ABSTRACT Recreational abuse of toluene-containing volatile inhalants by adolescents is a significant public health problem. The mechanisms underlying the abuse potential of such substances

remain unclear, but could involve increased activity in mesoaccumbal dopamine (DA) afferents innervating the nucleus accumbens (ACB). Here, using _in vitro_ electrophysiology, we show that

application of behaviorally relevant concentrations of toluene directly stimulates DA neurons in the ventral tegmental area (VTA), but not surrounding midbrain regions. Toluene stimulation

of VTA neurons persists when synaptic transmission is reduced. Moreover, unlike _non_-DA neurons, the magnitude of VTA DA neuron firing does not decline during longer exposures designed to

thereby enhancing DA release in the ACB. The finding that toluene stimulates mesoaccumbal neurotransmission by activating VTA DA neurons directly (independently of transynaptic inputs)

provide insights into the neural substrates that may contribute to the initiation and pathophysiology of toluene abuse. SIMILAR CONTENT BEING VIEWED BY OTHERS ETHANOL BLOCKS A NOVEL FORM OF

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Article Open access 21 November 2024 ACUTE AND PROTRACTED ABSTINENCE FROM METHAMPHETAMINE BIDIRECTIONALLY CHANGES INTRINSIC EXCITABILITY OF INDIRECT PATHWAY SPINY PROJECTION NEURONS IN THE

DORSOMEDIAL STRIATUM Article Open access 15 July 2022 INTRODUCTION Inhalant abuse represents a major health problem because of its association with future drug use (NSDUH, 2003) and severe

neurological/cognitive deficits (Filley, 2004). Many of these products (ie, spray paint, glue, etc.) including toluene and the abuse of pure toluene solvent, has been reported (Flanagan and

Ives, 1994). Although overall drug use by teenagers has declined since 1998, inhalant use has increased (NSDUH, 2003). Inhalant use begins in preadolescence and continues well into adulthood

(Rosenberg et al, 2002; NSDUH, 2003; Wu and Ringwalt, 2006; Cairney et al, 2005). Dopaminergic afferents arising from the ventral tegmental area (VTA) and projecting to the nucleus

accumbens (ACB) are crucial elements in the neural circuits that mediate arousal, motivation, and reinforcement (Wise, 2002). Increased dopamine (DA) neurotransmission within this

mesoaccumbal pathway mediates the reinforcing and psychomotor stimulant effects of drugs of abuse (Wise, 2002). In experimental animals, toluene inhalation produces reinforcing effects

(Bespalov et al, 2003; Blokhina et al, 2004; Funada et al, 2002; Weiss et al, 1979; Lee et al, 2006) and stimulates locomotor activity (Bowen, 2006; Riegel et al, 2003). Depletion of DA in

the ACB attenuates this latter effect (Riegel et al, 2003). Toluene also induces c-FOS activation in both the VTA and ACB (Lo and Chen, 2005), and increases the firing and bursting of VTA DA

neurons _in vivo_ (Riegel and French, 1999a). Although acute toluene exposure increases TTX-sensitive DA release in the dorsal striatum (Stengard et al, 1994), similar effects have not been

reported in the ACB (Kondo et al, 1995; Gerasimov et al, 2002b). Therefore, some investigators have concluded that a DA-independent (Spanagel and Weiss, 1999) or an, as yet, undetermined

(Hyman and Malenka, 2001) mechanism mediates the actions of toluene. Such conclusions, if incorrect, may confound future work seeking to identify the mechanisms underlying toluene abuse and

hamper the development of prevention strategies for toluene addiction (Beyer et al, 2001; Schiffer et al, 2006). DA neurons are spontaneously active (Grace and Bunney, 1983) and their

increased action potential firing stimulates DA release in the ACB (Wise, 2002). DA firing is influenced also by multiple mediators including various trans-synaptic inputs, local (GABAergic)

interneurons, and somatodendritic DA released from VTA DA neurons (Adell and Artigas, 2004; Marinelli et al, 2006; Beckstead et al, 2004). Therefore, toluene may influence one or more

neurotransmitter systems and the effects observed may vary depending upon the treatment regimen employed and the region or subregion examined. Indeed, the VTA is a heterogeneous structure

composed of DA and non-DA (ie, _γ_-GABA) neurons (Ikemoto and Wise, 2004; Van Bockstaele and Pickel, 1995). Furthermore, recent work has shown that infusion of ethanol and other drugs into

the posterior VTA elicits positive reinforcing effects, whereas anterior VTA infusion does not (Zangen et al, 2006; Rodd et al, 2004b; Ikemoto and Wise, 2004). Previous studies investigating

toluene did not determine where toluene acts to alter the activity of midbrain DA neurons, and if these changes are sufficient to increase DA release in the ACB (Riegel and French, 1999a).

The present studies determined whether _in vivo_ behaviorally relevant concentrations of toluene directly stimulate mesoaccumbal neurotransmission by a direct activation of VTA DA neurons.

_In vitro_ brain-slice experiments revealed that toluene displays anatomical specificity for VTA DA neurons that persists in treatments that reduce synaptic transmission and during longer,

escalating exposures designed to emulate ‘huffing’. Dual-probe microdialysis studies in awake animals revealed that perfusion of toluene into the VTA increases extracellular DA

concentrations in both the VTA and distal ACB. Parts of this data have been presented previously in preliminary form (Riegel and French, 2002). METHODS TOLUENE A stock solution of toluene in

polyoxyethylene-sorbitan mono-oleate (Tween-80, final concentration 0.02%) was prepared immediately before use with care taken to avoid any solvent loss. For electrophysiological

recordings, the stock solution was diluted in oxygenated aCSF (see below) and delivered (2 ml/min) to the recording chamber (35°C) by a gravity-feed (plasticizer-free) perfusion system.

Toluene was bath applied using two separate protocols. In _single applications_, each concentration of toluene was applied to the bath for 3 min to determine reversibility. Existing studies

indicate that inhalant abuse is characterized by a rapid intoxication (Meredith et al, 1989) and a related steep rise in brain toluene concentrations (Gerasimov et al, 2002a; Ameno et al,

1992; Kishi et al, 1988). Thus, as with other drugs (Balster and Schuster, 1973), the abuse liability of toluene may be affected by pharmacokinetic properties (Gerasimov et al, 2002a;

Lammers et al, 2005) and supplemental ‘huffs’ may be required to preserve the ‘high’ reported by users (NSDUH, 2003). Therefore, to better model these conditions, a _staircase paradigm_ was

employed. In this paradigm, escalating concentrations of toluene (seven concentrations at 3 min/concentration) were applied to the bath and the total duration of toluene exposure was

increased (21 min). Concentrations of toluene in perfusate samples taken from the recording chamber were determined by gas chromatograph (Riegel and French, 1999a) and were adjusted to match

_in vivo_ concentrations (4–79 μg/ml) observed in the blood of rodents (Riegel and French, 1999a) and humans exposed to toluene (Garriott et al, 1981). The drug reached the recording

chamber in ∼30 s, and the fluid in the chamber completely turned over in 3 min. For microdialysis, known aliquots of the toluene stock solution were diluted in aCSF and sealed immediately

just before reverse-dialysis. Data generated under conditions similar to those of the present study have shown that 10–30% of the drug concentration in the perfusion buffer reaches brain

tissue surrounding the dialysis probe (Yim and Gonzales, 2000; Gonzales et al, 1998). Thus, perfusate concentrations of toluene (1, 3, 10 mM) are expected to generate _in situ_

concentrations of 0.1, 0.3, and 1 mM, which are in accord with the _in vitro_ concentrations employed (23–822 μM) and previous _in vivo_ studies (for a discussion see Riegel et al, 2003).

_IN VITRO_ EXTRACELLULAR ELECTROPHYSIOLOGY SLICE PREPARATION Brain slices were prepared from male Sprague–Dawley rats (PD 19-40) according to protocols approved by the University of Arizona

IACUC (Wang and French, 1993b). Control experiments in brain slices prepared from older male Sprague–Dawley rats (PD 60) showed analogous (see below) toluene-induced changes in firing

(_n_=9, data not shown). Coronal slices (400 μm) containing the VTA were incubated in oxygenated aCSF composed of (in mM) 124 NaCl, 2.5 KCl, 1.25 KH2PO4, 2.4 MgSO4, 2.5 CaCl2, 25.7 NaHCO3,

and 10 D-glucose. Where indicated, aCSF was altered by lowering Ca2+ and increasing Mg2+ to either 0.5 mM Ca2+/10 mM Mg2+ (Media-1) or 0.25 mM Ca2+/7.25 mM Mg2+ (Media-2). Flow rates were

kept at 2 ml/min to insure adequate infusion of the altered media, consistent with previous published studies (Bondy and Harrington, 1979; Washio and Inouye, 1978; Alderdice and Volle, 1978;

Scholfied, 1981). Incubation with such media for 5 min or greater has been shown to block synaptic transmission (Bondy and Harrington, 1979; Washio and Inouye, 1978; Alderdice and Volle,

1978; Scholfied, 1981; Hutter and Kostial, 1954). Extracellular electrophysiological recordings were made with glass microelectrodes filled with a solution of 2% pontamine sky-blue in 0.5 M

sodium acetate (Yin and French, 2000). Action potential signals were amplified, and filtered (1–10 kHz) (Wang and French, 1993a; Marinelli et al, 2006). RECORDINGS Recording sites were found

with a dissection microscope and confirmed histologically _post hoc_ (see below: ‘Localization of recording sites and microdialysis probes’). All locations were anatomically differentiated

by reference to the rat atlas (Paxinos and Watson, 1988). VTA neurons were identified according to conventional pharmacological and electrophysiological criteria (Grace and Bunney, 1983;

Grace and Bunney, 1984; Johnson and North, 1992; Cameron et al, 1997; Marinelli et al, 2006). DA neurons possess: (1) bi- or tri-phasic action potentials with a somatodendritic notch, (2)

action potential lasting >2 ms, (3) ∼0.5 Hz firing rates, (4) inhibition to DA (50–100 μM), and (5) insensitivity to 5-HT (60 μM). Non-DA (type-II) neurons possess: (1) biphasic

positive–negative action potentials lacking an S-D notch, (2) action potential lasting <2 ms, (3) 1–2 Hz firing rates, and (4) insensitivity to inhibition by DA (50–100 μM) and 5-HT (60 

μM). _Non_-DA (type-III) neurons displayed: (1) positive–negative action potentials lacking a somatodendritic notch, (2) action potential lasting >2 ms, (3) ∼1.5–2 Hz firing rates, and

(4) inhibition to DA (50–100 μM) and 5-HT (60 μM). Consistent with previous electrophysiological studies, neurons in the interpeduncular nucleus (IPN) were classified by anatomical location

(Lena et al, 1993; Takagi, 1984) and DA neurons in the retrorubral field (RRF) were identified by electrophysiological/pharmacological criteria analogous to those used for VTA DA neurons

(Deutch et al, 1988). The identification of neurons in the rostral interstitial nucleus (RIN) was based upon _post hoc_ histological analysis (below). The _duration of inactivation_ was

defined as an episode of reduced action potential amplitude, below noise level, which sometimes occurred immediately after a period of pronounced neuronal excitation. Although the

reversibility of toluene's actions permitted multiple challenges, only one cell was recorded from each brain slice. _IN VIVO_ MICRODIALYSIS SURGERY Male Sprague–Dawley rats (PD 70-80)

were anesthetized with equithesin (1% pentobarbital, 2% magnesium sulfate, 4% chloral hydrate, 42% propyleneglycol, 11% ethanol, 3 ml/kg i.p.). Microdialysis guide cannulae (CMA/11, CMA

microdialysis, North Chelmsford, MA) were inserted stereotaxically (David Kopf instruments, Topanga, CA) and secured to the skull using stainless-steel screws and dental acrylic. Membrane

probe length (VTA: 1 mm; ACB: 2 mm) was adjusted for stereotaxic location (VTA: AP −5.6, _L_ −0.4, _V_ −7.8; ACB: AP +1.7, _L_ −1.4, _V_ −6.4), where AP, _L_, and, _V_ denote mm from bregma

(Paxinos and Watson, 1988). After surgery, the animals were housed individually and allowed to recover for 2–4 days before experiments were started. MICRODIALYSIS Microdialysis was performed

as described previously (Zapata and Shippenberg, 2005). The evening before the experiment, probes were inserted into the guide cannulae connected to a perfusion system consisting of 1 ml

gastight syringes mounted on microdialysis pumps (CMA/102) and fitted with FEP tubing (CMA microdialysis) connecting the probes through dual-channel, quartz-lined, low-resistance swivels.

Probes were perfused overnight (0.3 μl/min) and 1 h before experiments (1 μl/min) with aCSF (in mM: 145 NaCl, 2.8 KCl, 1.2 CaCl2, 1.2 MgCl2, 0.25 ascorbic acid, 5.4 D-glucose, pH 6.5–7.0

adjusted with NaOH). During experiments, the VTA superfusate alternated between aCSF, vehicle (Tween-80, final concentration in aCSF 0.02%), or toluene (1, 3, and 10 mM; 1 h/concentration).

Dialysis fractions were collected every 15 min, frozen (−80°C), and analyzed for DA content using HPLC-electrochemical detection within 48 h (Zapata and Shippenberg, 2005). Dialysate DA

concentrations were quantified using an external standard curve. The detection limit of DA in dialysate was 0.3 nM. LOCALIZATION OF RECORDING SITES AND MICRODIALYSIS PROBES The anterior

(−5.2 to −5.6 mm posterior to bregma) and posterior (−5.6 to −6.04 mm posterior to bregma) divisions of the VTA were defined as in other studies (Rodd et al, 2005; Zangen et al, 2006;

Ikemoto and Wise, 2004; Carlezon et al, 2000; Carlezon and Nestler, 2002; Rodd-Henricks et al, 2000). For electrophysiology, recording sites were confirmed by iontophoresis of pontamine

sky-blue dye at experiment completion (Wang and French, 1993a). Then, as described elsewhere (Johnson et al, 1996), brain slices were resectioned (5–10 μm), stained with cresyl violet, and

examined by light microscopy. Microdialysis probe placement was confirmed after brains were removed, frozen, and sectioned on a cryostat (30 μm) as previously described (Zapata and

Shippenberg, 2005). Only when probe location was confirmed in both the VTA and ACB (Paxinos and Watson, 1988), was data from that animal included for further analysis. DATA ANALYSIS For

electrophysiology, only one neuron per slice was tested. Changes in neuron firing rates are expressed as a change from baseline (Hz). Significant differences between treatments were

determined by a Student's paired _t_-test. All depicted action potential waveforms are averaged (⩾4 spikes/trace). For microdialysis, basal DA levels in dialysate were determined in

each animal and represent the average of four samples collected before vehicle perfusion and toluene exposure. For each concentration of toluene, four consecutive 15 min dialysate samples

were obtained. The averaged values at each toluene concentration were compared to the effect of vehicle. Statistical significance was determined using a repeated measure one-way ANOVA and

the Newman–Keuls test for _post hoc_ comparisons. DRUGS AND REAGENTS Toluene (analytical grade), benzene (reagent grade), and carbon disulfide (reagent grade) were purchased from Fischer

Scientific (Tustin, CA). DA and serotonin were purchased from Sigma Chemicals (St Louis, MO) and baclofen from RBI (Natick, MA). RESULTS _IN VITRO_ EXTRACELLULAR ELECTROPHYSIOLOGY Initial

experiments using single applications determined whether toluene affects the excitability of VTA DA neurons in an isolated brain slice preparation. Short 3 min applications of toluene

stimulated VTA DA neurons. In all cases, the increase in firing was concentration-dependent and firing rates returned to baseline upon washout (Figure 1a) (maximum 3.4±0.3 Hz above baseline;

_n_=17; _p_<0.05, data not shown). The maximum increase in firing was greater in the anterior VTA (Figure 1d: 4.6±0.4 Hz above baseline; _n_=7), than the posterior VTA (Figure 1d:

2.8±0.3 Hz above baseline; _n_=10; _p_<0.01). These _in situ_ results resembled previous _in vivo_ studies, with respect to both the basal properties of DA neurons (Table 1) and the

reversible, toluene-induced increase in firing (Riegel and French, 1999a). In non-DA neurons, toluene stimulated the firing of type-II (Figure 1b; _n_=5) and type-III cells (_n_=7) (data not

shown, but a sample type-III cell is shown in Figure 3b). At both anterior and posterior VTA locations, toluene induced similar increases in non-DA neuron firing. So, the data were grouped

(Figure 1d: maximum 3.21±0.63 Hz above baseline; _n_=12). The stimulation of non-DA cells _in vitro_ differs from the inhibition of firing previously observed _in vivo_ (Riegel and French,

1999b), and may reflect the loss of monosynaptic glutamate inputs to neurons in a slice preparation (Tzschentke, 2001). To determine whether toluene increased VTA firing by altering

neurotransmitter release onto DA cells, we tested a low-Ca2+, high-Mg2+ aCSF media, previously shown to inhibit synaptic transmission (Brodie et al, 1990; Katz, 1970; Katz and Miledi, 1970).

The stimulatory properties of toluene (370 μM) were still apparent under these conditions (Figure 1c: 370 μM, single applications). As basal firing rates can vary under low Ca2+ conditions,

initial results with toluene in media-1 (0.5 mM Ca2+/10 mM Mg2+; Figure 1d: 1.73±0.86 Hz above baseline; _n_=5) were confirmed with experiments in media-2 (0.25 mM Ca2+/7.25 mM Mg2+; Figure

1d: 2.32±1.01 Hz above baseline; _n_=5). The magnitude of the toluene-induced increase in firing did not differ between media (Figure 1d; _p_>0.05, ANOVA), suggesting that toluene

directly activates VTA neurons. To determine if the stimulatory properties of toluene were unique to the VTA, three adjacent midbrain structures were tested using single applications. In the

IPN, the basal firing rate was 11.0±2.70 Hz (Table 1). Toluene inhibited the firing of neurons in the IPN (Figure 2a). The response was robust and concentration-dependent (Figure 2d:

maximum, −10.2±0.71 Hz below baseline; _n_=7). However, transient (<30 s) and concentration-independent increases were also noted in some neurons in the IPN (Figure 2a: <4 Hz, _n_=2).

In contrast, toluene did not significantly alter neuronal activity in the RRF (Figure 2b) or RIN (Figure 2c) (Figure 2e; maximum, 0.43±0.39 Hz above baseline, _n_=12; _p_>0.05). Basal

firing characteristics of neurons in the IPN and RRF are summarized in Table 1, and resemble those reported previously (Takagi, 1984; Deutch et al, 1988). The characteristics of neurons in

the RIN are shown (Table 1), although characterization of this region is lacking. Thus, these results demonstrate that behaviorally relevant concentrations of toluene (single applications)

selectively stimulate VTA neurons and that the increase in firing is similar in both DA and non-DA neurons. Behavioral responses to solvents are determined in part by the pattern of exposure

(Lammers et al, 2005). Therefore, prolonged _in vitro_ exposures were investigated. Using a staircase paradigm, we observed that cumulative applications of toluene stimulated VTA DA neurons

(Figure 3a; 22–823 μM) and non-DA neurons (Figure 3b; 22–633 μM). The increases in firing were concentration-dependent and reversed upon washout (Figure 3a and b). At DA neurons, the

efficacy of toluene did not change as a function of exposure paradigm. Thus, maximal activity under staircase conditions (Figure 3c: 4.07±0.44 Hz above baseline at 822 μM toluene, first

application, _n_=21) resembled firing under conditions using single applications (Figure 1d: 3.41±0.37 Hz above baseline at 740 μM toluene, _n_=17). In contrast, in non-DA neurons under

staircase conditions, the maximal efficacy of toluene declined ∼40% (Figure 3c: 2.19±0.32 Hz above baseline; at 633 μM, first application, _n_=9) relative to single applications (Figure 1d:

3.21±0.63 above baseline at 740 μM, _n_=12). The diminished efficacy is unlikely to be owing to tissue accumulation of toluene, because at comparable concentrations, the magnitude of the

increase in DA cell firing was similar during both short (single applications: 3 min) and long (staircase: 21 min) exposures. Furthermore, the attenuation of firing was observed only in

non-DA neurons during cumulative staircase conditions. More prolonged staircase exposure to toluene (>21 min) inactivated VTA DA (Figure 3a) and non-DA (Figure 3b) neurons and briefly

limited further firing. As noted in previous studies (Riegel and French, 1999a), this stimulation-induced inactivation was not owing to acute, irreversible cell death, because in all

instances, firing recovered to baseline when the perfusion media were replaced with toluene-free aCSF. With first applications, toluene inactivated non-DA neurons 2–3 times longer than DA

neurons (Figure 3a and b). To investigate if the toluene-induced inactivation of non-DA neurons was additive, the staircase paradigm was reapplied after firing resumed. The efficacy and

duration of inactivation during the two repetitive episodes were noted for comparison. In DA neurons (Figure 3a), maximal firing (Figure 3c; episode-1: 4.00±0.42 Hz above baseline, _n_=17;

episode-2: 4.09±0.49 Hz above baseline, _n_=9) and periods of inactivity (Figure 3d: episode-1: 2.50±1.11 min, _n_=17; episode-2: 2.82±0.96 min, _n_=9) remained the same. In non-DA neurons

(Figure 3b), however, maximal efficacy declined (Figure 3c episode-1: 2.2±0.2 Hz above baseline, _n_=9; episode-2: 1.3±0.3 Hz above baseline, _n_=3) and the duration of inactivation

increased (Figure 3d; episode-1: 11.3±1.6 min, _n_=9, _p_<0.01; episode-2: 28.2±2.3 min, _n_=3; _p_<0.001). Any assessment of toluene during a third episode was not possible, because

of the limitation of recording from a single neuron for longer than 2.5 h. Higher concentrations of toluene were not investigated because of the plateauing of neuronal stimulation. The

decline in efficacy and increase in the period of inactivation was only observed in non-DA neurons. Toluene (<1 mM) stimulated VTA DA neuron firing repeatedly without a significant

decline in efficacy. _IN VIVO_ MICRODIALYSIS Given these findings, we determined whether toluene can stimulate VTA DA neurons directly _in vivo_, and if so, whether this effect is sufficient

to increase mesoaccumbal DA release. Dual-probe microdialysis was used to measure concentrations of somatodendritic DA release in the VTA, an indirect marker of VTA DA neuron activity, and

DA released from DA terminals in the ACB. DA RELEASE IN THE VTA Toluene was infused directly into the VTA by reverse dialysis at concentrations of 1, 3, or 10 mM (see Methods). In the VTA,

infusion of vehicle resulted in a non significant shift (<15%) in basal dialysate DA concentrations (Figure 4a: 0.67±0.09 nM; _n_=9). In contrast, infusion of toluene increased

concentrations of somatodendritic VTA-DA (Figure 4a and b: F3,24=13.21, _p_<0.0001). A significant increase was observed in response to 3 and 10 mM toluene (Figure 4b; _p_<0.001 _vs_

vehicle). Significant increases in VTA DA were observed when toluene was infused into anterior or posterior VTA regions (Figure 4b; antVTA: F3,12=5.376, _p_=0.014; post VTA: F3,9=8.561,

_p_=0.0055 ANOVA). The anatomical location of all dialysis probes is shown in Figure 5. DA RELEASE IN THE ACB The basal concentration of ACB DA in dialysate was 1.82±0.43 nM. DA

concentrations in the ACB did not change following intra-VTA vehicle perfusion. However, reverse-dialysis of toluene into the VTA significantly increased DA levels in the ACB (Figure 4c and

d; _n_=11, F3,43=3.561, _p_=0.026, ANOVA). All toluene-induced increases in DA reversed within 60 min. The ACB-DA increases were significant, when toluene was infused into the posterior VTA

(Figure 4d; F3,18=4.772, _p_=0.013, ANOVA), but not the anterior VTA (Figure 4c and d; F3,9=1.613, _p_=0.250, ANOVA). This topographical selectivity (posterior VTA _vs_ anterior VTA)

parallels that previously observed in behavioral studies in which microinjection techniques were used to assess the effects of other drugs of abuse including both alcohol, and cannabinoids

(Zangen et al, 2006; Zangen et al, 2002; Ikemoto and Wise, 2004; Rodd-Henricks et al, 2000, 2002; Rodd et al, 2005). To confirm functional connectivity between the posterior VTA and ACB

probe placements, baclofen was infused into the VTA and DA levels after toluene washout and ACB DA levels were determined (Westerink et al, 1996). Baclofen produced a robust decline in ACB

DA concentrations (35±8% of vehicle; _n_=11; data not shown). Perfusion of toluene into regions anterior (_n_=5), dorsal (_n_=7), lateral (_n_=2), or ventral (_n_=2) to the VTA failed to

alter ACB-DA (Figure 4e and f) demonstrating the regional selectivity of this effect. Collectively, these data show that the effects of toluene on ACB-DA levels are specific to the posterior

VTA. DISCUSSION The present results indicate that toluene directly stimulates DA cell firing, thereby facilitating DA release in the ACB. Dopaminergic innervation of the ACB is a key

substrate for the reinforcing effects of other drugs of abuse (Wise, 2002). Nevertheless, despite intensive study, the neural substrates mediating the CNS actions of toluene have remained

elusive. Previous behavioral studies indicated that the locomotor activating effects of toluene require intact mesoaccumbal DA transmission (Riegel et al, 2003). Moreover, toluene increases

the activity of VTA DA neurons _in vivo_ (Riegel and French, 1999a). To date, however, it remained unclear whether toluene stimulated these neurons by direct actions on neurons _within the

VTA_ and if such activation was sufficient to increase DA release from distal DA terminals in the ACB. TOLUENE STIMULATES VTA NEURONS, LEADING TO DA RELEASE IN THE ACB Our data support the

premise that toluene-containing inhalants activate mesoaccumbal neurotransmission via direct actions on VTA DA neurons. First, toluene stimulated DA neurons in the VTA slice, a preparation

in which transynaptic inputs to DA cells are severed (Tzschentke, 2001). Similar effects were observed in slices incubated in aCSF containing low Ca2+/high Mg2+, conditions in which synaptic

transmission is inhibited (Katz, 1970; Katz and Miledi, 1970). Identical media were used previously to study the site of action for other drugs of abuse (Brodie et al, 1990; Sanghera et al,

1984; Trulson et al, 1987) including toluene (in other regions) (Magnusson et al, 1998). These findings suggest that toluene increases DA neuronal activity post-synaptically and

independently of afferent input. Second, _in vivo_ microdialysis showed that direct intra-VTA perfusion of toluene enhanced DA concentrations in the VTA and ACB. As VTA DA neuron cell bodies

are the only known source of DA in this region (Adell and Artigas, 2004), the former effect most likely reflects the well-described increase in somatodendritic DA release that occurs as a

consequence of increased firing of DA neurons (Adell and Artigas, 2004). Other drugs of abuse also increase somatodendritic DA release (Adell and Artigas, 2004). Work, however, from several

laboratories has shown that the VTA is functionally heterogeneous (Ikemoto and Wise, 2004; Ford et al, 2006). Experimental animals will work to obtain infusions of ethanol, opiates,

cannabinoids, or psychostimulants into the posterior VTA, whereas infusions into the anterior VTA are without effect (Rodd-Henricks et al, 2000, 2002; Rodd et al, 2005; Rodd et al, 2004b;

Zangen et al, 2006). Interestingly and consistent with this heterogeneity, microdialysis revealed that toluene was more effective in increasing ACB-DA concentrations when infused directly

into the posterior than in the anterior VTA. Third, the effects of toluene were anatomically specific. Unlike VTA DA cells, bath application of toluene did not alter DA neuronal firing in

the RRF or RIN. Furthermore, firing of GABAergic neurons in the IPN was inhibited. Similarly, reverse-dialysis of toluene into regions adjacent to the VTA did not alter ACB-DA levels.

Anterior VTA perfusion of toluene failed to change ACB-DA, a finding consistent with anatomical studies showing that most DA neurons in this region do not innervate the ACB (Westerink et al,

1998; Tzschentke, 2001; Brog et al, 1993). However, there is an apparent trend to a delayed response, the magnitude of which may have been masked by a downward baseline drift in this

experimental group. This delayed response may reflect diffusion of toluene from the probe to more posterior VTA regions. The toluene-induced stimulation of anterior VTA DA neurons may be

related to the reported toluene-induced increase in DA release in the prefrontal cortex (Gerasimov et al, 2002b). Taken together, these findings indicate that by stimulating posterior VTA DA

cells, toluene increases extracellular DA concentrations in the distal ACB. CONCENTRATION DEPENDENCE AND NEUROBEHAVIORAL IMPLICATIONS Toluene is rapidly absorbed, distributed, and

eliminated from brain tissue (Gerasimov et al, 2002a; Kishi et al, 1988). Behavioral effects of toluene depend on both the brain concentrations and the pattern of exposure (Kishi et al,

1993; Lammers et al, 2005). Although inhalation exposure is relevant to inhalant abuse, the site (or sites) of action can be difficult to interpret. In both of our experimental approaches,

toluene was delivered directly to the VTA. The concentrations of toluene employed were those previously shown to be achieved in humans (Garriott et al, 1981) and experimental animals (see

discussion in Riegel et al, 2003). To simulate this exposure pattern _in vitro_, VTA slices were perfused with repeated, rapidly rising, escalating concentrations of toluene (staircase

paradigm). Extracellular recordings preserve action potential firing for long periods of time and are correlated with DA release in the ACB (Marinelli et al, 2006; Westerink et al, 1996). We

observed that VTA firing increased only over a narrow concentration range. At lower concentrations (<100 μM), toluene increased DA cell firing rates, but ACB DA concentrations were

unaltered. As noted elsewhere (Wise, 2002; Yeomans, 1989), the stimulation of unmyelinated fibers may have been insufficient to detect increased synaptic overflow of DA in distal terminals.

At intermediate concentrations, toluene increased both DA neuron firing and DA concentrations in the ACB. As discussed above (see Methods), retro-dialysis results in an estimated 10-fold

reduction in drug tissue concentration. Therefore, there is good concordance between the current results and previous _in vivo_ experiments, where blood toluene concentrations of 300 μM

stimulated VTA DA neurons (Riegel and French, 1999a). The firing of VTA DA neurons inactivated temporarily when toluene concentrations reached ∼1 mM. This inactivation persisted until

concentrations decreased, and is consistent with previous _in vivo_ inhalation studies showing inactivation of DA neurons when blood toluene concentrations exceeded ∼900 μM (Riegel and

French, 1999a). Thus, it is plausible that the lack of toluene-induced DA release in previous _in vivo_ studies results from an inactivation of VTA DA neurons induced by higher toluene

concentrations (Kondo et al, 1995; Gerasimov et al, 2002b). The lack of an accumbal DA response to 10 mM toluene may seem surprising given that this concentration increased extracellular DA

concentrations in the VTA (ie, somatodendritic release). The mechanisms mediating the differential effects of the higher toluene concentration on VTA and ACB DA levels are unclear.

Importantly, however, DA clearance in the VTA is less efficient than in terminal areas (Cragg and Greenfield, 1997). Thus, DA tissue concentrations may remain elevated even after suppression

of cell firing in the VTA. It is known that depolarization inhibits DA transporter function (Sonders et al, 1997). Sustained depolarization by toluene may further inhibit VTA DA uptake

resulting in impaired DA clearance in this region and an elevation of VTA DA levels. Interestingly, other organic solvents (eg, ethanol) inhibit DA uptake (Robinson et al, 2005). Therefore,

direct effects of toluene on transporter activity cannot be ruled out. In the staircase paradigm, VTA non-DA neurons were also stimulated. However, unlike DA cells, these neurons displayed

an inactivation threshold that became lower with repeated stimulation. Some VTA non-DA cells are GABA projection neurons (Cameron et al, 1997; Van Bockstaele and Pickel, 1995), which

innervate striatal cholinergic interneurons (Pickel and Chan, 1990) and cerebellar nuclei (Ikai et al, 1992). Although the functional role of these neurons is not completely understood (Van

Bockstaele and Pickel, 1995), their activation by low concentrations of toluene may contribute to TTX-sensitive increases in extracellular levels of GABA in these regions (Stengard and

O’Connor, 1994; Stengard et al, 1993) and to decreases in striatal ACh release (Stengard, 1994) during toluene inhalation. Other VTA non-DA cells are GABAergic interneurons (type-II) that

tonically inhibit VTA DA neurons _in vivo_ (Johnson and North, 1992). Although the mechanism underlying the shift in the inactivation point remains unclear, increases in the firing activity

of DA over GABA neurons would be expected to facilitate mesoaccumbal output. Finally, regarding the cellular targets, studies in cell expression systems indicate that toluene disrupts the

activity of numerous ion channels (Cruz et al, 1998; Bale et al, 2005; Beckstead et al, 2000), Ca2+ signaling (Westerink and Vijverberg, 2002; Westerink et al, 2002; Meulenberg and

Vijverberg, 2003), ATPases (Calderon-Guzman et al, 2005), and G-proteins (Tsuga et al, 1999; Tsuga and Honma, 2000; Tsuga et al, 2002). Additional studies will be needed to determine if one

or more of these mechanisms may be contributing to the toluene-induced excitation of VTA DA neurons. In conclusion, our results indicate that toluene selectively activates VTA DA neurons

projecting to the ACB. They identify a narrow concentration range within which toluene directly stimulates mesoaccumbal neurotransmission. Such findings suggest that previous conclusions,

that DA-independent mechanisms underlie the rewarding properties of inhalants, may be premature. Furthermore, given the alarming increase in inhalant abuse among youth (NSDUH, 2003),

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Google Scholar  Download references ACKNOWLEDGEMENTS We thank Drs Karten, Krause, Beckstead, Ford, and Williams for critically reading the manuscript. Support for A Riegel was provided by

NIDA T-334180. A Zapata and T Shippenberg were supported by the National Institute on Drug Abuse, Intramural Research Program. AUTHOR INFORMATION Author notes * Arthur C Riegel and Agustin

Zapata: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, USA Arthur C Riegel 

& Edward D French * Vollum Institute, Oregon Health & Science University, Portland, OR, USA Arthur C Riegel * US Department of Health and Human Services, Intramural Research Program,

Integrative Neuroscience Section, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA Agustin Zapata & Toni S Shippenberg Authors * Arthur C Riegel View

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Shippenberg, T. _et al._ The Abused Inhalant Toluene Increases Dopamine Release in the Nucleus Accumbens by Directly Stimulating Ventral Tegmental Area Neurons. _Neuropsychopharmacol_ 32,

1558–1569 (2007). https://doi.org/10.1038/sj.npp.1301273 Download citation * Received: 24 May 2006 * Accepted: 06 October 2006 * Published: 10 January 2007 * Issue Date: 01 July 2007 * DOI:

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currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * inhalant abuse * ventral tegmental area (VTA) * nucleus

accumbens (ACB) * electrophysiology * microdialysis * dopamine (DA)