS (2007) Brain edema and breakdown of the blood-brain barrier during methamphetamine intoxication: critical role of brain hyperthermia. Eur J Neurosci 26: 1242–53. [DOI] [PubMed] [Google Scholar]
  50. Kosenko E, Llansola M, Montoliu C, Monfort P, Rodrigo R, Hernandez-Viadel M, Erceg S, Sánchez-Perez AM, Felipo V (2003) Glutamine synthetase activity and glutamine content in brain: modulation by NMDA receptors and nitric oxide. Neurochemistry International 43: 493–499. [DOI] [PubMed] [Google Scholar]
  51. Krasnova IN, Justinova Z, Cadet JL (2016) Methamphetamine addiction: involvement of CREB and neuroinflammatory signaling pathways. Psychopharmacology (Berl) 233: 1945–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Mashayekhi V, Eskandari MR, Kobarfard F, Khajeamiri A, Hosseini MJ (2014) Induction of mitochondrial permeability transition (MPT) pore opening and ROS formation as a mechanism for methamphetamine-induced mitochondrial toxicity. Naunyn Schmiedebergs Arch Pharmacol 387: 47–58. [DOI] [PubMed] [Google Scholar]
  53. Maximino C, Herculano AM (2010) A review of monoaminergic neuropsychopharmacology in zebrafish. Zebrafish 7: 359–378. [DOI] [PubMed] [Google Scholar]
  54. Mi G, Gao Y, Yan H, Jin X, Ye E, Liu S, Gong Z, Yang H, Yang Z (2016) l-Scoulerine attenuates behavioural changes induced by methamphetamine in zebrafish and mice. Behav Brain Res 298: 97–104. [DOI] [PubMed] [Google Scholar]
  55. Muskiewicz DE, Resendiz-Gutierrez F, Issa O, Hall FS (2020) Synthetic psychoactive cathinones: hypothermia and reduced lethality compared to methamphetamine and methylenedioxymethamphetamine. Pharmacol Biochem Behav 191: 172871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Namiki M, Mori T, Sawaguchi T, Ito S, Suzuki T (2005) Underlying mechanism of combined effect of methamphetamine and morphine on lethality in mice and therapeutic potential of cooling. Journal of pharmacological sciences 99: 168–176. [DOI] [PubMed] [Google Scholar]
  57. Numachi Y, Ohara A, Yamashita M, Fukushima S, Kobayashi H, Hata H, Watanabe H, Hall FS, Lesch KP, Murphy DL, Uhl GR, Sora I (2007) Methamphetamine-induced hyperthermia and lethal toxicity: role of the dopamine and serotonin transporters. Eur J Pharmacol 572: 120–8. [DOI] [PubMed] [Google Scholar]
  58. Oja SS, Saransaari P, Korpi ER (2017) Neurotoxicity of ammonia. Neurochemical research 42: 713–720. [DOI] [PubMed] [Google Scholar]
  59. Richards JR, Albertson TE, Derlet RW, Lange RA, Olson KR, Horowitz BZ (2015) Treatment of toxicity from amphetamines, related derivatives, and analogues: a systematic clinical review. Drug Alcohol Depend 150: 1–13. [DOI] [PubMed] [Google Scholar]
  60. Richards JR, Johnson EB, Stark RW, Derlet RW (1999) Methamphetamine abuse and rhabdomyolysis in the ED: a 5-year study. Am J Emerg Med 17: 681–5. [DOI] [PubMed] [Google Scholar]
  61. Riley AL, Nelson KH, To P, Lopez-Arnau R, Xu P, Wang D, Wang Y, Shen HW, Kuhn DM, Angoa-Perez M, Anneken JH, Muskiewicz D, Hall FS (2020) Abuse potential and toxicity of the synthetic cathinones (i.e., “Bath salts”). Neurosci Biobehav Rev 110: 150–173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Riordan SM, Williams R (1997) Treatment of hepatic encephalopathy. New England Journal of Medicine 337: 473–479. [DOI] [PubMed] [Google Scholar]
  63. Romanelli F, Smith KM (2006) Clinical effects and management of methamphetamine abuse. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 26: 1148–1156. [DOI] [PubMed] [Google Scholar]
  64. Sabol KE, Yancey DM, Speaker HA, Mitchell SL (2013) Methamphetamine and core temperature in the rat: ambient temperature, dose, and the effect of a D2 receptor blocker. Psychopharmacology (Berl) 228: 551–61. [DOI] [PubMed] [Google Scholar]
  65. Schurer S, Klingel K, Sandri M, Majunke N, Besler C, Kandolf R, Lurz P, Luck M, Hertel P, Schuler G, Linke A, Mangner N (2017) Clinical Characteristics, Histopathological Features, and Clinical Outcome of Methamphetamine-Associated Cardiomyopathy. JACC Heart Fail 5: 435–445. [DOI] [PubMed] [Google Scholar]
  66. Shih T-H, Horng J-L, Hwang P-P, Lin L-Y (2008) Ammonia excretion by the skin of zebrafish (Danio rerio) larvae. American Journal of Physiology-Cell Physiology 295: C1625–C1632. [DOI] [PubMed] [Google Scholar]
  67. Singh RA, Kosten TA, Kinsey BM, Shen X, Lopez AY, Kosten TR, Orson FM (2012) Dose-dependent changes in the locomotor responses to methamphetamine in BALB/c mice: low doses induce hypolocomotion. Pharmacol Biochem Behav 103: 230–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Soares E, Pereira FC (2019) Pharmacotherapeutic strategies for methamphetamine use disorder: mind the subgroups. Expert Opin Pharmacother 20: 2273–2293. [DOI] [PubMed] [Google Scholar]
  69. Sonsalla P, Nicklas W, Heikkila R (1989) Role for excitatory amino acids in methamphetamine-induced nigrostriatal dopaminergic toxicity. Science 243: 398–400. [DOI] [PubMed] [Google Scholar]
  70. Sonsalla PK, Gibb JW, Hanson GR (1986) Roles of D1 and D2 dopamine receptor subtypes in mediating the methamphetamine-induced changes in monoamine systems. Journal of Pharmacology and Experimental Therapeutics 238. [PubMed] [Google Scholar]
  71. Spanos LJ, Yamamoto BK (1989) Acute and subchronic effects of methylenedioxymethamphetamine [(+/−)MDMA] on locomotion and serotonin syndrome behavior in the rat. Pharmacol Biochem Behav 32: 835–40. [DOI] [PubMed] [Google Scholar]
  72. Spina MB, Cohen G (1989) Dopamine turnover and glutathione oxidation: implications for Parkinson disease. Proceedings of the National Academy of Sciences 86: 1398–1400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Staszewski RD, Yamamoto BK (2006) Methamphetamine-induced spectrin proteolysis in the rat striatum. J Neurochem 96: 1267–76. [DOI] [PubMed] [Google Scholar]
  74. Tackie-Yarboi E, Wisner A, Horton A, Chau TQT, Reigle J, Funk AJ, McCullumsmith RE, Hall FS, Williams FE, Schiefer IT (2020) Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish. ACS Chem Neurosci 11: 2761–2773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Treatment CfSA (1999) Medical Aspects of Stimulant Use Disorders. In: Disorders TfSU (ed) (Treatment Improvement Protocol (TIP) Series). Substance Abuse and Mental Health Services Administration (US), Rockville (MD), pp 1–42 [Google Scholar]
  76. Walzl A, Kramer N, Mazza G, Rosner M, Falkenhagen D, Hengstschläger M, Schwanzer-pfeiffer D, Dolznig H (2012) A simple and cost efficient method to avoid unequal evaporation in cellular screening assays, which restores cellular metabolic activity. International Journal of Applied Science and Technology 2. [Google Scholar]
  77. Wijetunga M, Seto T, Lindsay J, Schatz I (2003) Crystal methamphetamine‐associated cardiomyopathy: tip of the iceberg? Journal of Toxicology: Clinical Toxicology 41: 981–986. [DOI] [PubMed] [Google Scholar]
  78. Willson C (2019) Sympathomimetic amine compounds and hepatotoxicity: Not all are alike-Key distinctions noted in a short review. Toxicol Rep 6: 26–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Won S, Hong RA, Shohet RV, Seto TB, Parikh NI (2013) Methamphetamine‐associated cardiomyopathy. Clinical cardiology 36: 737–742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Xu W, Zhu JP, Angulo JA (2005) Induction of striatal pre- and postsynaptic damage by methamphetamine requires the dopamine receptors. Synapse 58: 110–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. Yamamoto BK, Moszczynska A, Gudelsky GA (2010) Amphetamine toxicities Classical and emerging mechanisms. Annals of the New York Academy of Sciences 1187: 101. [DOI] [PMC free article] [PubMed] [Google Scholar]

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