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Toluene’s effects on activity and extracellular dopamine in the mouse are altered by GABAA antagonism
Accepted Manuscript Title: TOLUENE’s EFFECTS ON ACTIVITY AND EXTRACELLULAR DOPAMINE IN THE MOUSE ARE ALTERED BY GABAA ANTAGONISM Authors: Sean P. Callan, Aaron K. Apawu, Tiffany A. Mathews, Scott E. Bowen PII: DOI: Reference:
S0304-3940(17)30206-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.004 NSL 32692
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
17-11-2016 16-2-2017 2-3-2017
Please cite this article as: Sean P.Callan, Aaron K.Apawu, Tiffany A.Mathews, Scott E.Bowen, TOLUENE’s EFFECTS ON ACTIVITY AND EXTRACELLULAR DOPAMINE IN THE MOUSE ARE ALTERED BY GABAA ANTAGONISM, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2017.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TOLUENE’S EFFECTS ON ACTIVITY AND EXTRACELLULAR DOPAMINE IN THE MOUSE ARE ALTERED BY GABAA ANTAGONISM Sean P. Callana, Aaron K. Apawub, Tiffany A. Mathewsb, and Scott E. Bowena*.
a
Department of Psychology
Wayne State University 5057 Woodward Ave Detroit, MI, USA 48202 Email address: [email protected] Email address: [email protected]
b
Department of Chemistry
Wayne State University 5101 Cass Ave Detroit, MI, USA, 48202
Highlights
A method of exposing freely moving mice to solvents while performing microdialysis. Acute toluene exposure increases caudate extracellular dopamine concentration. Toluene-induced increases in extracellular dopamine persist for at least an hour. GABA antagonism increased extracellular dopamine and locomotor activity in mice.
Abstract The abuse of inhalants like toluene continues to be widespread around the world, especially among children and teenagers. Despite its frequency of misuse, the dynamics between dopamine (DA) and gamma-aminobutyric acid (GABA) in response to toluene exposure remains unclear. To further decipher toluene’s actions, we used a dynamic exposure system in combination with microdialysis to examine in vivo the effects of acutely inhaled toluene on DA release within the mouse caudate putamen (CPu). Results show that toluene inhalation produced increases in DA levels and locomotor activity. In mice that were pretreated with the GABAA antagonist, bicuculline, there was no change in the locomotor response during toluene but activity was potentiated following toluene exposure. Bicuculline pretreatment increased extracellular DA levels during toluene exposure, suggesting that DA and GABA-releasing neuron interaction may play a role in the rewarding properties of toluene.
Keywords: Toluene, Microdialysis, Dopamine, Caudate Putamen, Mouse
Introduction The deliberate inhalation of volatile chemicals, like toluene, for intoxication is one of the most under-appreciated and under-researched substance abuse concerns in the world [1-4]. While the neurochemical mechanisms that underlie toluene intoxication are not fully understood, previous research has shown that inhalation of high concentrations of toluene vapor (>5000 ppm) alters brain neurotransmitter dynamics, particularly in the dopamine (DA) system [for review see 5, 6-8]. Previously, we have demonstrated that acute toluene inhalation increases electrically stimulated DA release in the striatum, and that repeated exposure attenuates electrically stimulated DA release in the nucleus accumbens (NAc) of mice [9]. Stengard et al. [10] have also shown that acutely inhaled toluene increased DA levels in the rat striatum (CPu), while Riegel and colleagues [11] reported that sub-chronic injections of toluene (600 mg/kg, i.p.) increased levels of DA present in the rat CPu ex-vivo for several hours after the last toluene treatment. In addition to toluene’s effects on DA, in vitro research suggests that toluene administration enhances GABAA receptor function, reversibly increasing synaptic currents at those sites [12]. Bale et al.[13] reported that repeated in vitro application of toluene to neurons increased NMDA current amplitude but decreased the amplitude of GABA-activated currents. However, O’Leary-Moore et al.[14] demonstrated that acutely inhaled toluene reduced hippocampal GABA levels in juvenile rats. Despite evidence of the importance of DA and GABA in toluene’s action on the brain, particularly in the CPu, there has never been an examination of DA or GABA dynamics in the mouse striatum at the extracellular level during toluene exposure. Prior attempts at measuring dialysate in response to toluene exposure have focused on using the injection route of toluene administration [15], have used rats and have not
measured free locomotor behavior during vapor exposure. Instead, these studies have either eschewed activity measures [16], utilized a tethered exposure system [10], or did not take a measure of locomotor activity [17]. Measures of activity are important indices of drug action, and simultaneous collection of activity and dialysate would allow for direct comparison between the two techniques. As such, these experiments used a dynamic exposure system that allowed for simultaneous extracellular neurochemical analysis and locomotor behavior in awake, freely moving mice during toluene vapor exposure. Our results demonstrate a persistent increase in extracellular DA following toluene exposure independent of changes to locomotor activity, as well as a potential mediation of this effect by GABA activity. Materials and Methods Twenty-six male Swiss-Webster mice, ~postnatal day (PND) 30, were purchased from Harlan Laboratories (Portage, MI, USA). Mice were housed in a certified vivarium (Association for Assessment and Accreditation of Laboratory Animal Care; AAALAC) with ad libitum access to Rodent Lab Diet 5001 (PMI, Nutrition International, Inc., Brentwood, MO) and water in a temperature controlled (20 – 22oC) environment with a 12-h light cycle (0600 – 1800 h). The Wayne State University Institutional Animal Care and Use Committee (IACUC) approved all animal procedures which were in accordance with the NIH “Guide for the Care and Use of Laboratory Animals: Eighth edition” [18]. Mice were divided into five groups (N = 5-6 for each group) which included three toluene concentrations only (0, 4000, 8000 ppm) and two toluene concentrations + drug (0 or 4000 ppm; 10 μM GABAA agonist bicuculline). Group sizes were chosen based on our prior work with microdialysis [19-22]. Toluene exposures were conducted using a dynamic exposure similar to a system described previously [23]. Toluene was delivered into a chamber consisting
of a 37.8 L rectangular glass tank (51cm x 28cm x 12cm, 1428cm2 floor area) fitted with a Plexiglas® lid. The lid was equipped with two 1.2 cm access ports, each located at opposite ends, which allowed for delivery of toluene vapor, as well as, the collection of the dialysis samples. The mouse was restricted to a 30.4 cm x 20.32 cm section of the chamber through the insertion of two Plexiglas® walls. The microdialysis swivel was fixed to one Plexiglas® wall, which allowed injection, and collection tubes to exit the chamber through a porthole. Toluene vapor was generated by directing airflow through a bubbler that was immersed in a 500-ml solvent bath contained in a 1-L round-bottom flask. Air saturated with toluene exited the bath and was mixed with filtered laboratory (fresh) air that was then delivered to the exposure chamber for 30 min. Flow rates were maintained at 10 L/min and toluene concentration was determined using a single wavelength monitoring infrared (IR) spectrometer (Miran 1A, Foxboro Analytical, North Haven, CT). When toluene exposure was discontinued, any remaining toluene vapor was quickly removed from the chamber via a vacuum tube. The dynamic exposure system was housed under a fume hood, which also served to provide white background noise and isolation from the laboratory environment. During toluene exposure, animals were allowed to move around the interior of the chamber. Locomotor activity was measured within the exposure chamber via three sets of 16beam infrared (I/R) emitter–detector arrays (Med Associates, St. Albans, VT) mounted on Plexiglas bases around the sides of the exposure chambers. Interruptions of I/R beams resulted in an analog signal being recorded by automated activity software (Open Field Activity Software [SOF-811], Med Associates, St. Albans, VT). This automated measure of activity was transformed into 15-min blocks over the duration of the session to coincide with the restrictions to dialysate sampling rate.
Extracellular DA level was measured in vivo using microdialysis prior, during, and after acute toluene exposure. The stereotaxic surgery for microdialysis was performed as previously described [24]. Briefly, stereotaxic coordinates (AP +1, ML -1.6, DV -2.5) were determined using the mouse brain atlas [25] and mice were anesthetized with Isoflurane®. A CMA/7 guide cannula was implanted unilaterally into the ventral portion of the striatum (CPu). Following 72 hours of recovery, a CMA/7 dialysis probe (2 mm membrane length) was inserted into the cannula and perfused overnight with artificial cerebrospinal fluid (aCSF; 0.4 mM ascorbic acid, 126 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 2.4 mM CaCl2, 25 mM NaHCO3, 1.2 mM NaH2PO4, 11 mM D-glucose, pH 7.4) at a flow rate of 0.4 μL/min [21]. The next day, the flow rate was increased to 1.1 μL/min for a 1h equilibration period and dialysate samples were collected from freely moving mice at 15 min intervals. After collection of eight baseline dialysis samples, mice were exposed to toluene vapor for 30 min, followed by a 1-hour recovery period. Control animals (air-only) were placed in an identical chamber and dialysate was collected using the same schedule previously described. The influence of GABA on DA action was examined in separate groups of mice. Bicuculline (GABAA antagonist) was mixed into aCSF at a 10 μM concentration and was perfused directly into the CPu for 30 min prior to toluene administration. The 10 μM dose of bicuculline was selected based on prior published work with bicuculline administered via reverse dialysis [26, 27]. All collected dialysate samples were stored in a -80 o C freezer and analyzed within two weeks of collection as previously reported by our group [19]. High performance liquid chromatography (HPLC; LC-20AD pump; Shimadzu, Columbia, MD) was used to analyze samples by manually injecting 10 µL of dialysate samples into a 10 µL injection loop. The
mobile phase used to separate the analytes was the ESA MDTM mobile phase (which consisted of: 75 mM NaH2PO4, 3 mM 1-octanesulfonic acid, 0.125 mM EDTA, 9 % acetonitrile, and 0.2 0.5 % triethylamine; pH = 3.0) and was operated at a flow rate of 0.4 mL/min. The analytes were separated using a C18 column (Luna 100 x 3 mm, C18, 2.6 μM column; Phenomenex, Torrance, CA). DA was electrochemically detected using ESA 5014B microdialysis cell (E1 = -150 mV; E2 = +220 mV; ESA Coulochem III (Thermo-Fisher, Chelmsford, MA) with an in-line ESA 5020 guard cell (potential of guard cell: + 350 mv) positioned before the injection loop. Separation and quantification of the analytes on the HPLC system was controlled by LC Solutions Software (Shimadzu, Columbia, MD). The retention time of DA was at approximately 7 - 8 min. Integration and quantification of DA peak area were performed against known concentrations of DA standards (0, 2.5, 5, and 10 nM). Data analysis Data analysis was performed using SPSS (Version 23) software. Spontaneous locomotor data was analyzed using a Mixed-factorial ANOVA for both exposure and recovery. An alpha level of p < 0.05 determined statistical significance. Tukey’s honest significant difference posthoc contrasts were used to determine the locus of significant main effects and interactions. Individual fractions were assessed as single univariate ANOVAs. Two statistical outliers (one in exposure and one in post-exposure) were replaced with group means. For the neurochemical measurements, baseline DA levels were calculated by averaging DA levels in the eight baseline fractions. DA levels during the experimental phase were represented as a percent of that baseline average as previously described [21, 28] and analyzed using Mixed ANOVAs. For the bicuculline study, extracellular DA levels were analyzed using two 2 x 2 ANOVAs with concentration and bicuculline condition serving as the between-
subjects variables. In cases where sphericity was violated in the Mixed ANOVAs analyses, the Huynh-Feldt degrees of freedom correction was utilized. Results The animals placed overnight into the dynamic exposure system had a mean baseline locomotor count of 47.8 ± 14.1 cm (mean ± SEM) for the 1-hour acclimation period prior to toluene exposure. For locomotor activity, toluene exposure produced a significant betweensubjects effect, F (2, 22) = 13.62, p < 0.001, with post-hoc tests revealing that both 4,000- and 8,000-ppm concentrations were significantly elevated compared to air control (p’s < 0.01). No main effect was observed for bicuculline (p = 0.80) nor was there a toluene x bicuculline interaction (p = 0.54). There was a significant main effect of time, F (1, 22) = 25.16, p < 0.001, with animals having lower activity in the second fifteen minutes of exposure (Figure 1). A significant toluene x time interaction, F (2, 22) = 5.78, p < 0.05, was observed with 8,000 ppm toluene producing a greater decline in activity over time than 4,000 ppm of toluene. Neurochemically, toluene exposure significantly increased extracellular DA in the CPu (F2,21 = 7.67, p < 0.01, Figure 2). Post-hoc analysis revealed that the highest concentration of 8000-ppm toluene significantly elevated extracellular DA levels as compared to both air and 4000-ppm concentration (p < 0.05). No significant main effect of time was observed (p = 0.79) nor was there a significant time x concentration interaction (p = 0.08). Pretreatment with bicuculline had no significant effect on DA levels during toluene exposure (p = 0.67) and no significant interactions were found for bicuculline x time, or bicuculline x time x concentration effects (p’s = 0.18 and 0.18, respectively). However, univariate examination of the fractions suggested that the variance during the first 15-min exposure period might have been suppressing a possible difference during the second 15-min exposure period. Indeed, univariate analysis of
the two fractions during toluene exposure revealed no effect of toluene (p = 0.40) or bicuculline (p = 0.41) during the first 15-min exposure period. However, during the second 15-min exposure period there was a significant effect of toluene concentration (F1,17 = 5.79, p < 0.05) with the 4000 ppm conditions having higher extracellular DA (p < 0.05, Figure 2). Pretreatment with bicuculline also resulted in a higher extracellular DA level (F1,17 = 22.95, p < 0.001) than in animals not given bicuculline (Figure 2). Finally, there was a significant toluene concentration x bicuculline interaction (F1,17 = 17.09, p < 0.01) indicating that extracellular DA levels for the 4000 ppm toluene x bicuculline condition increased throughout exposure, while the two air controls and the 4,000 ppm condition showed slight decreases in DA over time (Figure 2). As seen in Figure 3, a significant between-subjects effect of toluene was observed for locomotor activity following toluene exposure, F (2, 22) = 7.52, p < 0.01. Post-hoc tests showed that 4,000 ppm, but not 8,000 ppm, was significantly elevated over air controls. A significant toluene X bicuculline interaction was also observed, F (1, 22) = 20.43, p < 0.001, whereby bicuculline reduced activity in air controls, but enhanced activity in toluene exposed animals. There was a main effect of time, F (3, 66) = 17.55, p < 0.001, with animals showing less activity in later time points (Figure 3). There was also a significant time x toluene interaction, F (6, 66) = 3.90, p < 0.01. Following toluene exposure, a significant increase in DA levels (F2,21 = 8.39, p < 0.01, Figure 4), with the 8000 ppm concentration of toluene significantly elevating DA levels as compared to the air control and 4000-ppm groups (p < 0.05; Figure 4). There was no main effect of time (p = 0.65) on DA levels, nor was there a time x concentration interaction. Pretreatment with bicuculline had no effect on DA levels (p = 0.55) and there were no significant interactions for bicuculline x concentration (p = 0.22), bicuculline x time (p = 0.30), or bicuculline x time x
concentration (p = 0.26). Univariate analyses of the four recovery fractions revealed that toluene exposure significantly increased extracellular DA in the 3rd (F2,21 = 7.37, p < 0.05) and 4th (F2,21 = 3.57, p < 0.05) 15 minute fractions. Post-hoc analyses for both effects reveal that the 8,000ppm toluene group was significantly elevated compared to controls. Discussion The present study utilized a dynamic exposure system combined with microdialysis, which allowed for assessment of both behavioral and neurochemical changes in mice during toluene vapor exposure. To our knowledge, this is the first report of the effects of toluene on mouse locomotor activity and extracellular DA using a dynamic exposure system. Increases in mouse locomotor activity were observed during toluene exposure to both 4000 and 8000 ppm and this effect occurred during the first 15 min of exposure and again during the recovery period. At the highest toluene concentration (8000 ppm), locomotor activity was suppressed with animals displaying little to no motor activity during the second 15 min of exposure (Figure 1), though animals quickly recovered following exposure (Figure 3). This biphasic effect of toluene is similar to what has been reported for toluene in static exposure systems [29-31], as well as for several other abused inhalants [23], and abused depressant drugs and ethanol [32, 33]. The results obtained here represent the first time toluene-induced DA effects have been observed in a mouse model. The highest exposure of 8000-ppm toluene significantly increased extracellular DA levels by ~340% (as compared to controls) and this increase persisted throughout both the exposure and recovery periods (Figure 4). The increase in extracellular CPu DA is generally consistent with findings from other reports of toluene exposure in rodents [10, 11], although the present findings do differ from prior reports in several ways. For example, Stengard et al. (2004) demonstrated that two hours of toluene (2000 ppm) exposure in rats
increased extracellular DA by 147% within the striatum [10]. Our results demonstrate that 30 min of toluene did not significantly elevate DA until exposures reached 8000 ppm. One possible explanation for this discrepancy is that toluene blood concentrations have been shown to rise for up to 2 hours before becoming asymptotic (Gospe and Al-Bayati [34]). As such, it is entirely plausible that the difference between the current findings with 4000 ppm and those of Stengard, Hoglund [10] with 2000 ppm is a function of the interaction between duration of exposure and concentration. Noteworthy is the persistent elevation of extracellular DA for the hour following 8,000ppm toluene exposure. This continued increase of extracellular DA suggests that toluene or its metabolites may have a secondary effect on the DA system in addition to any primary actions occurring during toluene exposure. Although we did not measure blood concentrations of toluene in the present work, Koga’s work suggests that the half-life of toluene in mice is as low as 23 min [35], indicating that the persistent increase in extracellular DA is unlikely to be stimulated by toluene alone. While the exact mechanism remains unclear, one could hypothesize that toluene may be interfering with DA reuptake via the DA transporter. However, in our previous report, fast-scan cyclic voltammetry demonstrated that acute toluene exposure increased electrically stimulated DA release with no effect on DA uptake (Apawu et al., 2015). This suggests that uptake is unlikely to be the cause of this effect. Another possible explanation is that toluene exposure may transiently impede DA metabolism by impairing monoamine oxidase (MAO) or catechol-omethyltransferase (COMT) (e.g., Riegel et al., 2006). Alternatively, toluene’s metabolism into benzyl alcohol may also affect DA dynamics (Needham and Houslay, 1982), though to our knowledge there has never been an investigation of whether benzyl alcohol itself increases
extracellular DA in the CPu. Regardless of action, these findings suggest that toluene exposure creates long-term changes in brain neurochemistry and future research should investigate the impact of acute toluene exposure on the metabolites of dopamine (DOPAC and 3-MT) as well as the effect of benzyl alcohol on extracellular DA. Indeed, the apparent effect of toluene on the CPu DA might provide an explanation for seemingly transient nature of solvent misuse, as this critical region for the planning of drug seeking behavior does not appear to be as strongly impacted by toluene inhalation as other brain regions [e.g., the NAc; 15, 17]. While prior work has focused on the role of NMDA antagonism and DA in examination of toluene’s locomotor stimulating properties [36], to our knowledge this report represents the first time GABA antagonism and DA have been investigated together following toluene exposure. Pretreatment with the GABAA antagonist bicuculline interacted with toluene exposure—suppressing activity in controls during recovery, while potentiating activity in toluene exposed animals during the same time period. Bicuculline also increased extracellular DA level during toluene exposure, suggesting that GABA receptors in the CPu may have an inhibitory effect on dopaminergic neurons. Extracellular levels of DA in the CPu may be moderated by additional mechanisms, including internal cellular actions, afferent pathway signaling, and neurotransmission from neurons within the CPu [reviewed by 37]. In response to toluene administration, it is possible that DA receptor functioning may vary between the CPu, NAc, and other regions due to the influences of these outside factors. For example, evidence suggests that negative modulation of GABA receptors reduces intracranial self-stimulation in response to toluene vapor, suggesting that our obtained effect may be specific to the CPu [38]. The implications of the current finding are contrary to Stengard and O'Connor [39] who reported that administration of 2000-ppm toluene did not decrease striatal GABA in rats, as would be
expected given our current results. However, it is possible that 2000-ppm toluene, even when given for 2 hours, is not sufficient to depress striatal GABA levels. It is important to note that unilateral infusion of bicuculline may have contributed to the high variance observed in the locomotor response, as only one hemisphere was affected by the GABA antagonism. Future studies should investigate the effect of bilateral CPu infusion of bicuculline prior to toluene exposure on locomotor behavior and extracellular DA. In summary, our report extends previous findings of toluene exposure in rodents by combining methodologies that allowed for collection of dialysate samples while recording behavioral activity levels from awake and freely moving mice during toluene exposure. This dynamic exposure system preserves the route of administration preferred by human abusers of toluene while not restricting the animal via a body tether. This combination allows for the investigation of novel hypotheses, including the locomotor-stimulating properties of acute toluene exposure, which were previously untestable due to hardware limitations inherent in an inhalant microdialysis experiment. Our results suggest that CPu GABA may moderate the locomotor response to toluene exposure. We have also demonstrated a protracted increased in extracellular DA in the striatum. Finally, our results suggest that the DA dynamics of acute toluene inhalation may be related to GABAergic functioning, and future studies are warranted to delineate the relative contributions of each neurotransmitter to toluene’s actions within the striatum.
References 1. 2.
3. 4.
5. 6. 7.
8. 9.
10.
11.
12. 13.
14.
15.
Cruz, S.L., The latest evidence in the neuroscience of solvent misuse: an article written for service providers. Subst Use Misuse, 2011. 46 Suppl 1: p. 62-7. Cruz, S.L. and S.E. Bowen, Inhalant Abuse, in Neural Mechanisms of Action of Drugs of Abuse and Natural Reinforcers, M.M. Ubach, R. Mondragon-Ceballos, Editor. 2008, Research Signpost: Kerala, India. p. 61-87. Howard, M.O., et al., Inhalant use and inhalant use disorders in the United States. Addict Sci Clin Pract, 2011. 6(1): p. 18-31. Bowen, S.E. and S.L. Cruz, Inhalants: addiction and toxic effects in the human, in The effects of drug abuse on the human nervous system, B. Madras and M. Kuhar, Editors. 2014, Academic Press: Oxford, UK. p. 553-569. Bowen, S.E., et al., The last decade of solvent research in animal models of abuse: mechanistic and behavioral studies. Neurotoxicol Teratol, 2006. 28(6): p. 636-47. Balster, R.L., Neural basis of inhalant abuse. Drug Alcohol Depend, 1998. 51(1-2): p. 207-14. Cruz, S.L. and R. Balster, Neuropharmacology of Inhalants, in Biological Research on Addiction:Comprehensive Addictive Behaviors and Disorders, P.M. Miller, Editor. 2013, Elsevier Inc.: Academic Press,: San Diego. p. 637-645. Bowen, S.E., M.O. Howard, and E.L. Garland, Inhalant Use Disorders in the United States. 2016. Apawu, A.K., T.A. Mathews, and S.E. Bowen, Striatal dopamine dynamics in mice following acute and repeated toluene exposure. Psychpharmacology, 2015. 232: p. 173184. Stengard, K., G. Hoglund, and U. Ungerstedt, Extracellular dopamine levels within the striatum increase during inhalation exposure to toluene: a microdialysis study in awake, freely moving rats. Toxicol Lett, 1994. 71(3): p. 245-55. Riegel, A.C., et al., Repeated exposure to the abused inhalant toluene alters levels of neurotransmitters and generates peroxynitrite in nigrostriatal and mesolimbic nuclei in rat. Ann N Y Acad Sci, 2004. 1025: p. 543-51. Beckstead, M.J., et al., Glycine and gamma-aminobutyric acid(A) receptor function is enhanced by inhaled drugs of abuse. Mol Pharmacol, 2000. 57(6): p. 1199-205. Bale, A.S., et al., Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene. Neuroscience, 2005. 130(1): p. 197-206. O'Leary-Moore, S.K., et al., Region-dependent alterations in glutamate and GABA measured by high-resolution magnetic resonance spectroscopy following acute binge inhalation of toluene in juvenile rats. Neurotoxicol Teratol, 2007. 29(4): p. 466-75. Riegel, A.C., et al., The abused inhalant toluene increases dopamine release in the nucleus accumbens by directly stimulating ventral tegmental area neurons. Neuropsychopharmacology, 2007. 32(7): p. 1558-69.
16.
17. 18.
19.
20.
21.
22.
23. 24. 25.
26.
27.
28. 29.
30. 31.
Koga, Y., et al., Toluene Inhalation Increases Extracellular Noradrenaline and Dopamine in the Medial Prefrontal Cortex and Nucleus Accumbens in Freely-Moving Rats. The Journal of the Kyushu Dental Society, 2007. 61(1): p. 39-54. Gerasimov, M.R., et al., Toluene inhalation produces regionally specific changes in extracellular dopamine. Drug Alcohol Depend, 2002. 65(3): p. 243-51. NRS, Guide for the care and use of laboratory animals. 2011, National Research Council (U.S.). Institute for Laboratory Animal Research (U.S.), National Academies Press: Washington, D.C. p. xxv, 220 p. Birbeck, J.A., M. Khalid, and T.A. Mathews, Potentiated striatal dopamine release leads to hyperdopaminergia in female brain-derived neurotrophic factor heterozygous mice. ACS Chem Neurosci, 2014. 5(4): p. 275-81. Bosse, K.E., et al., Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8‐ deficient mice. Journal of Neurochemistry, 2015. 135(6): p. 1218-1231. Bosse, K.E. and T.A. Mathews, Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice. Neurosci Lett, 2011. 489(3): p. 172-6. Bosse, K.E. and T.A. Mathews, Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice. Neuroscience letters, 2011. 489(3): p. 172-176. Bowen, S.E. and R.L. Balster, Effects of inhaled 1,1,1-trichloroethane on locomotor activity in mice. Neurotoxicol Teratol, 1996. 18(1): p. 77-81. Khalid, M., R.A. Aoun, and T.A. Mathews, Altered striatal dopamine release following a sub-acute exposure to manganese. J Neurosci Methods, 2011. 202(2): p. 182-91. Franklin, K.B.J. and G. Paxinos, Paxinos and Franklin's The mouse brain in stereotaxic coordinates. Third edition. ed. 2008, Amsterdam: Academic Press, an imprint of Elsevier. 256. Zackheim, J. and E.D. Abercrombie, Thalamic regulation of striatal acetylcholine efflux is both direct and indirect and qualitatively altered in the dopamine-depleted striatum. Neuroscience, 2005. 131(2): p. 423-36. Whitehead, K.J., S. Rose, and P. Jenner, Involvement of intrinsic cholinergic and GABAergic innervation in the effect of NMDA on striatal dopamine efflux and metabolism as assessed by microdialysis studies in freely moving rats. Eur J Neurosci, 2001. 14(5): p. 851-60. Mathews, T.A., et al., No role of the dopamine transporter in acute ethanol effects on striatal dopamine dynamics. Synapse, 2006. 60(4): p. 288-94. Tegeris, J.S. and R.L. Balster, A comparison of the acute behavioral effects of alkylbenzenes using a functional observational battery in mice. Fundam Appl Toxicol, 1994. 22(2): p. 240-50. Bowen, S.E., S. Kimar, and S. Irtenkauf, Comparison of toluene-induced locomotor activity in four mouse strains. Pharmacol Biochem Behav, 2010. 95(2): p. 249-57. Conti, A.C., et al., Investigation of calcium-stimulated adenylyl cyclases 1 and 8 on toluene and ethanol neurobehavioral actions. Neurotoxicol Teratol, 2012. 34(5): p. 481488.
32.
33.
34. 35. 36. 37. 38.
39.
Matchett, J.A. and C.K. Erickson, Alteration of ethanol-induced changes in locomotor activity by adrenergic blockers in mice. Psychopharmacology (Berl), 1977. 52(2): p. 2016. Smoothy, R. and M.S. Berry, Time course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice. Psychopharmacology (Berl), 1985. 85(1): p. 57-61. Gospe, S.M., Jr. and M.A.S. Al-Bayati, A comparison of oral and inhalation exposures to toluene. Journal of the American College of Toxicology, 1994. 13(1): p. 21-32. Koga, K., [Distribution, metabolism and excretion of toluene in mice (author's transl)]. Nihon Yakurigaku Zasshi, 1978. 74(6): p. 687-98. Lo, P.S., et al., Acute neurobehavioral effects of toluene: involvement of dopamine and NMDA receptors. Toxicology, 2009. 265(1-2): p. 34-40. Kreitzer, A.C. and R.C. Malenka, Striatal plasticity and basal ganglia circuit function. Neuron, 2008. 60(4): p. 543-54. Tracy, M.E., M.L. Banks, and K.L. Shelton, Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice. Psychopharmacology (Berl), 2016. 233(4): p. 715-25. Stengard, K. and W.T. O'Connor, Acute toluene exposure decreases extracellular gamma-aminobutyric acid in the globus pallidus but not in striatum: a microdialysis study in awake, freely moving rats. Eur J Pharmacol, 1994. 292(1): p. 43-6.
Figure 1 – Locomotor activity during the 30 minute exposure period. Toluene concentrations of 4,000 and 8,000 ppm increased locomotor activity as compared to air controls during the first 15 min of exposure; bicuculline had no effect on locomotor activity alone or in combination with toluene exposure (left panel). Toluene no longer increased locomotor activity during the second half of the toluene exposure (right panel); bicuculline was still without effect.
Figure 2. The effect of acute toluene exposure and GABAA antagonism on extracellular DA levels in the mouse CPu (data are represented as percent of baseline DA) during the 30 minute exposure period. A) No difference in extracellular DA levels in the mouse CPu during the first half of the 30 min toluene exposure (4000 or 8000 ppm). Bicuculline pretreatment had no effect on extracellular DA. B) As seen in the right panel, only the 8,000-ppm concentration of toluene significantly increased DA in the mouse CPu during the second half of the 30 min toluene exposure, while bicuculline pretreatment had no effect on extracellular DA. Figure 3 – Locomotor activity data during the one hour (in four 15-min bins) recovery period following acute toluene exposure. As seen in the panels, 4,000 ppm of toluene significantly elevated locomotor activity over baseline. Additionally, bicuculline interacted with toluene, suppressing activity in controls, while potentiating activity in toluene-exposed animals. Figure 4 – Shown are extracellular DA in the CPu for the one hour (in four 15-min bins) recovery period following exposure. Animals exposed to 8,000-ppm toluene vapor showed a significant increase in DA as compared to air controls. There was no effect of bicuculline pretreatment. Data are represented as percent of baseline DA.
S0304-3940(17)30206-9 http://dx.doi.org/doi:10.1016/j.neulet.2017.03.004 NSL 32692
To appear in:
Neuroscience Letters
Received date: Revised date: Accepted date:
17-11-2016 16-2-2017 2-3-2017
Please cite this article as: Sean P.Callan, Aaron K.Apawu, Tiffany A.Mathews, Scott E.Bowen, TOLUENE’s EFFECTS ON ACTIVITY AND EXTRACELLULAR DOPAMINE IN THE MOUSE ARE ALTERED BY GABAA ANTAGONISM, Neuroscience Letters http://dx.doi.org/10.1016/j.neulet.2017.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
TOLUENE’S EFFECTS ON ACTIVITY AND EXTRACELLULAR DOPAMINE IN THE MOUSE ARE ALTERED BY GABAA ANTAGONISM Sean P. Callana, Aaron K. Apawub, Tiffany A. Mathewsb, and Scott E. Bowena*.
a
Department of Psychology
Wayne State University 5057 Woodward Ave Detroit, MI, USA 48202 Email address: [email protected] Email address: [email protected]
b
Department of Chemistry
Wayne State University 5101 Cass Ave Detroit, MI, USA, 48202
Highlights
A method of exposing freely moving mice to solvents while performing microdialysis. Acute toluene exposure increases caudate extracellular dopamine concentration. Toluene-induced increases in extracellular dopamine persist for at least an hour. GABA antagonism increased extracellular dopamine and locomotor activity in mice.
Abstract The abuse of inhalants like toluene continues to be widespread around the world, especially among children and teenagers. Despite its frequency of misuse, the dynamics between dopamine (DA) and gamma-aminobutyric acid (GABA) in response to toluene exposure remains unclear. To further decipher toluene’s actions, we used a dynamic exposure system in combination with microdialysis to examine in vivo the effects of acutely inhaled toluene on DA release within the mouse caudate putamen (CPu). Results show that toluene inhalation produced increases in DA levels and locomotor activity. In mice that were pretreated with the GABAA antagonist, bicuculline, there was no change in the locomotor response during toluene but activity was potentiated following toluene exposure. Bicuculline pretreatment increased extracellular DA levels during toluene exposure, suggesting that DA and GABA-releasing neuron interaction may play a role in the rewarding properties of toluene.
Keywords: Toluene, Microdialysis, Dopamine, Caudate Putamen, Mouse
Introduction The deliberate inhalation of volatile chemicals, like toluene, for intoxication is one of the most under-appreciated and under-researched substance abuse concerns in the world [1-4]. While the neurochemical mechanisms that underlie toluene intoxication are not fully understood, previous research has shown that inhalation of high concentrations of toluene vapor (>5000 ppm) alters brain neurotransmitter dynamics, particularly in the dopamine (DA) system [for review see 5, 6-8]. Previously, we have demonstrated that acute toluene inhalation increases electrically stimulated DA release in the striatum, and that repeated exposure attenuates electrically stimulated DA release in the nucleus accumbens (NAc) of mice [9]. Stengard et al. [10] have also shown that acutely inhaled toluene increased DA levels in the rat striatum (CPu), while Riegel and colleagues [11] reported that sub-chronic injections of toluene (600 mg/kg, i.p.) increased levels of DA present in the rat CPu ex-vivo for several hours after the last toluene treatment. In addition to toluene’s effects on DA, in vitro research suggests that toluene administration enhances GABAA receptor function, reversibly increasing synaptic currents at those sites [12]. Bale et al.[13] reported that repeated in vitro application of toluene to neurons increased NMDA current amplitude but decreased the amplitude of GABA-activated currents. However, O’Leary-Moore et al.[14] demonstrated that acutely inhaled toluene reduced hippocampal GABA levels in juvenile rats. Despite evidence of the importance of DA and GABA in toluene’s action on the brain, particularly in the CPu, there has never been an examination of DA or GABA dynamics in the mouse striatum at the extracellular level during toluene exposure. Prior attempts at measuring dialysate in response to toluene exposure have focused on using the injection route of toluene administration [15], have used rats and have not
measured free locomotor behavior during vapor exposure. Instead, these studies have either eschewed activity measures [16], utilized a tethered exposure system [10], or did not take a measure of locomotor activity [17]. Measures of activity are important indices of drug action, and simultaneous collection of activity and dialysate would allow for direct comparison between the two techniques. As such, these experiments used a dynamic exposure system that allowed for simultaneous extracellular neurochemical analysis and locomotor behavior in awake, freely moving mice during toluene vapor exposure. Our results demonstrate a persistent increase in extracellular DA following toluene exposure independent of changes to locomotor activity, as well as a potential mediation of this effect by GABA activity. Materials and Methods Twenty-six male Swiss-Webster mice, ~postnatal day (PND) 30, were purchased from Harlan Laboratories (Portage, MI, USA). Mice were housed in a certified vivarium (Association for Assessment and Accreditation of Laboratory Animal Care; AAALAC) with ad libitum access to Rodent Lab Diet 5001 (PMI, Nutrition International, Inc., Brentwood, MO) and water in a temperature controlled (20 – 22oC) environment with a 12-h light cycle (0600 – 1800 h). The Wayne State University Institutional Animal Care and Use Committee (IACUC) approved all animal procedures which were in accordance with the NIH “Guide for the Care and Use of Laboratory Animals: Eighth edition” [18]. Mice were divided into five groups (N = 5-6 for each group) which included three toluene concentrations only (0, 4000, 8000 ppm) and two toluene concentrations + drug (0 or 4000 ppm; 10 μM GABAA agonist bicuculline). Group sizes were chosen based on our prior work with microdialysis [19-22]. Toluene exposures were conducted using a dynamic exposure similar to a system described previously [23]. Toluene was delivered into a chamber consisting
of a 37.8 L rectangular glass tank (51cm x 28cm x 12cm, 1428cm2 floor area) fitted with a Plexiglas® lid. The lid was equipped with two 1.2 cm access ports, each located at opposite ends, which allowed for delivery of toluene vapor, as well as, the collection of the dialysis samples. The mouse was restricted to a 30.4 cm x 20.32 cm section of the chamber through the insertion of two Plexiglas® walls. The microdialysis swivel was fixed to one Plexiglas® wall, which allowed injection, and collection tubes to exit the chamber through a porthole. Toluene vapor was generated by directing airflow through a bubbler that was immersed in a 500-ml solvent bath contained in a 1-L round-bottom flask. Air saturated with toluene exited the bath and was mixed with filtered laboratory (fresh) air that was then delivered to the exposure chamber for 30 min. Flow rates were maintained at 10 L/min and toluene concentration was determined using a single wavelength monitoring infrared (IR) spectrometer (Miran 1A, Foxboro Analytical, North Haven, CT). When toluene exposure was discontinued, any remaining toluene vapor was quickly removed from the chamber via a vacuum tube. The dynamic exposure system was housed under a fume hood, which also served to provide white background noise and isolation from the laboratory environment. During toluene exposure, animals were allowed to move around the interior of the chamber. Locomotor activity was measured within the exposure chamber via three sets of 16beam infrared (I/R) emitter–detector arrays (Med Associates, St. Albans, VT) mounted on Plexiglas bases around the sides of the exposure chambers. Interruptions of I/R beams resulted in an analog signal being recorded by automated activity software (Open Field Activity Software [SOF-811], Med Associates, St. Albans, VT). This automated measure of activity was transformed into 15-min blocks over the duration of the session to coincide with the restrictions to dialysate sampling rate.
Extracellular DA level was measured in vivo using microdialysis prior, during, and after acute toluene exposure. The stereotaxic surgery for microdialysis was performed as previously described [24]. Briefly, stereotaxic coordinates (AP +1, ML -1.6, DV -2.5) were determined using the mouse brain atlas [25] and mice were anesthetized with Isoflurane®. A CMA/7 guide cannula was implanted unilaterally into the ventral portion of the striatum (CPu). Following 72 hours of recovery, a CMA/7 dialysis probe (2 mm membrane length) was inserted into the cannula and perfused overnight with artificial cerebrospinal fluid (aCSF; 0.4 mM ascorbic acid, 126 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 2.4 mM CaCl2, 25 mM NaHCO3, 1.2 mM NaH2PO4, 11 mM D-glucose, pH 7.4) at a flow rate of 0.4 μL/min [21]. The next day, the flow rate was increased to 1.1 μL/min for a 1h equilibration period and dialysate samples were collected from freely moving mice at 15 min intervals. After collection of eight baseline dialysis samples, mice were exposed to toluene vapor for 30 min, followed by a 1-hour recovery period. Control animals (air-only) were placed in an identical chamber and dialysate was collected using the same schedule previously described. The influence of GABA on DA action was examined in separate groups of mice. Bicuculline (GABAA antagonist) was mixed into aCSF at a 10 μM concentration and was perfused directly into the CPu for 30 min prior to toluene administration. The 10 μM dose of bicuculline was selected based on prior published work with bicuculline administered via reverse dialysis [26, 27]. All collected dialysate samples were stored in a -80 o C freezer and analyzed within two weeks of collection as previously reported by our group [19]. High performance liquid chromatography (HPLC; LC-20AD pump; Shimadzu, Columbia, MD) was used to analyze samples by manually injecting 10 µL of dialysate samples into a 10 µL injection loop. The
mobile phase used to separate the analytes was the ESA MDTM mobile phase (which consisted of: 75 mM NaH2PO4, 3 mM 1-octanesulfonic acid, 0.125 mM EDTA, 9 % acetonitrile, and 0.2 0.5 % triethylamine; pH = 3.0) and was operated at a flow rate of 0.4 mL/min. The analytes were separated using a C18 column (Luna 100 x 3 mm, C18, 2.6 μM column; Phenomenex, Torrance, CA). DA was electrochemically detected using ESA 5014B microdialysis cell (E1 = -150 mV; E2 = +220 mV; ESA Coulochem III (Thermo-Fisher, Chelmsford, MA) with an in-line ESA 5020 guard cell (potential of guard cell: + 350 mv) positioned before the injection loop. Separation and quantification of the analytes on the HPLC system was controlled by LC Solutions Software (Shimadzu, Columbia, MD). The retention time of DA was at approximately 7 - 8 min. Integration and quantification of DA peak area were performed against known concentrations of DA standards (0, 2.5, 5, and 10 nM). Data analysis Data analysis was performed using SPSS (Version 23) software. Spontaneous locomotor data was analyzed using a Mixed-factorial ANOVA for both exposure and recovery. An alpha level of p < 0.05 determined statistical significance. Tukey’s honest significant difference posthoc contrasts were used to determine the locus of significant main effects and interactions. Individual fractions were assessed as single univariate ANOVAs. Two statistical outliers (one in exposure and one in post-exposure) were replaced with group means. For the neurochemical measurements, baseline DA levels were calculated by averaging DA levels in the eight baseline fractions. DA levels during the experimental phase were represented as a percent of that baseline average as previously described [21, 28] and analyzed using Mixed ANOVAs. For the bicuculline study, extracellular DA levels were analyzed using two 2 x 2 ANOVAs with concentration and bicuculline condition serving as the between-
subjects variables. In cases where sphericity was violated in the Mixed ANOVAs analyses, the Huynh-Feldt degrees of freedom correction was utilized. Results The animals placed overnight into the dynamic exposure system had a mean baseline locomotor count of 47.8 ± 14.1 cm (mean ± SEM) for the 1-hour acclimation period prior to toluene exposure. For locomotor activity, toluene exposure produced a significant betweensubjects effect, F (2, 22) = 13.62, p < 0.001, with post-hoc tests revealing that both 4,000- and 8,000-ppm concentrations were significantly elevated compared to air control (p’s < 0.01). No main effect was observed for bicuculline (p = 0.80) nor was there a toluene x bicuculline interaction (p = 0.54). There was a significant main effect of time, F (1, 22) = 25.16, p < 0.001, with animals having lower activity in the second fifteen minutes of exposure (Figure 1). A significant toluene x time interaction, F (2, 22) = 5.78, p < 0.05, was observed with 8,000 ppm toluene producing a greater decline in activity over time than 4,000 ppm of toluene. Neurochemically, toluene exposure significantly increased extracellular DA in the CPu (F2,21 = 7.67, p < 0.01, Figure 2). Post-hoc analysis revealed that the highest concentration of 8000-ppm toluene significantly elevated extracellular DA levels as compared to both air and 4000-ppm concentration (p < 0.05). No significant main effect of time was observed (p = 0.79) nor was there a significant time x concentration interaction (p = 0.08). Pretreatment with bicuculline had no significant effect on DA levels during toluene exposure (p = 0.67) and no significant interactions were found for bicuculline x time, or bicuculline x time x concentration effects (p’s = 0.18 and 0.18, respectively). However, univariate examination of the fractions suggested that the variance during the first 15-min exposure period might have been suppressing a possible difference during the second 15-min exposure period. Indeed, univariate analysis of
the two fractions during toluene exposure revealed no effect of toluene (p = 0.40) or bicuculline (p = 0.41) during the first 15-min exposure period. However, during the second 15-min exposure period there was a significant effect of toluene concentration (F1,17 = 5.79, p < 0.05) with the 4000 ppm conditions having higher extracellular DA (p < 0.05, Figure 2). Pretreatment with bicuculline also resulted in a higher extracellular DA level (F1,17 = 22.95, p < 0.001) than in animals not given bicuculline (Figure 2). Finally, there was a significant toluene concentration x bicuculline interaction (F1,17 = 17.09, p < 0.01) indicating that extracellular DA levels for the 4000 ppm toluene x bicuculline condition increased throughout exposure, while the two air controls and the 4,000 ppm condition showed slight decreases in DA over time (Figure 2). As seen in Figure 3, a significant between-subjects effect of toluene was observed for locomotor activity following toluene exposure, F (2, 22) = 7.52, p < 0.01. Post-hoc tests showed that 4,000 ppm, but not 8,000 ppm, was significantly elevated over air controls. A significant toluene X bicuculline interaction was also observed, F (1, 22) = 20.43, p < 0.001, whereby bicuculline reduced activity in air controls, but enhanced activity in toluene exposed animals. There was a main effect of time, F (3, 66) = 17.55, p < 0.001, with animals showing less activity in later time points (Figure 3). There was also a significant time x toluene interaction, F (6, 66) = 3.90, p < 0.01. Following toluene exposure, a significant increase in DA levels (F2,21 = 8.39, p < 0.01, Figure 4), with the 8000 ppm concentration of toluene significantly elevating DA levels as compared to the air control and 4000-ppm groups (p < 0.05; Figure 4). There was no main effect of time (p = 0.65) on DA levels, nor was there a time x concentration interaction. Pretreatment with bicuculline had no effect on DA levels (p = 0.55) and there were no significant interactions for bicuculline x concentration (p = 0.22), bicuculline x time (p = 0.30), or bicuculline x time x
concentration (p = 0.26). Univariate analyses of the four recovery fractions revealed that toluene exposure significantly increased extracellular DA in the 3rd (F2,21 = 7.37, p < 0.05) and 4th (F2,21 = 3.57, p < 0.05) 15 minute fractions. Post-hoc analyses for both effects reveal that the 8,000ppm toluene group was significantly elevated compared to controls. Discussion The present study utilized a dynamic exposure system combined with microdialysis, which allowed for assessment of both behavioral and neurochemical changes in mice during toluene vapor exposure. To our knowledge, this is the first report of the effects of toluene on mouse locomotor activity and extracellular DA using a dynamic exposure system. Increases in mouse locomotor activity were observed during toluene exposure to both 4000 and 8000 ppm and this effect occurred during the first 15 min of exposure and again during the recovery period. At the highest toluene concentration (8000 ppm), locomotor activity was suppressed with animals displaying little to no motor activity during the second 15 min of exposure (Figure 1), though animals quickly recovered following exposure (Figure 3). This biphasic effect of toluene is similar to what has been reported for toluene in static exposure systems [29-31], as well as for several other abused inhalants [23], and abused depressant drugs and ethanol [32, 33]. The results obtained here represent the first time toluene-induced DA effects have been observed in a mouse model. The highest exposure of 8000-ppm toluene significantly increased extracellular DA levels by ~340% (as compared to controls) and this increase persisted throughout both the exposure and recovery periods (Figure 4). The increase in extracellular CPu DA is generally consistent with findings from other reports of toluene exposure in rodents [10, 11], although the present findings do differ from prior reports in several ways. For example, Stengard et al. (2004) demonstrated that two hours of toluene (2000 ppm) exposure in rats
increased extracellular DA by 147% within the striatum [10]. Our results demonstrate that 30 min of toluene did not significantly elevate DA until exposures reached 8000 ppm. One possible explanation for this discrepancy is that toluene blood concentrations have been shown to rise for up to 2 hours before becoming asymptotic (Gospe and Al-Bayati [34]). As such, it is entirely plausible that the difference between the current findings with 4000 ppm and those of Stengard, Hoglund [10] with 2000 ppm is a function of the interaction between duration of exposure and concentration. Noteworthy is the persistent elevation of extracellular DA for the hour following 8,000ppm toluene exposure. This continued increase of extracellular DA suggests that toluene or its metabolites may have a secondary effect on the DA system in addition to any primary actions occurring during toluene exposure. Although we did not measure blood concentrations of toluene in the present work, Koga’s work suggests that the half-life of toluene in mice is as low as 23 min [35], indicating that the persistent increase in extracellular DA is unlikely to be stimulated by toluene alone. While the exact mechanism remains unclear, one could hypothesize that toluene may be interfering with DA reuptake via the DA transporter. However, in our previous report, fast-scan cyclic voltammetry demonstrated that acute toluene exposure increased electrically stimulated DA release with no effect on DA uptake (Apawu et al., 2015). This suggests that uptake is unlikely to be the cause of this effect. Another possible explanation is that toluene exposure may transiently impede DA metabolism by impairing monoamine oxidase (MAO) or catechol-omethyltransferase (COMT) (e.g., Riegel et al., 2006). Alternatively, toluene’s metabolism into benzyl alcohol may also affect DA dynamics (Needham and Houslay, 1982), though to our knowledge there has never been an investigation of whether benzyl alcohol itself increases
extracellular DA in the CPu. Regardless of action, these findings suggest that toluene exposure creates long-term changes in brain neurochemistry and future research should investigate the impact of acute toluene exposure on the metabolites of dopamine (DOPAC and 3-MT) as well as the effect of benzyl alcohol on extracellular DA. Indeed, the apparent effect of toluene on the CPu DA might provide an explanation for seemingly transient nature of solvent misuse, as this critical region for the planning of drug seeking behavior does not appear to be as strongly impacted by toluene inhalation as other brain regions [e.g., the NAc; 15, 17]. While prior work has focused on the role of NMDA antagonism and DA in examination of toluene’s locomotor stimulating properties [36], to our knowledge this report represents the first time GABA antagonism and DA have been investigated together following toluene exposure. Pretreatment with the GABAA antagonist bicuculline interacted with toluene exposure—suppressing activity in controls during recovery, while potentiating activity in toluene exposed animals during the same time period. Bicuculline also increased extracellular DA level during toluene exposure, suggesting that GABA receptors in the CPu may have an inhibitory effect on dopaminergic neurons. Extracellular levels of DA in the CPu may be moderated by additional mechanisms, including internal cellular actions, afferent pathway signaling, and neurotransmission from neurons within the CPu [reviewed by 37]. In response to toluene administration, it is possible that DA receptor functioning may vary between the CPu, NAc, and other regions due to the influences of these outside factors. For example, evidence suggests that negative modulation of GABA receptors reduces intracranial self-stimulation in response to toluene vapor, suggesting that our obtained effect may be specific to the CPu [38]. The implications of the current finding are contrary to Stengard and O'Connor [39] who reported that administration of 2000-ppm toluene did not decrease striatal GABA in rats, as would be
expected given our current results. However, it is possible that 2000-ppm toluene, even when given for 2 hours, is not sufficient to depress striatal GABA levels. It is important to note that unilateral infusion of bicuculline may have contributed to the high variance observed in the locomotor response, as only one hemisphere was affected by the GABA antagonism. Future studies should investigate the effect of bilateral CPu infusion of bicuculline prior to toluene exposure on locomotor behavior and extracellular DA. In summary, our report extends previous findings of toluene exposure in rodents by combining methodologies that allowed for collection of dialysate samples while recording behavioral activity levels from awake and freely moving mice during toluene exposure. This dynamic exposure system preserves the route of administration preferred by human abusers of toluene while not restricting the animal via a body tether. This combination allows for the investigation of novel hypotheses, including the locomotor-stimulating properties of acute toluene exposure, which were previously untestable due to hardware limitations inherent in an inhalant microdialysis experiment. Our results suggest that CPu GABA may moderate the locomotor response to toluene exposure. We have also demonstrated a protracted increased in extracellular DA in the striatum. Finally, our results suggest that the DA dynamics of acute toluene inhalation may be related to GABAergic functioning, and future studies are warranted to delineate the relative contributions of each neurotransmitter to toluene’s actions within the striatum.
References 1. 2.
3. 4.
5. 6. 7.
8. 9.
10.
11.
12. 13.
14.
15.
Cruz, S.L., The latest evidence in the neuroscience of solvent misuse: an article written for service providers. Subst Use Misuse, 2011. 46 Suppl 1: p. 62-7. Cruz, S.L. and S.E. Bowen, Inhalant Abuse, in Neural Mechanisms of Action of Drugs of Abuse and Natural Reinforcers, M.M. Ubach, R. Mondragon-Ceballos, Editor. 2008, Research Signpost: Kerala, India. p. 61-87. Howard, M.O., et al., Inhalant use and inhalant use disorders in the United States. Addict Sci Clin Pract, 2011. 6(1): p. 18-31. Bowen, S.E. and S.L. Cruz, Inhalants: addiction and toxic effects in the human, in The effects of drug abuse on the human nervous system, B. Madras and M. Kuhar, Editors. 2014, Academic Press: Oxford, UK. p. 553-569. Bowen, S.E., et al., The last decade of solvent research in animal models of abuse: mechanistic and behavioral studies. Neurotoxicol Teratol, 2006. 28(6): p. 636-47. Balster, R.L., Neural basis of inhalant abuse. Drug Alcohol Depend, 1998. 51(1-2): p. 207-14. Cruz, S.L. and R. Balster, Neuropharmacology of Inhalants, in Biological Research on Addiction:Comprehensive Addictive Behaviors and Disorders, P.M. Miller, Editor. 2013, Elsevier Inc.: Academic Press,: San Diego. p. 637-645. Bowen, S.E., M.O. Howard, and E.L. Garland, Inhalant Use Disorders in the United States. 2016. Apawu, A.K., T.A. Mathews, and S.E. Bowen, Striatal dopamine dynamics in mice following acute and repeated toluene exposure. Psychpharmacology, 2015. 232: p. 173184. Stengard, K., G. Hoglund, and U. Ungerstedt, Extracellular dopamine levels within the striatum increase during inhalation exposure to toluene: a microdialysis study in awake, freely moving rats. Toxicol Lett, 1994. 71(3): p. 245-55. Riegel, A.C., et al., Repeated exposure to the abused inhalant toluene alters levels of neurotransmitters and generates peroxynitrite in nigrostriatal and mesolimbic nuclei in rat. Ann N Y Acad Sci, 2004. 1025: p. 543-51. Beckstead, M.J., et al., Glycine and gamma-aminobutyric acid(A) receptor function is enhanced by inhaled drugs of abuse. Mol Pharmacol, 2000. 57(6): p. 1199-205. Bale, A.S., et al., Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene. Neuroscience, 2005. 130(1): p. 197-206. O'Leary-Moore, S.K., et al., Region-dependent alterations in glutamate and GABA measured by high-resolution magnetic resonance spectroscopy following acute binge inhalation of toluene in juvenile rats. Neurotoxicol Teratol, 2007. 29(4): p. 466-75. Riegel, A.C., et al., The abused inhalant toluene increases dopamine release in the nucleus accumbens by directly stimulating ventral tegmental area neurons. Neuropsychopharmacology, 2007. 32(7): p. 1558-69.
16.
17. 18.
19.
20.
21.
22.
23. 24. 25.
26.
27.
28. 29.
30. 31.
Koga, Y., et al., Toluene Inhalation Increases Extracellular Noradrenaline and Dopamine in the Medial Prefrontal Cortex and Nucleus Accumbens in Freely-Moving Rats. The Journal of the Kyushu Dental Society, 2007. 61(1): p. 39-54. Gerasimov, M.R., et al., Toluene inhalation produces regionally specific changes in extracellular dopamine. Drug Alcohol Depend, 2002. 65(3): p. 243-51. NRS, Guide for the care and use of laboratory animals. 2011, National Research Council (U.S.). Institute for Laboratory Animal Research (U.S.), National Academies Press: Washington, D.C. p. xxv, 220 p. Birbeck, J.A., M. Khalid, and T.A. Mathews, Potentiated striatal dopamine release leads to hyperdopaminergia in female brain-derived neurotrophic factor heterozygous mice. ACS Chem Neurosci, 2014. 5(4): p. 275-81. Bosse, K.E., et al., Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8‐ deficient mice. Journal of Neurochemistry, 2015. 135(6): p. 1218-1231. Bosse, K.E. and T.A. Mathews, Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice. Neurosci Lett, 2011. 489(3): p. 172-6. Bosse, K.E. and T.A. Mathews, Ethanol-induced increases in extracellular dopamine are blunted in brain-derived neurotrophic factor heterozygous mice. Neuroscience letters, 2011. 489(3): p. 172-176. Bowen, S.E. and R.L. Balster, Effects of inhaled 1,1,1-trichloroethane on locomotor activity in mice. Neurotoxicol Teratol, 1996. 18(1): p. 77-81. Khalid, M., R.A. Aoun, and T.A. Mathews, Altered striatal dopamine release following a sub-acute exposure to manganese. J Neurosci Methods, 2011. 202(2): p. 182-91. Franklin, K.B.J. and G. Paxinos, Paxinos and Franklin's The mouse brain in stereotaxic coordinates. Third edition. ed. 2008, Amsterdam: Academic Press, an imprint of Elsevier. 256. Zackheim, J. and E.D. Abercrombie, Thalamic regulation of striatal acetylcholine efflux is both direct and indirect and qualitatively altered in the dopamine-depleted striatum. Neuroscience, 2005. 131(2): p. 423-36. Whitehead, K.J., S. Rose, and P. Jenner, Involvement of intrinsic cholinergic and GABAergic innervation in the effect of NMDA on striatal dopamine efflux and metabolism as assessed by microdialysis studies in freely moving rats. Eur J Neurosci, 2001. 14(5): p. 851-60. Mathews, T.A., et al., No role of the dopamine transporter in acute ethanol effects on striatal dopamine dynamics. Synapse, 2006. 60(4): p. 288-94. Tegeris, J.S. and R.L. Balster, A comparison of the acute behavioral effects of alkylbenzenes using a functional observational battery in mice. Fundam Appl Toxicol, 1994. 22(2): p. 240-50. Bowen, S.E., S. Kimar, and S. Irtenkauf, Comparison of toluene-induced locomotor activity in four mouse strains. Pharmacol Biochem Behav, 2010. 95(2): p. 249-57. Conti, A.C., et al., Investigation of calcium-stimulated adenylyl cyclases 1 and 8 on toluene and ethanol neurobehavioral actions. Neurotoxicol Teratol, 2012. 34(5): p. 481488.
32.
33.
34. 35. 36. 37. 38.
39.
Matchett, J.A. and C.K. Erickson, Alteration of ethanol-induced changes in locomotor activity by adrenergic blockers in mice. Psychopharmacology (Berl), 1977. 52(2): p. 2016. Smoothy, R. and M.S. Berry, Time course of the locomotor stimulant and depressant effects of a single low dose of ethanol in mice. Psychopharmacology (Berl), 1985. 85(1): p. 57-61. Gospe, S.M., Jr. and M.A.S. Al-Bayati, A comparison of oral and inhalation exposures to toluene. Journal of the American College of Toxicology, 1994. 13(1): p. 21-32. Koga, K., [Distribution, metabolism and excretion of toluene in mice (author's transl)]. Nihon Yakurigaku Zasshi, 1978. 74(6): p. 687-98. Lo, P.S., et al., Acute neurobehavioral effects of toluene: involvement of dopamine and NMDA receptors. Toxicology, 2009. 265(1-2): p. 34-40. Kreitzer, A.C. and R.C. Malenka, Striatal plasticity and basal ganglia circuit function. Neuron, 2008. 60(4): p. 543-54. Tracy, M.E., M.L. Banks, and K.L. Shelton, Negative allosteric modulation of GABAA receptors inhibits facilitation of brain stimulation reward by drugs of abuse in C57BL6/J mice. Psychopharmacology (Berl), 2016. 233(4): p. 715-25. Stengard, K. and W.T. O'Connor, Acute toluene exposure decreases extracellular gamma-aminobutyric acid in the globus pallidus but not in striatum: a microdialysis study in awake, freely moving rats. Eur J Pharmacol, 1994. 292(1): p. 43-6.
Figure 1 – Locomotor activity during the 30 minute exposure period. Toluene concentrations of 4,000 and 8,000 ppm increased locomotor activity as compared to air controls during the first 15 min of exposure; bicuculline had no effect on locomotor activity alone or in combination with toluene exposure (left panel). Toluene no longer increased locomotor activity during the second half of the toluene exposure (right panel); bicuculline was still without effect.
Figure 2. The effect of acute toluene exposure and GABAA antagonism on extracellular DA levels in the mouse CPu (data are represented as percent of baseline DA) during the 30 minute exposure period. A) No difference in extracellular DA levels in the mouse CPu during the first half of the 30 min toluene exposure (4000 or 8000 ppm). Bicuculline pretreatment had no effect on extracellular DA. B) As seen in the right panel, only the 8,000-ppm concentration of toluene significantly increased DA in the mouse CPu during the second half of the 30 min toluene exposure, while bicuculline pretreatment had no effect on extracellular DA. Figure 3 – Locomotor activity data during the one hour (in four 15-min bins) recovery period following acute toluene exposure. As seen in the panels, 4,000 ppm of toluene significantly elevated locomotor activity over baseline. Additionally, bicuculline interacted with toluene, suppressing activity in controls, while potentiating activity in toluene-exposed animals. Figure 4 – Shown are extracellular DA in the CPu for the one hour (in four 15-min bins) recovery period following exposure. Animals exposed to 8,000-ppm toluene vapor showed a significant increase in DA as compared to air controls. There was no effect of bicuculline pretreatment. Data are represented as percent of baseline DA.