Neurotoxicology and Teratology 73 (2019) 9–14 Contents lists available at ScienceDirect Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera Brief communication Repeated toluene exposure alters the synaptic transmission of layer 5 medial prefrontal cortex T ⁎ Silvia L. Cruz, Mayra Torres-Flores, Emilio J. Galván Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Calzada de los Tenorios No. 235, México City 14330, Mexico ARTICLE INFO ABSTRACT Keywords: Toluene is an organic solvent commonly misused by inhalation among adolescents to experience psychoactive Inhalants effects. Repeated toluene exposure produces several cognitive deficits, including working memory impairment in Toluene which the medial prefrontal cortex (mPFC) plays a central role. Among other effects, toluene antagonizes NMDA Dopamine receptors, enhance GABAA receptor-mediated responses and increases dopamine release. We have recently re- Synaptic plasticity ported that animals repeatedly exposed to toluene show increased mPFC excitability; however, alterations in Medial prefrontal cortex synaptic transmission, including long-term synaptic plasticity of glutamatergic responses have not been studied thus far. Here we used extracellular recordings to determine the effects of repeated toluene exposure (8000 ppm for 30 min, twice a day, for ten days) on the synaptic transmission converging on prelimbic layer 5 pyramidal neurons of the mPFC in adolescent male Wistar rats. Repeated toluene exposure increased mPFC's synaptic strength and reduced the inhibitory transmission assessed by input-output curves and paired-pulse inhibition protocols, respectively. Both toluene and a selective D1 receptor antagonist blocked the ability of exogenous dopamine to induce synaptic potentiation. Repeated toluene exposure also altered the ability of NMDA to induce synaptic depression of excitatory transmission. Taken together, the changes in synaptic strength and impairment of the NMDA-mediated plasticity of the mPFC demonstrate a series of synaptic modifications of the glutama- tergic transmission that may underlie the cognitive impairment resulting from repeated toluene exposure. 1. Introduction of repeated toluene inhalation include memory impairment (Huerta- Rivas et al., 2012) and increased excitability (Armenta-Resendiz et al., Inhalant misuse is the intentional inhalation of products containing 2018). Prolonged exposure to toluene is also associated with neuronal substances that are volatile at room temperature and have psychoactive structural changes. Inhalation to 8000 ppm toluene for 10 days in- effects (Balster et al., 2009). Toluene, in its pure form or as an in- creases the expression of GABAA α1 subunits, NR1 and NR2B subunits gredient of many commercial products, is the most commonly misused of the NMDA receptor (Williams et al., 2005). solvent. When inhaled at high concentrations (typically 5000 to Few studies have analyzed the electrophysiological alterations 12,000 ppm; Bukowski, 2001) for brief periods, toluene produces mind- caused by high toluene concentrations. Wayman and Woodward altering effects. This intentional exposure pattern contrasts with occu- (2018), showed that a single 10,500 ppm toluene inhalation session pational exposure, characterized by extended inhalation periods (8 h/ significantly reduces the firing output of layer 5/6 prelimbic neurons day, 5 days/week) to very low solvent concentrations (Bowen et al., projecting to the nucleus accumbens core. More recently, we showed 2006). that 20 exposure sessions to 8000 ppm toluene increase the excitability Depending on its concentration, toluene can act on several mole- of prelimbic layer 5 pyramidal neurons of the medial prefrontal cortex cular targets. At a low millimolar range, toluene inhibits NMDA re- (mPFC) in adolescent rats. This hyperexcitability is characterized by ceptors in a non-competitive manner (Bale et al., 2005; Cruz et al., enhanced action potential discharge mediated by a decrease in the slow 1998), is a positive allosteric modulator of GABAA and glycine receptors component of the hyperpolarization current and increased glutama- (Bale et al., 2005; Beckstead et al., 2000) and increases dopamine re- tergic activity (Armenta-Resendiz et al., 2018). lease in the mesocortical system (Gerasimov et al., 2002). Other effects To the best of our knowledge, there are no studies analyzing ⁎ Corresponding author at: Departamento de Farmacobiología, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Calzada de los Tenorios No. 235, Col. Granjas Coapa C.P. 14330, Mexico. E-mail address: [email protected] (E.J. Galván). https://doi.org/10.1016/j.ntt.2019.02.002 Received 1 December 2018; Received in revised form 26 February 2019; Accepted 27 February 2019 Available online 28 February 2019 0892-0362/ © 2019 Elsevier Inc. All rights reserved. S.L. Cruz, et al. Neurotoxicology and Teratology 73 (2019) 9–14 whether synaptic transmission alterations exist in the mPFC of adoles- responses were amplified with an Axopatch 200B; the analog signals cent animals repeatedly exposed to toluene. Here, we show that re- were sampled by a Digidata 1440A interface coupled to pCLAMP 10 peated exposure to toluene increases the mPFC synaptic strength and software (Molecular Devices, Foster City, CA). The fEPSP latency was reduces the inhibitory transmission impinging on prelimbic mPFC layer calculated from the end of the electrical stimulus to the beginning of the 5 pyramidal in adolescent rats. We also show that toluene blocked the fEPSP sink. Paired-pulse inhibition was expressed as the ratio between ability of exogenous dopamine to induce synaptic potentiation. the second and the first fEPSP of the pair (S2/S1). To corroborate the participation of the GABAergic inhibition, picrotoxin (50 μM; see 2. Materials and methods Fig. 1C) was perfused at the end of the PPI protocol. Dopamine (DA) perfusion (100 μM, 10 min) was performed in the presence of 0.4 mM of 2.1. Animals ascorbic acid to avoid fast oxidation of DA; NMDA (20 μM) was bath perfused for 3 min. A total of 47 male Wistar rats (postnatal days 30–37) provided form our vivarium were used. Our procedures complied with the Mexican Official Norm for utilization and care of laboratory animals “NOM-062- 2.6. Statistical and data analysis ZOO-1999”, the local Ethics Committee of our Institution (authoriza- tions: 0101-14 and 0090-14), the National Institutes of Health guide- A two-way ANOVA test was used to analyze data obtained from I-O lines (NIH, 2011) and ARRIVE guidelines. curves, FV-fEPSP curves and the effects of different ISIs on fEPSP and PPI ratios. One-Way ANOVA followed by Student-Newman-Keuls post- 2.2. Drugs hoc was used to compare the effect of toluene on the fEPSP slope during perfusion of DA. For further comparisons, we analyzed an early and late Toluene (99.8% HPLC grade), dopamine, L-ascorbic acid, S(−)-ra- effect of DA at minute 10th of DA perfusion and min 40th after DA clopride (+)-tartrate salt, R(+)-SCH-23390 hydrochloride, N-methyl-D- washout. Student's t-test was used for comparison of NMDA effects. All aspartate (NMDA), Kynurenic acid, and picrotoxin were purchased hypotheses were tested when α = 0.05. from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). Tetrodotoxin (TTX) was from Alomone Labs (Jerusalem, Israel). 3. Results 2.3. Toluene exposure 3.1. Repeated toluene exposure alters the synaptic efficacy of layer 5 mPFC The exposure method has been described in detail elsewhere (Armenta-Resendiz et al., 2018). Briefly, independent groups of rats Two evoked responses were evaluated, extracellular field potentials were exposed to air or 8000 ppm toluene for five consecutive days, left (fEPSP) and population spikes (PS). Whereas the fEPSP was sensitive to untreated during the weekend, and re-exposed to air or toluene for five the non-specific blocker of glutamate receptors, Kynurenic acid (Kyn additional days. Each session lasted 30 min, was conducted in 27-l static 2 mM), the inward deflection of the PS was abolished with the perfusion exposure chambers and was repeated twice daily (6 h apart). The of the sodium channel blocker TTX (0.5 μM; Fig. 1B). Next, we eval- amount of toluene injected into the chamber was calculated using the uated the input-output (I-O) relationship of the fEPSP (stimulation ideal gas equation for closed systems (Nelson, 1971). The nominal current vs. evoked fEPSP). In control, the maximal amplitude was concentration was confirmed with a photoionization detector (Pho- 1.3 ± 0.09 mV. The mPFC slices from animals repeatedly exposed to Check Tiger, Ion Science, LTD, Cambs, UK). Toluene concentration was toluene (hereafter referred as experimental group) reached a maximal chosen based on previous reports of its effect on animal behavior and value of 1.62 ± 0.1 mV (Fig. 1D). In both groups, the latency to the relevance to solvent intoxication (Armenta-Resendiz et al., 2018; evoked responses was constant at all the stimuli tested Bowen et al., 2006). (2.66 ± 0.03 ms). Computing the average rate of change of I-O curves (Villanueva-Castillo et al., 2017) showed that toluene exposure in- 2.4. Brain slice preparation creased the I-O relationship in 31.5%. Two-way ANOVA found changes in the I-O curve of control slices vs. the experimental group (F(1, Eighteen hours after the last solvent exposure, mPFC slices were 233) = 83.819; p = 0.001) and current intensity (F(12, 233) = 33.01; prepared, as described in detail elsewhere (Armenta-Resendiz et al., p < 0.001). Slices in which a presynaptic fiber volley (FV) preceded 2018). Briefly, after decapitation the brain was placed in frozen sucrose the postsynaptic response were independently analyzed to establish the solution consisting of (in mM): 210 sucrose, 25 NaHCO3, 2.8 KCl, 2 FV–fEPSP relationship. For a given FV, the evoked fEPSP response MgSO4, 1.25 NaH2PO4, 10 glucose, 4 MgCl2, and 1 CaCl2, bubbled with augmented 32.8% in the experimental group (F(12, 233) = 18.939; 95%:5% O2:CO2. The mPFC was dissected, and coronal slices (385 μm) p < 0.001; Fig.