Repeated toluene exposure increases the excitability of layer 5 pyramidal neurons in the prefrontal cortex of adolescent rats

Neurotoxicology and Teratology 68 (2018) 27–35

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Repeated toluene exposure increases the excitability of layer 5 pyramidal neurons in the prefrontal cortex of adolescent rats Monserrat Armenta-Resendiz, Silvia L. Cruz, Emilio J. Galván

T

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Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Calzada de los Tenorios No. 235, México City 14330, Mexico

A R T I C LE I N FO

A B S T R A C T

Keywords: Inhalants Solvents Toluene, neuronal excitability Medial prefrontal cortex

Despite serious health effects, volatile industrial products containing toluene are deliberately inhaled for their psychoactive actions, mainly among adolescents and young adults. Chronic toluene inhalation induces multiple alterations at the cellular and behavioral level; however, modifications of neuronal networks associated with the reward system after repeated toluene exposure are not thoroughly characterized. Here we used whole-cell recordings to determine the effects of repeated exposure to toluene (1000, 4000 or 8000 ppm for 30 min, twice a day, for ten days) on the neurophysiological properties of prelimbic layer 5 pyramidal neurons of the medial prefrontal cortex (mPFC) in adolescent male Wistar rats. Neurons from animals repeatedly exposed to toluene showed a concentration-dependent increase in action potential firing discharge. This increase was related to a reduction of the small-conductance calcium-activated potassium current (after-hyperpolarization current, IAHP) that controls the firing frequency of neurons. Likewise, toluene altered the kinetics of the action potential. The hyperexcitability seen in toluene-exposed animals was also associated with an increase in the glutamatergic spontaneous synaptic activity converging on mPFC neurons. In summary, repeated toluene exposure enhances the excitability of prelimbic layer 5 pyramidal neurons of the mPFC in adolescent rats.

1. Introduction Inhalant misuse, also referred to as inhalant abuse or volatile substance misuse, is the intentional inhalation of volatile substances to induce mind-altering effects (Balster et al., 2009; Rees et al., 1985). Inhalants are present in legal, inexpensive and readily accessible commercial products such as sprays, solvents, paint thinners, fuels and glues (Balster et al., 2009). In the inhalant abuse setting users are exposed to organic solvents at concentrations that are several thousand-fold higher (up to 15,000 ppm) than those found in the occupational setting (Bowen et al., 2006; Bukowski, 2001; Hathaway et al., 2004; Marjot and McLeod, 1989). Inhalant misuse is a public health problem that causes significant worldwide morbidity and mortality due to long-term toxicity, attention and memory impairment, sleep disturbances and other disorders (Real et al., 2015; Ridenour et al., 2007; Yücel et al., 2008). The most commonly inhaled substance among solvent users is toluene. The acute effects of toluene are similar to those produced by several central nervous system (CNS) depressant drugs (Balster, 1998). Toluene exposure in episodes that mimic binge patterns of intoxication

(1000–8000 ppm, for 15–30 min) produces antianxiety-like effects (Beasley et al., 2010; López-Rubalcava et al., 2000) decreases motor coordination (Bowen et al., 1996) and impairs cognition (Bale et al., 2005; Baydas et al., 2003; Huerta-Rivas et al., 2012; Win-Shwe et al., 2012). Inhalant misuse is particularly relevant among adolescents and young adults (Hynes-Dowell et al., 2011; Johnston et al., 2016; MedinaMora and Real, 2008; Villatoro et al., 2011). This problem is worsened because adolescence represents the final transitional period of behavioral and cognitive maturation. During this stage, the brain undergoes multiple adjustments, including neuronal pruning, neurotransmission refinement and regional brain maturation (Crews and Boettiger, 2009; Spear, 2000). The prefrontal cortex (PFC) is one of the brain areas that exhibit more signs of maturation during adolescence (Blakemore, 2008; Casey et al., 2008; Sturman and Moghaddam, 2011). This region integrates stimuli from multiple cortical and subcortical structures and functions, such as planning processing, working memory, impulse control, and risk assessment. The prelimbic region of mPFC is involved in learning and decision making and modulates the activity of the nucleus accumbens, a subcortical structure critically involved in the

⁎ 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.2018.04.006 Received 6 February 2018; Received in revised form 26 April 2018; Accepted 27 April 2018 Available online 30 April 2018 0892-0362/ © 2018 Elsevier Inc. All rights reserved.

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injection ports and a fan projecting into the chamber. The liquid solvent was delivered with a syringe through an injection port onto a piece of paper suspended below the fan, which was then turned on for rapid solvent evaporation and dispersion. We used the following equation for calculating the amount of solvent to be injected into the chamber to achieve specific concentrations (Nelson, 1971): Vl = ((MW Cppm Vs)/ ρ) ((P(10−6) / RT) where: Vl = volume of the solvent injected into the closed system to achieve the desired concentration in ppm (Cppm); MW = molecular weight (toluene = 92.14 g mol−1); Vs = volume of the closed-loop system (exposure chamber: 27 l); ρ = density (toluene = 0.865 g ml−1), P = pressure (0.77 atm in Mexico City), R = ideal gas constant (0.082 l atm mol−1 K−1), T = 293 K. Accordingly, the amounts of toluene injected into the chamber were 92 μl for 1000 ppm, 368 μl for 4000 ppm and 736 μl for 8000 ppm. Nominal concentrations were confirmed using a photoionization detector (PhoCheck Tiger, Ion Science, LTD, Cambs, UK). The actual concentrations varied from the 1st and the 30th minute after injection as follows: for a theoretical 1000 ppm concentration = from 1221 to 922 ppm; for 4000 ppm = from 4559 to 3580; for 8000 ppm = from 8802 to 7468 (Fig. 1, Supplementary figure). Toluene concentrations were chosen based on previous reports in animals (Beyer et al., 2001; Callan et al., 2017; Rivera-García et al., 2015) and relevance to solvent intoxication (Bowen et al., 2006; Bukowski, 2001; Hathaway and Proctor, 2004).

reward circuit (Euston et al., 2012; Rushworth et al., 2011). The negative impact of substance abuse on the PFC has been widely documented for ethanol (Ward et al., 2014), but studies using toluene are scarce despite similarities in action mechanisms between these drugs. Both ethanol and toluene increase cortical dopamine release (Riegel et al., 2007; Williams et al., 2005), are non-competitive NMDA receptor antagonists (Cruz et al., 1998) and exert positive allosteric GABAA receptor modulation (Beckstead et al., 2000). In addition, toluene modulates a wide range of ligand-operated neurotransmitter receptors and voltage-sensitive ion channels, which include, but are not limited to sodium, potassium and calcium channels (reviewed in Beckley and Woodward, 2013; Cruz et al., 2014). Perfusion of toluene (1 to 3 mM) on acute mPFC slices does not modify passive properties or the intrinsic excitability of mPFC neurons from adolescent rats (Beckley and Woodward, 2011). On the other hand, a recent study found that brief toluene inhalation (10,500 ppm) transiently alters, in a regional-specific manner, mPFC excitability (Wayman and Woodward, 2017). The same group of researchers found that repeated (6) pairings of 3000 ppm toluene exposure that induced a conditioned place preference: a) increased the evoked firing of infralimbic mPFC pyramidal neurons that project to the nucleus accumbens (NAc) core; b) decreased evoked firing of infralimbic pyramidal neurons projecting to the NAc shell, and c) had no effect on the excitability of prelimbic neurons regardless of the NAc projection target of the recorded neuron (Wayman and Woodward, 2018). Here, we investigate the effects of a sub-chronic exposure to toluene on the intrinsic excitability of cortical neurons of the prelimbic mPFC in adolescent rats. Our data provide evidence that toluene increases the excitability of prelimbic mPFC pyramidal neurons in a concentration-dependent manner by reducing the amplitude and duration of the slow after-hyperpolarization potential and increasing spontaneous synaptic activity.

2.4. Brain slice preparation Rats were deeply anesthetized with sodium pentobarbital (50 mg/ kg i.p.) and decapitated 18 h after the 20th solvent exposure. The brains were quickly removed and placed in frozen sucrose solution containing (in mM): 210 sucrose, 25 NaHCO3, 10 glucose, 2.8 KCl, 2 MgSO4, 1.25 NaH2PO4, 4 MgCl2 and 1 CaCl2, bubbled with 95%:5% O2: CO2. The modification in the Ca2+/Mg2+ ratio and the substitution of sucrose for D-glucose were used to minimize the neuronal damage following the mechanical slicing. The medial PFC (mPFC) was dissected out and cut into 385 μm coronal slices using a vibratome (Leica VT1000 S, Nussloch, Germany). Slices were incubated for 30 min at 35 °C and then stabilized for no < 1 h at room temperature in an incubation solution of the following composition (in mM): 125 NaCl, 2.5 KCl, 1.2 NaH2PO4, 25 NaHCO3, 10 glucose, 4 MgCl2 and 1 CaCl; bubbled with 95%:5% CO2:O2. Slices were individually placed in the recording chamber and superfused at a rate of 3–5 ml/min with artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 3.0 KCl, 1.25 NaH2PO4, 25 NaHCO3, 1 CaCl2, 2 MgCl2, and 10 D-glucose. The time elapsed between the last toluene exposure and the beginning of the experiment was 20 ± 1 h, which allowed for drug elimination.

2. Materials and methods 2.1. Animals A total of 50 adolescent male Wistar rats (postnatal day (PN) 25–37) were used in this study. Animals were bred and housed under a 12:12 h light/dark cycle in a climate-controlled room at 22 ± 2 °C at our local animal facility with ad libitum access to food and water under continuous veterinary supervision. The experimental procedures were carried out in strict accordance with the Mexican Official Norm for utilization and care of laboratory animals “NOM-062-ZOO-1999” and complied with the local Ethics Committee of our Institution (authorization numbers 0101-14 and 0090-14), the National Institutes of Health guidelines (NIH, 2011) and ARRIVE guidelines (Kilkenny et al., 2013) for animal research.

2.5. Whole cell recordings

2.2. Drugs

Layer 5 pyramidal cells located in the prelimbic sub-region of mPFC were visually identified using an upright infrared-differential interference contrast (IR-DIC) microscope (Nikon Eclipse FN1, Nikon Instruments, USA). Recordings were obtained using an Axopatch-1D amplifier (Molecular Devices, USA). The analog signals acquired during our experiments were real-time filtered via a Humbug noise eliminator and additional P-Clamp digital filters (8-pole Bessel with cut-off 5) were used at convenience for each analyzed cell in order to increase the signal-to-noise ratio. Borosilicate glass electrodes (4–8 MΩ resistance) were filled with standard solution containing (in mM): 120 K-MeSO4, 10 NaCl, 10 KCl, 0.5 EGTA, 4 ATP-Mg, 0.3 GTP, 10 HEPES and 14 phosphocreatine; pH 7.2–7.35 and osmolarity 280–300 mOsm. In experiments intended to isolate the afterhyperpolarization current (IAHP) in voltage clamp mode, the EGTA concentration was reduced to 0.2 mM to favor amplitude and total area of the isolated current. The access resistance (≤25 MΩ) was monitored during the length of the experiments to ensure high-quality recordings and experiments were discarded if the resistance changed > 20% throughout recordings. For

Toluene (99.8% HPLC grade), 1(S),9(R)-(−)-bicuculline methiodide (3-Triphenyl methylaminomethyl) pyridine (UCL-2077) and 4-hydroxyquinoline-2-carboxylic acid (kynurenic acid) were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA). All drugs except toluene were superfused at a rate of 3–5 ml/min. 2.3. Toluene exposure Independent groups of animals were exposed to air, 1000, 4000 or 8000 ppm toluene for 30 min, twice a day, 6 h apart, for five consecutive days. Animals were left untreated for the weekend and then reexposed to air or toluene twice a day for another five days in a static exposure chamber as previously described (Rivera-García et al., 2015). Briefly, each animal was placed into a 27-l cylindrical jar that had a rubber gasket fixed to the rim of the jar to ensure a close-fitting seal. The jar was then hermetically closed with a Plexiglas cover that had 28

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acquisition, storage and off-line analysis. Data were further analyzed using GraphPad Prism (GraphPad Software Software, San Diego CA), version 5.

experiments involving the monitoring of NMDA and AMPA-mediated glutamatergic spontaneous synaptic activity, a cesium-based intracellular solution with the following composition (in mM) was used: 120 CsMeSO4, 10 KCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP and 0.3 Na2-GTP at pH 7.2–7.35. For sEPSCs detection, the threshold limit was established at 10 pA. Because compensation of series resistance in voltage clamp mode attenuates and slows the spontaneous synaptic activity, the apparent peak current and EPSC kinetics here reported must be taken cautiously. It is expected that the manual adjustment of series resistances (5–25 ΩOhms) causes a distortion, reducing the peak current and increasing in the rise time value of the synaptic events recorded. Despite this limitation, the spontaneous events were abolished entirely in the presence of kynurenic acid, confirming their glutamatergic nature. Under the present experimental conditions, it cannot be discarded contamination from synaptic events arriving from the dendritic inputs. The following battery of electrophysiological protocols was used to study the passive and active properties of mPFC pyramidal cells: A) voltage-current (IV) curve: the membrane potential was set to −70 mV by injecting direct current when needed. The I-V plot was constructed by applying 30 pA steps of current injection (1.2 s duration) until reaching the action potential (AP) threshold. Input resistance (Rn) was calculated as the slope of the linear portion of the IV curve corresponding to −30, 0 and 30 pA steps, and the resting membrane potential (RMP) as the y-intercept. The decay time constant was calculated as the exponential constant of the capacitive response to a single 30 pA pulse. B) Firing frequency: the membrane potential was held at −70 mV and depolarizing pulses of increasing intensities (0–300 pA in 20 pA steps; 1 s duration at 0.2 Hz) were applied. The rheobase current was determined as the current step required to eliciting one AP in current clamp mode at −70 mV. AP frequency was measured by calculating the number of APs in 1 s. C) Current ramp: somatic current injections (300 ms duration) were applied to cells held at −70 mV until the AP threshold was reached. AP amplitude (voltage difference between the threshold value and the peak), width, rise and decay time were calculated using a custom made template. D) The afterhyperpolarization measurements were determined in two ways. In current clamp mode, cells were held at −55 mV and a current step (50 ms, 1–3 nA) elicited a short AP burst (2–5) followed by an afterhyperpolarization. This depolarizing potential favors the activation and increases the driving force of the slow Ca2+-activated K+ currents, as negative potentials reduce the amplitude or reverse the direction of the current. In addition, this protocol stimulates both the medium and the slow component of the afterhyperpolarization current that becomes most prominent following a burst of spikes (Storm, 1989). From the resulting inward conductance, the maximal amplitude (in mV) and total area (mV/ms) were measured. To determine the amplitude and duration of the afterhyperpolarization current, cells were held in voltage clamp mode at −50 mV and a 100 mV step (15 ms) were applied. From the resulting outward current, maximal amplitude (pA) and area (pA/ ms) were measured in each experimental condition.

3. Results 3.1. Behavioral assessment of toluene effects The 8000 ppm concentration produced ataxia and sedation (Beyer et al., 2001), head twitches (Rivera-García et al., 2015) and nose irritation. After repeated toluene exposure, tolerance to the sedative effects of toluene occurred along with sensitization to locomotor activity (Batis et al., 2010), while irritation and head twitches prevailed. No significant differences in body weight were found among animals from control and toluene-exposed groups (mean ± S.E.M. body weight at the beginning of the exposure sessions: control = 74.8 ± 3.9 g, 8000 ppm toluene = 74.4 ± 3 g; body weight at the end of the exposure sessions: control = 134.6 ± 6.3 g, 8000 ppm toluene = 132 ± 4.3 g). 3.2. Effects of repeated exposure to toluene on prefrontal cortex neurons Somatic whole cell recordings were performed in 175 pyramidal neurons located in the prelimbic area of the mPFC. Neurons displayed a stable membrane potential after the initial break-in and overshooting APs. In control cells, the RMP was −74.3 ± 0.86 mV with an input resistance (RN) of 132 ± 11 MΩ; spontaneous synaptic activity was barely evident. In response to a depolarizing current step (220 pA for 1200 ms), the neurons of control animals displayed three different firing patterns (Elston et al., 2005; Satake et al., 2008). Specifically, 62 out of 87 neurons exhibited a regular spiking fire pattern (RS), 7, short bursts of action potentials (intrinsic burst, IB) and 8, a single AP followed by a strong adaptation (adaptive firing, AF; Fig. 1A). Only the IB neurons exhibited a hyperpolarizing sag conductance when a negative current step (330 pA for 1200 ms) was applied. This proportion was similar in cells treated with 1000, 4000 and 8000 ppm toluene (61 RS, 8 IB, 9 AF; Fig. 1B). Because the regular spiking fire pattern was prevalent among the recorded cells, subsequent experiments were performed using only this neuronal subpopulation. We then determined the modulatory actions of toluene on the passive and active properties of mPFC neurons from animals exposed to 0, 1000, 4000 or 8000 ppm toluene. The RMP of control cells and of those from toluene-treated animals were similar (drug effect: F(3,88) = 0.43, P = 0.73). Toluene treatment increased the RN (drug effect: F(3,88) = 2.35, P = 0.02), reduced the membrane time constant (drug effect: F(3,88) = 11.9, P < 0.0001) and decreased the rheobase current to evoke APs (drug effect: F(3,88) = 9.7, P < 0.001). These effects were statistically significant at 8000 ppm (Fig. 1C). Further analysis of the IV relationship showed that the group exposed to 8000 ppm toluene exhibited increased membrane rectification in response to the injection of negative current pulses (percentage of rectification in the 8000 ppm exposed group = 18.35 ± 0.14%; drug effect: t(38) = 3.3; P = 0.004 (Fig. 1D).

2.6. Statistical and data analysis The effects of toluene on passive and active properties of mPFC neurons were analyzed with a one-way analysis of variance (ANOVA) followed by Dunnett (versus control) or Tukey test (among groups) post hoc tests. Comparisons among the kinetic parameters of the 1st, 5th and 10th AP in tonic bursts were done using repeated measures ANOVA. IAHP parameters of control and toluene-exposed neurons were compared before and after administration of UCL 2077 with the paired Student's ttest. The spontaneous synaptic activity in control and 8000 ppm-treated neurons was compared using the two-sample Kolmogorov-Smirnov test with XLSTAT 2018 (Addinsoft., New York, NY). All hypotheses were tested when α = 0.05. pClamp 10.5 software (Axon Instruments Inc. Foster City, CA, USA) was used for stimulus generation, data display,

3.3. Repeated exposure to toluene modifies the firing frequency of pyramidal neurons Fig. 2A exemplifies the effect of toluene on the firing frequency of mPFC from animals exposed to 4000 or 8000 ppm toluene. The neurons were current-clamped at −70 mV and current steps (50 pA, 1000 ms) were somatically injected. The 1000 and 4000 ppm toluene-exposed groups did not exhibit significant changes in the firing frequency when compared to control cells (Fig. 2A and B, gray circles), but the 8000 ppm-exposed group exhibited a significant increase in the firing discharge (AP number in control evoked with 300 pA (arrow in Fig. 2B, 29

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Fig. 1. Firing pattern and passive membrane properties of mPFC layer 5 prelimbic neurons. A) Sample recordings of firing patterns observed with a 150 pA, 1 s current step in control neurons. B) The proportion of cells showing different firing discharge patterns in control and toluene (1000 ppm, 4000 ppm, and 8000 ppm)-exposed animals. C) Bar graphs (mean ± S.E.M. and individual data) summarizing the passive properties of the experimental groups. RMP: resting membrane potential; Rn: input resistance for control neurons (ncells/animals = 30/10) and neurons from animals treated with 1000 ppm toluene (ncells/animals = 16/5), 4000 ppm toluene (ncells/animals = 16/5) or 8000 ppm toluene (ncells/animals = 30/10).*P < 0.05, ***P < 0.001; Dunnett's test. D) Averaged I/V plot for control neurons (ncells/ animals = 20/10) and neurons from animals treated with 1000 ppm toluene (ncells/animals = 16/5), 4000 ppm toluene (ncells/animals = 16/5) or 8000 ppm toluene (ncells/animals = 20/10). Each point represents the mean ± S.E.M. **P < 0.01; Student's t-test. The right panels show representative tracings of the negative portion of the I-V curve showing the inward rectification.

kinetics are shown in Fig. 3A and summarized in Table 1). Next, we visually inspected the AP waveforms with a series of ten consecutive phase plots (voltage trace derivatives against the instantaneous membrane potential; Fig. 3B) and found that toluene modified the waveform and significantly decreased AP threshold (t(18) = 13.06, P < 0.0001). The change in the AP threshold was confirmed with a depolarizing current ramp (0–250 pA/75 ms), which showed that toluene caused a shift in the start of nonlinearity in the slope resistance to a more negative membrane potential (Fig. 3C).

left panel = 15.4 ± 1.27; 1000 ppm-group = 15 ± 2.0; 4000 ppm = 16.5 ± 2.2; 8000 ppm = 25.4 ± 1.3; drug effect: F(3,88) = 50.04, P < 0.0001). Consistent with this observation, the 8000 ppm-exposed group exhibited significantly increased instantaneous firing frequency (IFF) (control IFF = 15.5 ± 1.3 Hz; 1000 ppm = 15.9 ± 1.26; 4000 ppm = 16.4 ± 1.6; 8000 ppm = 29.1 ± 1.7 Hz; drug effect: F(3,88) = 25, P < 0.0001; Fig. 2B, right panel).

3.4. Repeated exposure to toluene alters action potential kinetics 3.5. Repeated exposure to toluene reduces the slow component of the afterhyperpolarization current

We then analyzed the mechanism by which repeated exposure to 8000 ppm toluene altered the intrinsic excitability of cortical neurons. First, we compared the 1st, 5th and 10th AP obtained from a tonic burst (depolarizing step: 300 pA, 1.2 s) in the control and toluene-exposed groups. There was a decrease in AP amplitude, rise and decay time constant in subsequent spikes in the toluene group with respect to controls. Toluene also increased the AP half-width (changes in AP

The effect of toluene exposure on the afterhyperpolarization that follows a train of APs is shown in Fig. 4. The injection of a depolarizing pulse evoked a brief AP burst in neurons current-clamped at −55 mV. After AP repolarization, a slow afterhyperpolarization (sAHP) lasting > 1 s was exposed (Sah 1996). Compared to control, toluene 30

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Fig. 2. Toluene increases the firing frequency of PFC neurons. A) Representative action potential (AP) trains evoked with increasing depolarizing pulses (bottom trace: 100 pA), (middle trace: 200 pA), (top trace: 300 pA) in cells clamped at −70 mV. B) Averaged AP number elicited by increasing current steps in control (Ctrl, ncells/animals = 20/8) and toluene-exposed cells (1000 ppm: ncells/animals = 16/5; 4000 ppm: ncells/animals = 16/5; 8000 ppm: ncells/animals = 20/8). The right panel summarizes changes in the instantaneous firing frequency as a function of the AP number. Each point represents the mean ± S.E.M. ***P < 0.001; Dunnett's test.

discharge is mediated by a decrease in the slow component of the afterhyperpolarization current. Toluene also increases glutamatergic activity converging on mPFC neurons as evidenced by changes in the frequency of spontaneous postsynaptic currents recorded in the presence of bicuculline. Beckley and Woodward (2011) studied the effects of toluene perfusion (0.1–3 mM) in the recording chamber and found no changes in the intrinsic excitability of neurons within the mPFC of adolescent rats. On the other hand, layer 5/6 prelimbic neurons projecting to NAc core from adolescent rats exposed only once to 10,500 ppm toluene exhibited reduced (not increased) evoked firing as compared to neurons from control rats exposed only to air (Wayman and Woodward, 2017). A recent study found that exposing adolescent rats to 3000 ppm toluene every other day for a total of 6 sessions increased evoked firing in infralimbic mPFC pyramidal neurons that project to the nucleus accumbens (NAc) core, decreased evoked firing in infralimbic neurons projecting to the NAc shell and had no effects on prelimbic neurons (Wayman and Woodward, 2018). These results contrast with our findings showing that repeated (20×) exposure to 8000 ppm toluene increases the neuronal excitability of prelimbic mPFC neurons. Although we did not identify the projection target of the recorded prelimbic neurons, our results suggest that plastic changes occurred with prolonged exposures and binge-like toluene concentrations that led to generalized hyperexcitability within the neuronal mPCF network. In support to this idea, Williams et al. (Williams et al., 2005) reported an increase in the mRNA expression of several receptor units including NR1 and NR2B (NMDA), α1 (GABAA) and GluR2/3 (AMPA) in the mPFC of adult rats daily exposed to 8000 ppm toluene for 30 min. It remains to be determined if similar changes occur in adolescent rats and their relevance in the responses analyzed in the present work. The enhanced firing discharge observed in the 8000 ppm tolueneexposed group in our study was accompanied by an increase in the input resistance, inward rectification of potassium conductance, and a decrease in the rheobase current and the slow component of the afterhyperpolarization that follows the action potential spike. It is well established that modulation of subthreshold conductances active near the RMP shapes passive properties and excitability of central neurons. Therefore, it is expected that an increase in the cell's input resistance accompanied with a reduction in the membrane time constant, yields to an increase in the firing output, a phenomenon that we systematically

significantly reduced the sAHP amplitude (t(38) = 4.89, P < 0.0001; Fig. 4A). Similar results were observed when the normalized total AHP area was analyzed (t(38) = 5.04, P < 0.0001; Fig. 4A). Next, the outward Ca2+-activated K+ current (IAHP) was isolated by combining somatic voltage clamp (at −45 mV) and injection of a depolarizing voltage command to +100 mV (50 ms duration) (Abel et al., 2004; Pedarzani et al., 2005). Under these conditions, the outward current decayed with a time constant (τAHP) of 919 ± 145 ms and mean amplitude of 328.1 ± 55.4 pA. Toluene significantly reduced the τAHP (336 ± 39.7 ms) and amplitude (101.7 ± 13.3 pA). Notably, the perfusion of the slow IAHP blocker UCL-2077 (Soh and Tzingounis, 2010; Zhang et al., 2010) partially inhibited the amplitude of the IAHP in control cells, but barely reduced the already inhibited IAHP of the toluene-exposed group (Fig. 4B and C). 3.6. Repeated exposure to toluene increases the spontaneous glutamatergic events on PFC neurons Spontaneous excitatory postsynaptic currents (sEPSC) were recorded for 5 min in voltage-clamped neurons (−70 mV) bath-perfused with the GABAA receptor antagonist bicuculline (10 μM). Compared to control, neurons exposed to toluene exhibited a significant increase in the sEPSC amplitude (control = 21.7 ± 0.2 pA; toluene = 28.5 ± 0.3 pA; P < 0.0001; Kolmogorov-Smirnov test) accompanied with a reduction in the inter-event frequency (control = 5 ± 0.14 Hz; toluene = 7.8 ± 0.15 Hz, P < 0.0001; Kolmogorov-Smirnov test; Fig. 5A and B). The nature of the glutamatergic transmission was assessed by perfusion of the non-selective NMDA and AMPA/kainate receptor antagonist kynurenic acid (Kyn; 2 mM). Perfusion of Kyn systematically abolished the spontaneous events of mPFC neurons. 4. Discussion Our results show that repeated toluene exposure increases the intrinsic excitability of layer 5 prelimbic neurons in the mPFC of adolescent rats in a concentration-dependent manner. Lower toluene exposures (1000–4000 ppm) scarcely altered intrinsic properties of mPFC neurons. Nevertheless, exposure to 8000 ppm significantly reduced the rheobase current, enhanced the firing output, and modified the AP kinetics of mPFC cells. Our data also revealed that the enhanced AP 31

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Fig. 3. Repeated toluene exposure modifies the action potential kinetics. A) Sample tracings of the 1st, 5th and 10th AP waveforms to show the AP changes induced by 8000 ppm toluene (Tol). B) Phase plots obtained from ten consecutive APs indicated by the arrows in the bottom panels. C) Representative voltage traces in response to the injection of a current ramp (0–250 pA/75 ms) in control cells (ncells/animals = 15/8) and in neurons exposed to toluene (ncells/animals = 15/8).

dendritic branching and total neuronal size (Pascual et al., 2011). Although we did not conduct morphological studies, together, the decrease in the membrane time constant and the abovementioned evidence, suggest that under our experimental paradigm, the repeated exposure to 8000 ppm toluene caused a reduction of the total area of mPFC neurons. Nevertheless, this assumption requires experimental confirmation. On the other hand, elevated tissue levels of dopamine and serotonin have been found in the mPFC after repeated toluene exposure (Gerasimov et al., 2003; Koga et al., 2007; Rivera-García et al., 2015). Because different ascending neuromodulatory systems, including dopaminergic and serotonergic, exert control on the firing discharge of mPFC neurons via modulation of the IAHP, we cannot discard that the reduced IAHP may result from dysfunction of these modulatory systems. Indeed, dopamine via D1-like receptors, and serotonin, via the 5HT2A/2C receptors (Andrade, 2011; Zhang and Arsenault, 2005) suppress the afterhyperpolarization and increase the firing discharge of mPFC neurons (Chen et al., 2007; Thurley et al., 2008). Based on these data we could hypothesize that dysregulation of the dopaminergic or serotonergic innervation is involved in the increased excitability associated with the reduction in IHAP observed in our experiments. Finally, our data also show that repeated exposure to toluene alters the kynurenic-acid sensitive, spontaneous synaptic activity converging

observed in the 8000 ppm exposed-group. Our results also support the notion that toluene modulates membrane conductances active near the RMP; probably, outward potassium conductances. Importantly, the modulation of potassium currents by toluene has been previously reported in oocytes (Del Re et al., 2006). However, if repeated exposure to toluene modulates potassium currents in central neurons, is an issue that requires further investigation. On the other hand, the analysis of the I-V relationships showed that 8000 ppm toluene increases rectification in the negative portion of the plots. This voltage range corresponds to the membrane potential at which the potassium inward rectifiers are active in the rodent prefrontal cortex (Day, 2005), which suggests that toluene modulates potassium inward rectifiers. Notably, several potassium channels, including inward rectifiers of the prelimbic cortex, have been shown to be modulated by chronic exposure to alcohol, cocaine, and toluene (Marty and Spigelman, 2012; Tillar et al., 2002). As previously stated, the repeated exposure to toluene decreased the membrane time constant. Changes in this electrophysiological parameter have been successfully used as a tool to assess neuronal degeneration (Isokawa, 1997). In agreement with this, morphological studies performed in hippocampal and cortical neurons have provided evidence that animals chronically exposed to toluene exhibit a reduction in both 32

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Table 1 Action potential kinetic parameters. 1st AP

5th AP

10th AP

One-way ANOVA

75 ± 1.1 67.9 ± 1.7 t(28) = 1.8; P = 0.08

73.2 ± 1.4 65.5 ± 2.2+ t(28) = 2.1; P = 0.04

71.2 ± 1.6⁎ 61.6 ± 2.3⁎⁎,+ t(28) = 2.5; P = 0.02

F(2,42) = 4.1; P = 0.0243 F(2,42) = 8.3; P = 0.0018

Max Rise slope (normalized) Ctrl 100 ± 4.2 Tol 90.4 ± 5 t(28) = 1.4; P = 0.15

89.6 ± 4.4⁎⁎⁎ 77.9 ± 3.9⁎⁎ t(28) = 1.9; P = 0.06

84.3 ± 4.2⁎⁎⁎ 69.4 ± 4.1⁎⁎⁎,++ t(28) = 2.5; P = 0.0093

F(2,42) = 67.7; P < 0.0001 F(2,42) = 20.6; P < 0.0001

Decay slope (normalized) Ctrl 100 ± 4 Tol 81.9 ± 5.4+ t(28) = 2.7; P = 0.01

83.2 ± 5.4⁎⁎⁎ 69.1 ± 4.4+ t(28) = 2.2; P = 0.04

81.3 ± 5.4⁎⁎⁎ 60.7 ± 4.2⁎⁎⁎,++ t(28) = 3.01; P = 0.006

F(2,42) = 27.5; P < 0.0001 F(2,42) = 40.6; P < 0.0001

1.33 ± 0.06⁎ 1.29 ± 0.05⁎ t(28) = 0.4; P = 0.69

1.36 ± 0.06⁎⁎ 1.47 ± 0.05⁎⁎,+ t(28) = 2.3; P = 0.04

F(2,42) = 54.5; P < 0.0001 F(2,42) = 29.2; P < 0.0001

Amplitude (mV) Ctrl Tol

Half-width (ms) Ctrl Tol

1.1 ± 0.04 1.13 ± 0.04 t(28) = 0.6; P = 0.55

+

P < 0.05. P < 0.01; t-Student. ⁎ P < 0.05. ⁎⁎ P < 0.01. ⁎⁎⁎ P < 0.001; vs 1st AP, repeated measures ANOVA followed by Dunnett's test. ++

Concerning the latter issue, preliminary data from our laboratory indicate that the repeated exposure to toluene alters both short- and long-term forms of synaptic plasticity in the mPFC of adolescent rats (Torres, Cruz, and Galván; unpublished observations).

on prelimbic neurons. In addition to the action potential-evoked neurotransmitter release, the spontaneous vesicular release has been proposed as a mechanism that contributes to the maintenance of dendritic structures. This form of calcium-dependent (and –independent; see Glitsch, 2008) activity may carry synaptic information that influences the information processing of the CNS. Thus, the persistent synaptic bombardment observed in the prelimbic neurons repeatedly exposed to 8000 ppm toluene suggests morphological alterations in the neuronal connections, intracellular calcium dysregulation and altered synaptic transmission in the mPFC.

5. Conclusions In conclusion, we have demonstrated that repeated exposure to high toluene concentrations increases the intrinsic excitability of prelimbic neurons of the adolescent rat brain. Such alteration implies changes in Fig. 4. Repeated exposure to toluene reduces the slow component of the afterhyperpolarization. A) Superimposed voltage traces acquired at −50 mV. Middle and right panels summarize the changes in the afterhyperpolarization peak amplitude and area (measured under the dashed line of the voltage traces) for control and 8000 ppm toluene (Tol), ***P < 0.001; Student's t-test. B) Left panel: current traces (averaged from 5 consecutive sweeps) evoked by a 15-ms voltage command from −50 mV to 50 mV in control conditions and in the presence of the slow IAHP channel blocker UCL-2077 (10 μM) in neurons from animals exposed to air (left) or 8000 ppm toluene (right). C) Changes in peak slow IAHP amplitude and normalized total area for the three experimental conditions. Each bar represents the mean ± S.E.M. (control: ncells/animals = 20/10; **P < 0.01, toluene: ncells/animals = 20/10); ***P < 0.001, Tukey test.

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Fig. 5. Repeated exposure to 8000 ppm toluene alters the spontaneous synaptic activity of prefrontal cortex neurons. A) Representative traces from a continuous voltage clamp acquisition (10 s each trace) at −70 mV in the presence of bicuculline (10 μM) and kynurenic acid (2 mM) in control and toluene-exposed neurons. B) Cumulative probability (P) distribution of amplitude (left panel) and interevent intervals (frequency; right panel) of the spontaneous glutamatergic events of control (black; ncells/ animals = 12/6) and toluene-(red; ncells/animals = 12/ 6) exposed cells. For each cumulative graph, the inset box plots summarize the changes in the amplitude and sEPSC frequency ***P < 0.0001; Kolmogorov–Smirnov test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the synaptic strength and plasticity capabilities of the mPFC. These modifications may partially represent the cellular mechanisms responsible for the cognitive deficits related to chronic toluene consumption that have been reported in both clinical studies and multiple animal models. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ntt.2018.04.006.

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