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Addiction Biology
ORIGINAL ARTICLE doi:10.1111/adb.12097

Key role of salsolinol in ethanol actions on

dopamine neuronal activity of the posterior
ventral tegmental area

Miriam Melis1, Ezio Carboni1,3,4, Pierluigi Caboni2 & Elio Acquas2,3,4

Departments of Biomedical Sciences1and Life and Environmental Sciences2, Centre of Excellence on Neurobiology of Addiction3 and INN—National Institute of
Neuroscience4, University of Cagliari, Cagliari, Italy

ABSTRACT

Ethanol excites dopamine (DA) neurons in the posterior ventral tegmental area (pVTA). This effect is responsible for
ethanol’s motivational properties and may contribute to alcoholism. Evidence indicates that catalase-mediated con-
version of ethanol into acetaldehyde in pVTA plays a critical role in this effect. Acetaldehyde, in the presence of DA,
condensates with it to generate salsolinol. Salsolinol, when administered in pVTA, excites pVTA DA cells, elicits DA
transmission in nucleus accumbens and sustains its self-administration in pVTA. Here we show, by using ex vivo
electrophysiology, that ethanol and acetaldehyde, but not salsolinol, failed to stimulate pVTA DA cell activity in mice
administered α-methyl-p-tyrosine, a DA biosynthesis inhibitor that reduces somatodendritic DA release. This effect was
specific for ethanol and acetaldehyde since morphine, similarly to salsolinol, was able to excite pVTA DA cells in
α-methyl-p-tyrosine-treated mice. However, when DA was bath applied in slices from α-methyl-p-tyrosine-treated mice,
ethanol-induced excitation of pVTA DA neurons was restored. This effect requires ethanol oxidation into acetaldehyde
given that, when H2O2-catalase system was impaired by either 3-amino-1,2,4-triazole or in vivo administration of
α-lipoic acid, ethanol did not enhance DA cell activity. Finally, high performance liquid chromatography-tandem mass
spectrometry analysis of bath medium detected salsolinol only after co-application of ethanol and DA in α-methyl-p-
tyrosine-treated mice. These results demonstrate the relationship between ethanol and salsolinol effects on pVTA DA
neurons, help to untangle the mechanism(s) of action of ethanol in this area and contribute to an exciting research
avenue prosperous of theoretical and practical consequences.

Keywords Acetaldehyde, catalase, dopamine, ethanol, salsolinol, pVTA.

Correspondence to: Elio Acquas, Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale, 72—I-09124 Cagliari, Italy.
E-mail: acquas@unica.it

INTRODUCTION transmission in the nucleus accumbens shell (Howard

Ethanol is the main pharmacological ingredient of alco-
holic drinks and one of the most known psychoactive
chemicals worldwide. Ethanol is also renowned for its
liability to cause alcoholism, a condition that still plagues
human civilization carrying high individual, family and
societal costs, concerning and pertaining to a large scien-
tific community from bench- to bed-side.
The mesolimbic dopamine (DA) system is involved in
psychopharmacological effects of ethanol as a conse-
quence of ethanol’s ability to excite DA neurons in poste-
rior ventral tegmental area (pVTA) (Gessa et al. 1985;
Brodie, Shefner & Dunwiddie 1990; Foddai et al. 2004;
Okamoto, Harnett & Morikawa 2006), to stimulate DA
et al. 2008) and to affect motivation (Di Chiara 1997;
Koob et al. 1998). These properties, in agreement with
clinical evidence that ethanol promotes DA release in
the ventral striatum and exerts psychostimulant effects
(Boileau et al. 2003), represent, in turn, potential
grounds for development of alcoholism (Söderpalm &
Ericson 2013).
Peripheral ethanol metabolism mainly occurs by
alcohol dehydrogenase activity. Aversive peripheral
effects (Escrig et al. 2012) and somatic symptoms of
flushing reaction, but also centrally mediated effects
(Correa et al. 2012) are ascribed to acetaldehyde result-
ing from ethanol oxidation. In the brain, ethanol is
converted into acetaldehyde by catalase (Cohen, Sinet &

© 2013 Society for the Study of Addiction Addiction Biology

2 Miriam Melis et al.
Heikkila 1980; Aragon, Spivak & Amit 1991; Aragon,
Rogan & Amit 1992; Gill et al. 1992; Zimatkin et al.
2006), whose inhibition prevents a number of ethanol-
mediated behavioral responses ranging from voluntary
consumption (Aragon & Amit 1992), to locomotion
(Sanchis-Segura et al. 1999, 2005) and acquisition of
conditioned place preference (Font, Miquel & Aragon
2008). Indeed, acetaldehyde has long been suggested to
account for ethanol’s psychopharmacological actions
(Davis & Walsh 1970; Davis, Walsh & Yamanaka 1970;
Brown, Amit & Rockman 1979). However, this issue
remained controversial (Deitrich 2004) mainly because
peripherally produced acetaldehyde displays high
reactivity (Davis & Walsh 1970; Myers, Ng & Singer
1982) and conditional ability to cross blood brain barrier
(Eriksson & Sippel 1977; Tabakoff, Anderson & Ritzman
1976; but see also Correa et al. 2012 for a throughout
discussion on this issue). Nevertheless, since brain
catalase-mediated ethanol metabolism was proven
(Aragon et al. 1991, 1992), pre-clinical studies have
shown that acetaldehyde is a neuroactive molecule with
its own psychopharmacological properties (Correa et al.
2012). In particular, in vivo (Foddai et al. 2004) and in
vitro (Melis et al. 2007) evidence demonstrated that acet-
aldehyde stimulates the firing of pVTA DA neurons,
sustains its self-administration (Myers et al. 1982;
Rodd-Henricks et al. 2002; Rodd et al. 2005; Peana et al.
2011), elicits conditioned place preference (Melis et al.
2007) and promotes in nucleus accumbens shell DA
release (Melis et al. 2007) and extracellular signal regu-
lated kinase phosphorylation (Vinci et al. 2010). Remark-
ably, acetaldehyde has a very short half-life (Myers et al.
1982; Correa et al. 2012), and is a highly reactive
molecule, which may condensate, either spontaneously
or enzymatically (Chen et al. 2011), with nucleo-
philic compounds, such as monoamines, to produce
tetrahydroisoquinolines. When condensates with DA,
acetaldehyde generates 1-methyl-6,7-dihydroxy-1,2,3,4-
tetrahydroisoquinoline (salsolinol) (Yamanaka, Walsh &
Davis 1970; Jamal et al. 2003).
Early studies suggested that salsolinol might have a
role in psychopharmacological effects of ethanol (Davis &
Walsh 1970; Davis et al. 1970) and the relationship
between these two compounds has long been debated
(Lee et al. 2010; Correa et al. 2012; Hipólito et al. 2012;
Deehan, Brodie & Rodd 2013). Indeed, brain salsolinol
quantification following in vivo ethanol administration is
still an unresolved issue (Lee et al. 2010). Accordingly,
salsolinol also found in chocolate (Melzig et al. 2000),
bananas (Riggin, McCarthy & Kissinger 1976) and alco-
holic beverages (Duncan & Smythe 1982), shows poor
ability to cross blood brain barrier (Eriksson & Sippel
1977; Origitano, Hannigan & Collins 1981; Correa et al.
2012). Conversely, salsolinol is self-administered in pVTA
© 2013 Society for the Study of Addiction
by rats (Rodd et al. 2008) and, when microinjected into
this brain area, increases locomotor activity (Hipólito
et al. 2010) and elicits DA release in nucleus accumbens
shell (Hipólito et al. 2011; Deehan et al. 2013). Further-
more, salsolinol stimulates firing rate of DA neurons ex
vivo (Xie & Ye 2012; Xie et al. 2012).
Hence, provided that ethanol has to be converted into
acetaldehyde in order to excite DA neurons in pVTA
(Melis et al. 2007), and that acetaldehyde in presence of
DA may condensate with it to produce salsolinol
(Yamanaka et al. 1970; Chen et al. 2011), we hypoth-
esized that salsolinol might be involved in ethanol
stimulatory action on pVTA DA cells. The present study
was designed to challenge this hypothesis by recording
pVTA DA cell firing in acute brain slices ex vivo in which
somatodendritic DA release was reduced (Cheramy, Leviel
& Glowinski 1981) by in vivo administration of α-methyl-
p-tyrosine (αMpT) or reserpine.
MATERIALS AND METHODS
In vivo drug administration
This study was carried out in accordance with the Italian
law D.L. 116/1992 which allows experiments on labora-
tory animals only after submission and approval of a
research project to the competent authorities, and with
the guidelines approved by the European Commission
(n°2007/526/CE). Any possible effort was made to mini-
mize animal pain and discomfort and to reduce the
number of experimental subjects. Male CD-1 mice
(Harlan Italy SpA, Udine, Italy) used in this study were
administered either αMpT [300 mg/kg, intraperitoneal
(i.p.)] (Sigma-Aldrich, Milan, Italy) or vehicle, at postna-
tal day 21, 2 hours before sacrifice or reserpine (5 mg/kg,
i.p.) (Sigma-Aldrich) or vehicle, at postnatal day 20, 24
hours before sacrifice. α-Lipoic acid (αLA) (100 mg/kg,
i.p., dissolved in 0.3 M Tris, pH 7.4) (Sigma-Aldrich) was
administered 30 minutes before sacrifice (Ledesma &
Aragon 2012).
Assessment of DA in striatal tissue
The striatum from both brain sides was dissected on a
saline-iced block by using a Zeiss 2000 stereo microscope
(Carl Zeiss Instruments, Milan, Italy). Tissue was imme-
diately weighted, suspended in 250 μl of ice-cold
perchloric acid (200 mM) and sonicated with eight
5-second pulses followed by 3-second pauses (Sonics
Vibra Cell, Newtown, CT, USA). The suspension was then
centrifuged at 9391 × g for 10 minutes at 4°C. The super-
natant was filtered through Spin-X 2 ml vials (Nylon
0.22 μm filter) (Corning-Costar, Sigma-Aldrich) and the
filtrate was diluted 1:25 with distilled water. Samples
of 20 μl were injected into a high performance liquid
Addiction Biology

chromatography (HPLC) apparatus, equipped with a
15 cm LC—18 DB reverse-phase column with 5 μm par-
ticle size (Supelco, Milan, Italy) and coupled with a
coulometric detector (ESA Coulochem II, Sunnyvale, CA,
USA) to quantitate DA. Electrodes were set at +150 mV
(oxidation) and −200 mV (reduction). The mobile phase
(CH3COONa: 120 mM; Citric acid: 20 mM; Na2EDTA:
100 mg/ml; pH: 4.93; MeOH: 5 % v/v) was pumped at
the flow rate of 1 ml/minute by a HPLC pump [Jasco
Europe, Cremella (LC), Italy]. The assay sensitivity for DA
was 5 fmol/injection. Data, given as mean ± standard
error (SEM), were compared and analyzed by utilizing
one-way analysis of variance (ANOVA) and, whereby
allowed by significant main effects, followed by Tukey’s
post hoc comparisons.
Ex vivo electrophysiology
Whole-cell patch clamp recordings from CD-1 mouse
pVTA DA cells were similar to those previously described
(Melis et al. 2010). Briefly, pups were deeply anesthetized
with halothane and sacrificed. A block of tissue contain-
ing the midbrain was rapidly dissected and sliced in the
horizontal plane (230 μm) with a vibratome (VT1000S,
Leica, Wetzlar, Germany) in ice-cold low-Ca2+ solution
containing (in mM): 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2
MgCl2, 0.625 CaCl2, 18 NaHCO3 and 11 glucose. Slices
were transferred to a holding chamber with artificial
cerebro-spinal fluid (aCSF; 37°C) saturated with 95% O2
and 5% CO2 containing (in mM): 126 NaCl, 1.6 KCl, 1.2
NaH2PO4, 1.2 MgCl2, 2.4 CaCl2, 18 NaHCO3 and 11
glucose. Slices (two per animal) were allowed to recover
for at least 1 hour before being placed (as hemislices) in
the recording chamber and superfused with the aCSF
saturated with 95% O2 and 5% CO2. Cells were visualized
with an upright microscope with infrared illumination
(Axioskop FS 2 plus, Zeiss, Jena, Germany), and whole-cell
current-clamp recordings (one per hemislice) were
made by using an Axopatch 200B amplifier (Molecular
Devices, Sunnyvale, CA, USA). Current-clamp experi-
ments were made with electrodes filled with a solution
containing the following (in mM): 144 KCl, 10 HEPES,
3.45 BAPTA, 1 CaCl2, 2.5 Mg2ATP, and 0.25 Mg2GTP
(pH 7.2–7.4, 275–285 mOsm). Data were filtered at
2 kHz, digitized at 10 kHz and collected on-line with
acquisition software (pClamp 8.2, Molecular Devices). DA
neurons were identified according to the already pub-
lished criteria (Melis et al. 2010): cell morphology and
anatomical location (that is medial to the medial terminal
nucleus of the accessory optic tract), large hyperpola-
rization activated current (Ih > 100 pA), slow pacemaker-
like firing rate (< 5 Hz), long action potential duration
(> 2 ms). Each slice received only a single drug exposure
with the exception of data represented in Fig. 5. Drugs
were applied in known concentrations to the superfusion
© 2013 Society for the Study of Addiction
From ethanol to salsolinol 3
medium. Ethanol, acetaldehyde, (±)-salsolinol, acetate,
DA (Sigma-Aldrich) and morphine (Salars, Milan, Italy)
were dissolved in distilled water, whereas 3-amino-1,2,4-
triazole (3-AT; Sigma-Aldrich) was dissolved in dimethyl
sulfoxide (DMSO) (Sigma-Aldrich). The final concentra-
tion of DMSO was < 0.01%. Ethanol, acetaldehyde and
salsolinol concentrations were selected on the basis of
previous studies (Okamoto et al. 2006; Melis et al. 2007;
Xie & Ye 2012; Xie et al. 2012). Action potential fre-
quency was analyzed off-line with MiniAnalysis software
(Synaptosoft Inc., Decatur, GA, USA). The averaged action
potential frequency 5 minutes immediately before the
drug administration was taken as baseline, and the aver-
aged frequency for the third minute period centered on
peak response was taken for drug effect. All numerical
data are given as mean ± SEM. Data were compared and
analyzed by utilizing two-way ANOVA for repeated meas-
ures (treatment × time), or one-way ANOVA for repeated
measures, when appropriate. The significance level
was established at P < 0.05 and, whereby allowed by sig-
nificant main effects, ANOVA was followed by either
Dunnett’s or Bonferroni’s multiple comparisons.
Sample collection and sample preparation procedure for
high performance liquid chromatography-tandem mass
spectrometry (HPLC-MS/MS) analysis
Samples of aCSF (400 μl) were collected at second minute
of ethanol application to a control slice (Fig. 1A), to a slice
from a αMpT-treated mouse (b in Fig. 4A) and at second
minute of co-application of ethanol and DA (d in Fig. 4A).
Samples were collected from the recording chamber under
constant perfusion flow rate of 2.5 ml/minute. Samples
were placed into 2.0 ml glass vials and evaporated under
a gentle nitrogen stream. The pellet was redissolved
with 100 μl of methanol containing 0.1% formic acid and
subjected to HPLC-MS/MS analysis.
HPLC—Electrospray injection (ESI)—MS/MS analysis
A Varian 1200 L triple-quadrupole tandem mass spec-
trometer (Varian Inc., Palo Alto, CA, USA) coupled with
a ProStar 410 autosampler, two ProStar 210 (Varian
Inc.) pumps and a 1200 L triple-quadrupole mass
spectrometer (Varian Inc.) was used with an ESI source.
The Varian MS workstation version 6.7 software was used
for data acquisition and processing. Chromatographic
separation was performed on a Phenomenex Column
Synergi MAX-RP 80A (4.6 mm × 150 mm i.d., 4 μm)
[Phenomenex Italy, Castel Maggiore (Bo), Italy]. The
mobile phase consisted of (1) methanol 5% (v/v) contain-
ing 0.1% formic acid and (2) double distilled water 95%
(v/v) containing ammonium formate 1 mM. The mobile
phase, previously degassed with high-purity helium, was
pumped at a flow rate of 0.2 ml/minute, the injection
Addiction Biology
4 Miriam Melis et al.
volume was 10 μl and total run time 12 minutes. ESI was
operated in the positive ion mode. The electrospray capil-
lary potential was set at 65 V, the needle at 5850 V and
the shield at 750 V. Nitrogen, at 48 mTorr and 375°C, was
used as a drying gas for solvent evaporation. Full-scan
spectrum was obtained in the ranges of 100–1000
atomic mass unit (amu) for salsolinol, scan time of 0.75
amu, scan width of 0.70 amu and detector at 1450 V. For
ESI, the atmospheric pressure ionization housing was
kept at 50°C. Parent compound eluting at 8.94 minutes
was subjected to collision-induced dissociation using
argon at 2.40 mTorr in the multiple reaction monitoring
positive mode. The observed mass transitions and colli-
© 2013 Society for the Study of Addiction
Figure 1 Ethanol, acetaldehyde and
salsolinol excite pVTA DA neuronal firing
rate ex vivo.
Time course graphs illustrating the aver-
aged effects of ethanol (100 mM) (a), acet-
aldehyde (10 nM) (b) and salsolinol (10 nM)
(c) on firing rate of DA cells of the pVTA.
Data are normalized to the baseline. The
black bars represent the time of drug
application. n = 6 for all groups. Filled circles
represent time points significant when
compared to baseline as revealed by
Dunnett’s test. Representative traces are
shown in the insets. Calibration bar: 15 mV,
125 ms. (d) Magnification of the onset of
effects represented in panels a, b and c.
Time 0 and arrow represent the time
drugs reached the recording chamber.
(e) Concentration-response relationships
for percentage increase in firing rate pro-
duced by acetaldehyde (circles) and salso-
linol (squares). Each point shows the
mean ± SEM of responses (n = 6)
sion energy used for quantitation of salsolinol were
180.1 > 115.1 (−30 V), 180.1 > 117.1 (−22 V), 180.1 >
145.1 (−18 V), 180.1 > 163.1 (−14 V). The scan time
was 1 second, and the detector multiplier voltage was
set to 2000 V, with an isolation width of m/z 1.2 for
quadrupole 1 and m/z 2.0 for quadrupole 3.
RESULTS
Acetaldehyde-induced excitation of DA neuronal firing
rate requires DA
In agreement with previous in vitro studies, we found that
ethanol (100 mM) (Brodie et al. 1990; Okamoto et al.
Addiction Biology

Figure 2 Acetate does not affect pVTA DA neuronal firing rate.
Time course graph illustrating the averaged effects of acetate
(10 nM) on firing rate of DA cells of the pVTA (n = 5). Data are
normalized to the baseline.The black bar represents the time of drug
application
2006; Melis et al. 2007), acetaldehyde (10 nM) (Melis
et al. 2007) and salsolinol (10 nM) (Xie & Ye 2012; Xie
et al. 2012) significantly stimulate pVTA DA cell firing
rate by 142 ± 7% (one-way ANOVA for repeated meas-
ures followed by Dunnett’s multiple comparison test:
F5,80 = 6.07, P = 0.0001), 252 ± 48% (one-way ANOVA
for repeated measures followed by Dunnett’s multiple
comparison test: F5,90 = 4.12, P = 0.0003) and 171 ±
26% (one-way ANOVA for repeated measures followed
by Dunnett’s multiple comparison test: F5,76 = 6.24,
P < 0.0001), respectively (Fig. 1A–C). Particularly, the
onset of the effects of ethanol, acetaldehyde and
salsolinol was similar (Fig. 1D) and, in addition, acetalde-
hyde and salsolinol revealed overlapping dose-response
curves in the range between 1 and 100 nM (two-way
ANOVA F1,18 = 0.29, P = 0.6) (Fig. 1E). Notably, acetate,
at a concentration similar to effective acetaldehyde and
salsolinol one (that is 10 nM), did not affect pVTA DA
cell activity (one way ANOVA F18,112 = 0.33, P = 0.99)
(Fig. 2).
We previously reported that acetaldehyde is essential
for ethanol-induced excitation of pVTA DA neurons (Melis
et al. 2007). However, whether or not acetaldehyde’s
effects on pVTA DA neurons are mediated by its condensa-
tion with DA is unknown yet. Hence, we applied acetalde-
hyde (10 nM) to slices obtained from mice administered
αMpT (300 mg/kg i.p., 2 hours before slice preparation), a
drug that inhibits DA synthesis and release (Cheramy et al.
1981) and depletes striatal DA content. Accordingly,
striatal tissue DA content was reduced following adminis-
tration of either αMpT (300 mg/kg i.p.) or reserpine
(5 mg/kg i.p.), a drug able to deplete monoamine vesi-
cular stores, by irreversibly inhibiting vesicular uptake,
(two-way ANOVA F2,33 = 169, P = 0.0001 followed by
© 2013 Society for the Study of Addiction
From ethanol to salsolinol 5
Tukey’s test, P < 0.05). In αMpT-treated mice, we
found that acetaldehyde was unable to stimulate DA cell
firing rate (Fig. 3A).Two-way ANOVA yielded a significant
main effect of treatment (F1,180 = 17.22, P = 0.002) dis-
closing that acetaldehyde requires the presence of
somatodendritically released DA (Cheramy et al. 1981) to
excite DA cells. Furthermore, in order to rule out the pos-
sibility that the lack of effects of acetaldehyde on pVTA DA
cells could be due to a non-specific effect of αMpT, we next
tested whether or not αMpT administration would prevent
salsolinol and morphine effects. As shown in Fig. 3B & C,
salsolinol (100 nM) and morphine (1 μM) significantly
stimulated DA cell firing rate in slices from αMpT-treated
mice similarly to control slices (two-way ANOVA F1,160 =
0,03, P = 0.87; F1,112 = 0,42, P = 0.53 for salsolinol and
morphine, respectively). This indicates that αMpT treat-
ment did neither affect basal firing rate of these cells
(αMpT 3.1 ± 0.6 and control 3.2 ± 0.5 Hz, n = 43 and 35,
respectively; P = 0.3) nor the ability of drugs, such as
salsolinol itself (Xie & Ye 2012; Xie et al. 2012) and mor-
phine, to excite them.
Ethanol-induced excitation of DA neuronal firing rate
requires its conversion into acetaldehyde in the
presence of DA
Since acetaldehyde requires DA to stimulate pVTA DA cell
firing, we hypothesized that it might condensate with DA
and generate salsolinol. Hence, we next applied ethanol
(100 mM) to slices obtained from mice administered
either αMpT (300 mg/kg i.p., 2 hours before slice prepa-
ration) or reserpine (5 mg/kg i.p., 24 hours before slice
preparation). Both treatments fully prevented ethanol
ability to excite DA neurons (Fig. 4) (αMpT: two-way
ANOVA F1,240 = 8,14, P = 0.012; reserpine: two-way
ANOVA F1,208 = 10,25, P = 0.006). Furthermore, we
tested the contribution of DA to the effect of ethanol
(100 mM). As shown in Fig. 5A (left), ethanol failed to
stimulate the firing rate of DA cells; however, in the pres-
ence of bath applied DA (10 nM) its ability to enhance
pVTA DA cell activity was fully restored (one-way ANOVA
followed by Bonferroni’s multiple comparisons test
F34, 175 = 2,34, P = 0.0002) [Fig. 5A (right)].
Since ethanol requires both acetaldehyde and DA to
increase pVTA DA neuronal activity, we next tested
whether or not application of catalase inhibitor, 3-AT
(1 mM) would prevent DA to restore ethanol effects in
αMpT-treated mice. Remarkably [Fig. 5B (right)], 3-AT
fully abolished DA ability to recover the stimulating prop-
erties of ethanol on DA cell firing rate in αMpT-treated
mice (one-way ANOVA followed by Bonferroni’s multiple
comparison test F31, 155 = 2.68, P = 0.0001). Accordingly,
in vivo administration of αLA (100 mg/kg i.p., 30
minutes before slice preparation), a hydrogen peroxide
Addiction Biology
6 Miriam Melis et al.

Figure 3 Acetaldehyde-induced excita-

tion of DA neuronal firing rate requires the
presence of DA.
(a) Time course graph illustrating the aver-
aged effects of acetaldehyde (10 nM) on
firing rate of DA cells of the pVTA in mice
administered αMpT. (b) Time course graph
illustrating the averaged effects of salsolinol
(100 nM) on firing rate of DA cells of the
pVTA in mice administered αMpT. (c) Time
course graph illustrating the averaged
effects of morphine (1 μM) on firing rate of
the pVTA DA cells in mice administered
αMpT. Data are normalized to the baseline.
n = 6 for panels a and b, and n = 5 for panel
c.The black bars represent the time of drug
application. Grey areas represent the mean
response observed in control mice. Repre-
sentative traces of the spontaneous activity
of a DA neuron during baseline (a), acet-
aldehyde, salsolinol or morphine (b) and
wash-out (c) of an αMpT-treated mouse
are shown in the insets. Calibration: 15 mV,
125 ms

scavenger (Ledesma & Aragon 2012), fully prevented P = 0.66), even in the presence of bath applied DA
ethanol-induced increase in firing rate of pVTA DA cells (Fig. 5C). Lastly, 3-AT did not prevent acetaldehyde
in both naïve (one-way ANOVA F19,76 = 1.55, P = 0.09) actions on pVTA DA cells (one-way ANOVA F3,19 = 16.08,
and αMpT-treated mice (two-way ANOVA F1,52 = 0.2, P < 0.0001) (Fig. 6).

Figure 4 Ethanol-induced excitation of DA neuronal firing rate requires the presence of DA.
Time course graph illustrating the averaged effects of ethanol (100 mM) on firing rate of DA cells of the pVTA in mice pretreated with either
αMpT (white circles) or reserpine (grey circles). Data are normalized to the baseline. n = 6 for all groups. The black bar represents the time of drug
application. Grey area represents the mean response observed in control mice. Representative traces of the spontaneous activity of a DA neuron
during baseline (a), ethanol (b) and wash-out (c) of an αMpT- or reserpine-treated mouse are shown in the inset. Calibration: 15 mV, 125 ms

© 2013 Society for the Study of Addiction Addiction Biology

 From ethanol to salsolinol 7

Figure 5 Ethanol-induced excitation of DA neuronal firing rate requires its oxidation into acetaldehyde and the presence of DA.
(a)Time course graph illustrating the averaged effects of ethanol (100 mM) on firing rate of DA cells of the pVTA in mice pretreated with αMpT.
Note that the effect of ethanol is restored in the presence of exogenous DA (10 nM). On the right-hand of the panel, representative traces of
the spontaneous activity of a DA neuron during baseline (a), ethanol (b), DA (c) and ethanol co-applied with DA (d) of an αMpT-treated mouse.
Calibration: 15 mV, 125 ms. (B) Time course graph illustrating the averaged effects of ethanol (100 mM) on firing rate of DA cells of the pVTA
in mice pretreated with αMpT.The effect of ethanol is abolished in the presence of DA (10 nM) when acetaldehyde formation is prevented
by 3-AT application (1 mM). On the right-hand of the panel, representative traces of the spontaneous activity of a DA neuron during baseline
(a), ethanol (b), DA and 3-AT (c), and ethanol co-applied with DA and 3-AT (d) of a αMpT-treated mouse. Calibration: 15 mV, 125 ms. Data are
normalized to the baseline. n = 6 for all groups. (C)Time course graph illustrating the averaged effects of ethanol (100 mM) on firing rate of DA
cells of the pVTA in naïve and αMpT-treated mice following systemic administration of αLA (100 mg/kg i.p., 30 minutes before slice preparation).
The effect of ethanol is abolished when acetaldehyde formation is prevented by αLA, even in the presence of DA (10 nM) applied to slices from
αMpT-treated mice. On the right-hand panel, representative traces of the spontaneous activity of a DA neuron during baseline (a), vehicle or
DA (b), and ethanol co-applied with DA (c) of naïve and αMpT-treated mice following αLA administration. Calibration: 15 mV, 125 ms. Data are
normalized to the baseline. n = 5 for both groups.The black bars represent the time of drug application

HPLC-MS/MS detects salsolinol in aCSF after
co-application of ethanol and DA to slices from
αMpT-treated mice
Overall, these results demonstrate that ethanol stimu-
lates the spontaneous activity of pVTA DA neurons
following its oxidation into acetaldehyde and subsequent
condensation of acetaldehyde with DA to generate
salsolinol. If salsolinol is, indeed, the molecule responsi-
ble of the effects observed on DA firing rate, one would
expect to detect it in the aCSF under the same conditions
(see above). Hence, we analyzed by HPLC-MS/MS the

© 2013 Society for the Study of Addiction Addiction Biology

8 Miriam Melis et al.

Figure 6 Acetaldehyde effects on pVTA DA cells are independent of catalase inhibition.

(a) Time course graph illustrating the averaged effects of acetaldehyde (10 nM) in the presence of 3-AT (1 mM, 5 minutes) on firing rate of
pVTA DA cells (n = 5) in naïve mice. Data are normalized to the baseline. The black bar represents the time of drug application. Grey area
represents the mean response observed in control mice. (b) Bar graph summarizing the effects observed in (a). Acetaldehyde (ACD)
stimulates the firing rate of pVTA DA cells also in the presence of 3-AT, which per se is ineffective on DA cell spontaneous activity (one-way
ANOVA followed by Dunnett’s multiple comparison test, F3,19 = 16.18, P < 0.0001).

Figure 7 HPLC-MS/MS detects salsolinol in aCSF after co-application of ethanol and DA to slices from αMpT-treated mice.
Representative HPLC-MS/MS ion chromatograms (mass transition: 180.1 > 117.1; collision energy −22 V) of (a) standard of salsolinol (56 nM)
in aCSF; (b) aCSF from recording chamber at second-minute application of ethanol (100 mM) in control mouse; (c) aCSF from recording
chamber at second-minute application of ethanol (100 mM) in αMpT-treated mouse; (d) aCSF from recording chamber at second-minute
co-application of ethanol (100 mM) and DA (10 nM) in αMpT-treated mouse.

aCSF after application of ethanol (as in Fig. 5A, left) and
after co-application of ethanol and DA (as in Fig. 5A,
right). As shown in Fig. 7, we detected salsolinol when
ethanol was applied in slices from naïve mice (B), and
when ethanol and DA were co-applied in slices from naïve
(not shown) and αMpT-treated mice (D). Particularly,
under conditions of ethanol and DA co-application
(n = 4), salsolinol was determined at a higher concentra-
tion (24 ± 3 nM) than we expected (≤10 nM) on the basis
of the stoichiometry of its formation (1:1 from the

© 2013 Society for the Study of Addiction Addiction Biology


ethanol-derived acetaldehyde and the added DA). One
possible explanation might be that undetermined, and
not necessarily overlapping, kinetics of acetaldehyde and
salsolinol production may take place in the recording
chamber. In addition, sample collection for this analysis
might have been carried out (second minute of ethanol
application) when salsolinol formation had already taken
place at a possibly faster rate than that of removal of DA
by the constant flow rate of perfusion in the recording
chamber.
DISCUSSION
We here provide evidence for ethanol’s two-step mecha-
nism of action for its excitatory effects on pVTA DA
neurons. This mechanism consists of its former catalase-
mediated oxidation, and subsequent condensation of
ethanol-derived acetaldehyde with endogenous DA to
generate salsolinol, which excites pVTA DA neurons (Xie
& Ye 2012; Xie et al. 2012).
Several observations support our conclusions. First,
somatodendritically released DA is required for ethanol
effects, since these were absent in mice administered
either αMpT or reserpine. Notably, ethanol effects were
restored when DA was applied stoichiometrically with
respect to the concentration at which acetaldehyde
increases DA cell firing rate (present results and Melis
et al. 2007). Thus, bath application of exogenous DA, at a
concentration that per se does not affect DA neuronal
activity, fully recovered ethanol’s stimulatory effects on
DA firing rate. While this observation appears in contrast
with Xie et al. (2012), one must consider that the known
slow action of reserpine (Callaway, Kuczenski & Segal
1989) and different experimental conditions (that is mice
systemically administered 24 hours before experiment
versus acute bath application to slices) might account
for this discrepancy. In addition, reserpine is known to
inhibit the monoamine vesicular carrier and, transiently,
increase cytoplasmic DA concentration. Thus, it is possi-
ble that in the early phases of its bath application, such as
reported by Xie et al. (2012), reserpine may not only have
failed to reduce somatodendritic DA release, but also—if
any—have increased it by reverting DA transporter activ-
ity (Sulzer, Maidment & Rayport 1993). Notably, both
salsolinol and morphine were still able to excite pVTA DA
neurons in αMpT-treated mice. Collectively, these results
indicate that somatodendritically released DA is required
for ethanol and acetaldehyde to stimulate pVTA DA cell
activity.
Second, ethanol-derived acetaldehyde (Cohen et al.
1980; Aragon et al. 1991, 1992; Gill et al. 1992;
Zimatkin et al. 2006) requires the presence of
extracellular DA to enhance DA cell firing rate. In fact,
even in the presence of exogenous DA, 3-AT-mediated
© 2013 Society for the Study of Addiction
From ethanol to salsolinol 9
inhibition of ethanol metabolism prevents ethanol’s
ability to excite DA neurons in slices from αMpT-treated
mice. Accordingly, in vivo administration of the H2O2
scavenger, αLA, abolishes ethanol actions on pVTA DA
cells in both naïve and αMpT-treated mice, thus support-
ing that ethanol oxidation is a limiting step for ethanol
behavioral actions (Ledesma & Aragon 2012; Ledesma
et al. 2013; Peana et al. 2013) even in the presence of
DA. Accordingly, 3-AT does neither affect spontaneous
activity of pVTA DA cells nor their response to acetalde-
hyde in naïve mice. In contrast, one must notice that,
while basal DA cell spontaneous activity is not affected
in αMpT-treated mice, a dysfunctional H2O2-catalase
system alters pVTA DA cell spontaneous activity and
their responses to ethanol in αMpT-treated mice. How-
ever, at this stage, we have no explanation for this phe-
nomenon. One possibility might be that, since H2O2 is
produced by DA neurons in an activity-dependent
fashion (Avshalumov et al. 2005), impairment of H2O2-
catalase system might result in compensatory meta-
bolic changes in DA cells of αMpT-treated mice. Finally,
acetaldehyde-induced excitation of pVTA DA neurons is
absent in αMpT-treated mice, thus substantiating our
current hypothesis that ethanol-derived acetaldehyde
requires DA to excite DA neurons.
Third, salsolinol was detected in the aCSF only when
ethanol and exogenous DA were co-applied in αMpT-
treated slices (Fig. 7). Notably, we applied ethanol at a
concentration that was previously reported to enhance
pVTA DA cell activity under similar experimental condi-
tions (Okamoto et al. 2006; Melis et al. 2007; Xie et al.
2012). Although we acknowledge that this concentra-
tion is likely higher than the one resulting from a
behaviorally relevant dose of ethanol in vivo, direct com-
parisons between concentrations of ethanol contingently
self-administered in pVTA in vivo (Rodd et al. 2004,
2005) and those examined ex vivo in acute brain slices
(Okamoto et al. 2006; Melis et al. 2007) could not be
attempted. Nonetheless, rats self-administer ethanol
directly in pVTA at concentrations up to 300 mg%,
which are equivalent to 66 mM. Furthermore, we applied
concentrations of ethanol and acetaldehyde with a
1000:1 ratio (that is 100 mM versus 100 nM, respec-
tively), which comply the ratio of those systemically
administered (that is g/kg versus mg/kg). Additionally,
we cannot rule out that ethanol acute actions (at
100 mM) on spontaneous activity of DA neurons might
be the net effect resulting from complex synaptic changes
at both inhibitory (M. Melis and A. Bonci, unpublished
observations) and excitatory inputs integrated with cell
membrane properties (Okamoto et al. 2006; Tateno &
Robinson 2011). Conversely, acetaldehyde (at nM con-
centrations) only acts by changing the intrinsic excitabil-
ity of DA neurons (Melis et al. 2007). Notably, the
Addiction Biology
10 Miriam Melis et al.
differences in terms of absolute values of acetaldehyde
between in vivo and ex vivo studies may appear not so
critical. In fact, similar absolute amounts (number of
moles) of acetaldehyde result in pVTA after its in vivo
self-administration (6–90 μM in 100 nl/active lever
press, that is between 6 × 10–13 and 9 × 10–12 moles) as in
Rodd et al. (2005) and after its bath application (10–
100 nM in 2.5 ml at the constant flow rate of 2.5 ml/
minute, that is between 1.25 × 10–11 and 1.25 × 10−10
moles/pVTA) as in the present ex vivo experiments. Fur-
thermore, in vivo sequential microinjections in pVTA of
6.9 × 10–11 moles of acetaldehyde over 10 minutes, result
in large increases of DA efflux in nucleus accum-
bens shell (Deehan et al. 2012).
The stimulatory action of salsolinol on firing rate of
DA cells has been ascribed to activation of μ-opioid recep-
tors and concurrent stimulation of excitatory afferents
(Xie & Ye 2012). Thus, provided that the aim of our
study was to demonstrate whether ethanol acts as
prodrug of salsolinol, we did not further address this
issue. Finally, ethanol-derived acetaldehyde via hypotha-
lamic β-endorphins (Chronwall 1985) might be also
involved in the effects on VTA DA neurons and their
behavioral output (Spanagel, Herz & Shippenberg
1992; Sanchis-Segura et al. 2005; Pastor & Aragon
2008). Notably, acetate, the second by-product of ethanol
metabolism, has no effect on DA’s spontaneous neuronal
activity, thus supporting the behavioral evidence that
acetate is not involved in DA-dependent effects of ethanol
(Pardo et al. 2013).
The ability of ethanol to stimulate the firing rate of DA
neurons in the pVTA has long been considered a critical
step for its psychomotor and motivational effects (Di
Chiara 1997; Koob et al. 1998; Boileau et al. 2003) and
for its ability to initiate and maintain ethanol taking
behaviors (Di Chiara 1997; Koob et al. 1998; Söderpalm
& Ericson 2013). Remarkably, the present results are in
line with an early clinical report suggesting that admin-
istration of αMpT prevents ethanol ability to elicit eupho-
ria (Ahlenius et al. 1973). An immediate implication of
this study would involve the potential role of salsolinol in
stimulating firing rate of pVTA DA cells, and in exerting
motivational effects when ingested (through foods and
beverages). However, the questioned ability of salsolinol
to cross blood brain barrier (Origitano et al. 1981; Correa
et al. 2012) greatly reduces the impact of this implica-
tion. Nevertheless, given that ethanol is the main ingre-
dient of alcoholic drinks, the present results not only
provide a significant contribution to the understanding of
ethanol’s mechanism of action on DA cells, but also make
a significant advance in the field of alcohol research.
Finally, they provide new insights to look into the
neurobiological basis of alcoholism and suggest exciting
avenues of future research.
© 2013 Society for the Study of Addiction
Acknowledgements
This study was supported by the grant from Regione
Autonoma della Sardegna (RAS), Italy, L.R. 7/2007,
CRP_537-CUP F71J090006200002 to E.A. The helpful
comments of P. J. Mackenzie and G. Zernig, and kind
assistance on HPLC-MS/MS analyses of A. Murgia and I.
Ibba are gratefully acknowledged.
Authors’ Contributions
MM contributed to design the experiments, performed
the electrophysiological experiments, analyzed data and
wrote the manuscript. EC performed the quantification of
dopamine content in the tissue by HPLC-ECD, analyzed
data and contributed to write the manuscript. PC per-
formed the identification of salsolinol by HPLC-MS/MS
in the aCSF collected from the recording chamber, and
contributed to write the manuscript. EA conceived and
designed the study, performed in vivo administrations and
wrote the manuscript. All authors have critically
reviewed content and approved final version submitted
for publication.
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