Neurochemistry International 38 (2001) 163±168

www.elsevier.com/locate/neuint

Evidence for a serotonin transporter de®cit in experimental acute

liver failure
Adrianna Michalak, Nicolas Chatauret, Roger F. Butterworth*
Neuroscience Research Unit, CHUM HoÃpital Saint-Luc, University of Montreal, 1058 St. Denis Street, Montreal, Que., Canada H2X 3J4
Received 13 January 2000; received in revised form 28 March 2000; accepted 31 March 2000

Abstract

It has been suggested that alterations of serotonin transport may be implicated in the pathogenesis of the neuropsychiatric
symptoms encountered in acute liver failure. In order to address this issue, microdialysate concentrations of serotonin, its
precursor L-tryptophan and metabolite 5-hydroxyindoleacetic acid (5-HIAA) as well as brain regional distribution of serotonin
transporter ([3H]-citalopram) sites were measured in rats with acute liver failure resulting from hepatic devascularization. A
signi®cant loss of [3H]-citalopram sites was observed in dorsal Raphe nucleus, in frontal and frontoparietal cortices as well as in
substantia nigra of rats with severe encephalopathy resulting from acute liver failure. In frontal cortex, this loss of transporter
binding sites was accompanied by signi®cant increases of L-tryptophan, serotonin and 5-HIAA concentrations in extracellular
¯uid. Pharmacological manipulation of the brain serotonin system could a€ord a novel therapeutic approach to the prevention
of the neuropsychiatric symptoms characteristic of acute liver failure in humans. 7 2001 Elsevier Science Ltd. All rights
reserved.

Keywords: Acute liver failure; Hepatic coma; Serotonin transporter; Quantitative receptor autoradiography; [3H]-citalopram

1. Introduction have been described in the brains of animals with

The pathophysiologic mechanisms responsible for
hepatic encephalopathy (HE) in acute liver failure
have not been fully elucidated but de®cits of neuro-
transmission rather than primary cerebral energy fail-
ure are currently considered to be implicated (Bates et
al., 1989; Butterworth, 1994). Alterations of both glu-
tamatergic (Michalak et al., 1996; Knecht et al., 1997;
Michalak and Butterworth, 1997) and noradrenergic
(Michalak et al., 1998) systems have been described
in HE in acute liver failure and there is a growing
body of evidence that the serotoninergic system may
also be implicated. For example, extracellular levels
of the serotonin precursor, L-tryptophan and
decreases in postsynaptic serotoninergic2A receptors
* Corresponding author. Tel.: +1-514-281-2444 x 5759; fax: +1-
514-281-2492/2107.
E-mail address: butterwr@medclin.umontreal.ca (R.F. Butter-
worth).
thioacetamide-induced acute liver failure (Kaneko et
al., 1998). In contrast, an increase in serotonin1A
receptor expression was reported in hippocampus of
rats with acute hyperammonemia (Alexander et al.,
1995). An increase of serotoninergic activity and a con-
tribution of serotoninergic mechanisms to the neurop-
sychiatric symptoms characteristic of HE in acute liver
failure were proposed by Yurdaydin et al. (1990,
1996). Furthermore, thioacetamide-induced acute liver
failure as well as liver devascularization result in
increases in release of serotonin and increased extra-
cellular brain concentrations of the serotonin metab-
olite 5-hydroxyindoleacetic acid (5-HIAA) (Kaneko et
al., 1998; Bergqvist et al., 1995) suggesting that
increased serotonin release in brain may be a conse-
quence of acute liver failure.
The purpose of the present study was to evaluate the
e€ects of acute liver failure induced by liver ischemia
on: (a) extracellular (dialysate) brain concentrations of
serotonin, and its precursor L-tryptophan and metab-

0197-0186/01/$ - see front matter 7 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 9 7 - 0 1 8 6 ( 0 0 ) 0 0 0 6 2 - 0
164 A. Michalak et al. / Neurochemistry International 38 (2001) 163±168
olite 5-HIAA; (b) brain serotonin transporter sites
assessed using quantitative receptor autoradiography
and the serotonin transporter ligand [3H]-citalopram.
2. Materials and methods
2.1. Materials
N-methyl-[3H]-citalopram (speci®c activity 85 Ci/
mmol) was obtained from New England Nuclear (Bos-
ton, MA). Fluoxetine-HCl was purchased from Tocris
Cookson (Ballwin, MO). Tris±HCl, o-phthaldialdehyde
reagent solution, 2-mercaptoethanol, NaCl, KCl,
CaCl2, ethylene±diamine tetraacetic acid (EDTA),
sodium octanesulfonate and amino acid standards
were from Sigma (St. Louis, MO). Sodium phosphate
(monobasic), methanol (high-performance liquid chro-
matography (HPLC)-grade), tetrahydrofuran (HPLC-
grade), acetonitrile and magnesium chloride were
obtained from Anachemia (Montreal, Que., Canada).
Fresh, double-distilled, deionized water was used for
preparation of bu€ers and standard solutions. The
mobile phase for HPLC was ®ltered through 0.45 mm
pore-size membrane ®lters (Millipore, Bedford, MA)
and degassed under vacuum before use. The micro-
dialysis system (CMA/Microdialysis AB, Stockholm,
Sweden; Bioanalytical Systems, West Lafayette, IN)
consisted of a microinjection pump (CMA/100), a syr-
inge selector (CMA/111), a temperature controller
(CMA/150), a microfraction controller (CMA/140)
and housing system for freely-moving animals (CMA/
120). Tritium-sensitive ®lms were purchased from
Amersham (Arlington Heights, IL).
2.2. Animal surgery
Male, Sprague±Dawley rats (weighing 175±200 g)
(Charles River, St. Constant, Que., Canada) were
anesthetized with halothane and an end-to-side portaca-
val anastomosis was performed using a modi®cation of
the technique of Lee and Fisher (1961) as previously
reported (Michalak et al., 1996). A Brief laparotomy was
performed, the inferior vena cava and portal vein were
isolated, the inferior vena cava was partially clamped
(anastomosis clamp; Roboz Instruments, Washington,
DC), an epileptical piece of vein (1.5 times the portal vein
diameter) was removed, the portal vein was ligated, cut,
and an end-to-side portacaval anastomosis (shunt: SH)
was performed under a dissecting microscope. Total sur-
gery time was approximately 15 min. Sham-operated rats
(SM), matched for weight, were anesthetized with
halothane, and the inferior vena cava and portal vein
were clamped for 15 min. After surgery, animals were
placed in individual cages under constant conditions
(temperature, humidity, light cycle) with free access
to rat chow and water.
Twenty-four hours later, an intracerebral guide can-
nula was stereotactically implanted. The rats were
anesthetized (halothane), positioned in a stereotaxic
frame (David Kopf Instruments, Tujunga, CA) and a
disposable plastic-siliconized intracerebral guide can-
nula was inserted (coordinates: +3.2 mm (sagittal),
ÿ1.5 mm (lateral), ÿ2.5 mm (depth) relative to bregma
(Paxinos and Watson, 1982), closed with a dummy
probe. Total surgery time was approximately 15 min.
Throughout the surgery, the temperature of animals
was maintained at 378C (CMA/150 temperature con-
troller). Following surgery, rats were maintained in a
microdialysis chamber (CMA/120 system for freely-
moving animals) with free access to rat chow and
water.
Hepatic artery ligation (HAL) or laparotomy
(sham:SM) was performed 48 h after portacaval ana-
stomosis or 24 h after intracerebral guide cannula im-
plantation, in reanesthetized (Halothane) animals. In
this way, four groups of rats were created: liver ische-
mia (SH + HAL) and three control groups (SH +
SM, SM + HAL, SM + SM). Aortic and peritoneal
catheters were implanted for arterial glucose monitor-
ing and 5% w/v glucose (or 0.9% w/v NaCl) adminis-
tered when necessary.
2.3. Microdialysis
The microdialysis probe (CMA/12; 2 mm long, 0.5
mm optical diameter) was inserted into brain
(described earlier) after hepatic artery ligation or sham
surgery. The probe was perfused at a constant ¯ow
rate of 2 ml/min with Ringer's solution (147 mM
NaCl, 4 mM KCl, 2.4 mM CaCl2 (Benveniste and
Huttemeir, 1990)) prepared daily using deionized
water, ®ltered through 0.4 mm pore-size Millipore ®l-
ters and degassed before use. After an initial stabiliz-
ation period (3 h), 20 min fractions were collected and
neurological status tested at each 20 min time point.
Samples were then frozen and stored at ÿ808C. At the
end of experiments (1 h in coma stage), rats were
decapitated, brains were removed, sectioned and
stained with cresyl violet for pathological probe place-
ment veri®cation.
All experiments were in accordance with the Prin-
ciples of the Guide for the Care and Use of Experimen-
tal Animals, of the Canadian Council on Animal Care,
Ottawa, and the Guiding Principles for Research Invol-
ving Animals and Humans (Recommendations from the
Declaration of Helsinki), approved by the Council of
the American Physiological Society.
2.4. Tryptophan analysis by high-performance liquid
chromatography
Tryptophan was measured in brain extracellular ¯uid
 A. Michalak et al. / Neurochemistry International 38 (2001) 163±168
at coma stage of HE. Dialysates were analyzed using a
Perkin±Elmer reverse-phase HPLC system with ¯uor-
escence detection and precolumn o-phthalaldehyde
derivatization (Lavoie et al., 1987). The HPLC system
consisted of a solvent delivery system (Perkin±Elmer,
series 400) coupled to a ®lter ¯uorometer (Perkin±
Elmer, LC-10 ¯uorescence detector; excitation 370 nm,
emission 418±700 nm). Sample injections were per-
formed using a 50 ml loop of the CMA3200 autosam-
pler with derivatization accessories. The column was a
reverse-phase Perkin±Elmer C18 0.5 ®tted with a
Vydec reverse-phase C18 guard column. The chromato-
graph was performed with a gradient between metha-
nol and sodium phosphate bu€er 50 mM (pH 5.25)
containing 2% v/v tetrahydrofuran (Hazell et al., 1993)
at a constant ¯ow rate of 1 ml/min for a further 10
min. The gradient was then run from 25% v/v metha-
nol/75% v/v bu€er over 5 min, before re-equilibration
for reuse. Chromatograms were computed using a pro-
grammable recording integrator (Perkin±Elmer, LC-
100). Concentrations of tryptophan in extracellular
¯uid samples were calculated by reference to the in-
ternal standard (homoserine) (Lavoie et al., 1987).
2.5. Measurement of serotonin and 5-HIAA
Twenty microliters of dialysate from rats in the four
experimental groups (above) was injected directly onto
the column. The mobile phase consisted of 0.1 M
sodium diphosphate (pH 4.7) containing 10% aceto-
nitrile, 1 mM sodium octanesultonate, and 0.1 mM
EDTA. The electrochemical detector was set at 0.72 V
versus Ag/HgCl reference electrode. 5-HT and 5-
HIAA concentrations were calculated by comparison
with corresponding standards.
2.6. Serotonin (5-HT) transporter autoradiography
Four groups of rats were used as described earlier.
Table 1
Extracellular brain concentrations of aromatic amino acids and of serotonin and 5-HIAA at coma stages of encephalopathy in rats with acute
(ischemic) liver failure vs. controla
Concentration (pg/20 ml)
Amino acid/monoamine/metabolite Sham/sham
(control)
Tyrosine 1:7720:35
Phenylalanine 2:5720:59
Tryptophan 0:6620:07
Serotonin 13:220:2
5-HIAA 48:523:0
a
Values represent mean 2 SEM of triplicate determinations from six animals per treatment group. Sham: sham-operated; shunt: portacaval
shunted; HAL: hepatic artery ligated. Values signi®cantly di€erent from control indicated by  p < 0:05,  p < 0:01 by analysis of variance with
post-hoc Tukey test.
165
Rats with liver ischemia (and their appropriate con-
trols) were decapitated at coma stages of HE de®ned
as the loss of corneal re¯ex. The brains were removed,
frozen in chilled isopentane and stored at ÿ808C.
Sagittal sections (20 mm) of brain were cut, mounted
onto gelatin-coated slides and stored at ÿ208C until
use.
For determination of [3H]-citalopram binding, sec-
tions were preincubated for 15 min at 258C in 50 mM
Tris±HCl bu€er (pH 7.4) containing 120 mM NaCl
and 5 mM KCl (Bennett±Clarke et al., 1996). Sections
were incubated for 120 min at room temperature
in the bu€er containing 2 nM (three times the Kd)
[3H]-citalopram (total binding). Nonspeci®c binding
(which was less than 20% of total binding in all cases)
was determined in the presence of 10 mM ¯uoxetine±
HCl. Unbound radioactivity was removed by four
washes in ice-cold bu€er and ®nally in ice-cold distilled
water. The sections were dried under a stream of cool
air.
Autoradiograms were generated by apposing dried
tissue sections and appropriate tritium standards to tri-
tium-sensitive ®lm, at 48C for 4 weeks. Densitometric
analyses of the ®lms were performed with an image
analyzer (MCID system; St. Catherines, Ont.,
Canada). The speci®c binding was calculated as the
di€erence between total and nonspeci®c binding. Tri-
tium standards were used to convert optical density
measurements to binding site densities expressed as
pmol/g of tissue.
2.7. Statistical analysis
Data are expressed as mean2SEM values from ®ve
to eight animals. Di€erences between groups at coma
stage of rats with liver ischemia and the three obligate
control groups of rats were compared using one-way
ANOVA (Tukey±Kramer multiple-comparisons test).
A p < 0:05 was considered signi®cant.
Shunt/sham Sham/HAL Shunt/HAL
(control) (control) (acute liver failure)
5:6221:17 1:9020:34 13:521:42
5:3721:00 3:9020:80 15:121:98
1:2820:11 0:8820:06 2:2620:11
12:620:46 12:220:3 14:120:2
54:1212:1 70:623:9 107:725:0
166 A. Michalak et al. / Neurochemistry International 38 (2001) 163±168

3. Results control rat and a rat at coma stages of encephalopathy

Rats with liver ischemia manifested a reproducible
sequence of neurological impairment progressing
through decreased spontaneous activity starting 4±6 h
after hepatic artery ligation, followed by loss of right-
ing re¯ex (precoma), and 8±16 h post-ischemia, a loss
of corneal re¯ex (coma). Rats from the three control
groups did not show any neurological abnormalities.
Concentrations of L-tryptophan, serotonin and 5-
HIAA in extracellular ¯uid of frontal cortex of
ischemic and control groups of rats are shown in
Table 1. Signi®cant increases of serotonin, L-trypto-
phan and 5-HIAA were observed at coma stages of
hepatic encephalopathy in rats with liver ischemia
compared to control rats.
Quantitative autoradiographic assessment of seroto-
nin transporter sites using the selective ligand [3H]-cita-
lopram revealed a region-selective distribution in the
brains of control rats. Of particular note was the pre-
dominance of serotonin transporter sites in substantia
nigra, dorsal tegmental nucleus, dorsal raphe nucleus
and thalamus. This regional distribution is similar to
that previously described (Blakely et al., 1991; Ho€-
man et al., 1991).
Serotonin transporter distribution in the brains of rats
with liver ischemia at coma stages of encephalopathy
compared with control rats is shown in Figs. 1 and 2.
Fig. 1 shows a representative autoradiogram of [3H]-cita-
lopram binding sites in sagittal brain sections from a
due to acute liver failure. Signi®cant selective decreases
of [3H]-citalopram binding sites were observed in 5 of
the 11 regions studied, namely substantia nigra, dorsal
tegmental nucleus, and dorsal raphe nucleus of the
rats (Fig. 2). In cerebral cortex, decreased [3H]-citalo-
pram binding following acute liver failure was con®ned
to frontal and frontoparietal regions.
4. Discussion
By controlling the magnitude and duration of post-
synaptic responses, carrier-mediated serotonin trans-
port into the presynaptic neuron (and/or perineuronal
astrocytes) is essential for the ®ne-tuning of serotoni-
nergic neurotransmission. It is not surprising, there-
fore, that alterations of serotonin transporter function
have been reported in a wide range of neuropsychiatric
conditions including depression, anxiety, bipolar dis-
orders and psychosis (Perry et al., 1982). Results of
the present study reveal that experimental acute liver
failure, a disorder characterized by severe neuropsy-
chiatric and motor disturbances, results in a signi®cant
loss of binding sites for the serotonin transporter
ligand [3H]-citalopram. Moreover, loss of binding sites
were particularly apparent in regions of the brain such
as frontal and frontoparietal cortex and substantia
nigra, regions of the brain known to express high den-
sities of serotonin nerve terminals.

Fig. 1. Representative autoradiograms of [3H]-citalopram binding sites in brain sections from rats at coma stage of hepatic encephalopathy due
to acute (ischemic) liver failure and control rats. SuG: superior colliculus (super®cial grey layer); CG: central grey; Fr: frontal cortex; Th: thala-
mus; Hyp: hypothalamus; MB: mamillary bodies; DR: dorsal raphe nucleus; DT: dorsal tegmental nucleus; Cer: cerebellum; SH: shunt; SM:
sham; AL: hepatic artery ligation; ALF: acute liver failure.
 A. Michalak et al. / Neurochemistry International 38 (2001) 163±168 167

In vivo cerebral microdialysis studies revealed a
modest, but statistically signi®cant increase of basal
extracellular concentrations of serotonin and of the
serotonin metabolite 5-HIAA at severe stages of
encephalopathy in frontal cortex of rats with
ischemic liver failure, coincident with the loss of
[3H]-citalopram sites. Previous reports described
increased, evoked release of serotonin and increased
extracellular brain concentrations of 5-HIAA in rats
with either ischemic liver failure (Bergqvist et al., 1995)
or thioacetamide-induced acute liver failure (Kaneko
et al., 1998). Increased brain concentrations of 5-
HIAA and of 5-HIAA/serotonin ratios have also been
reported in autopsied brain tissue from patients with
acute liver failure who died in hepatic coma (Al Mar-
dini et al., 1993) suggesting that altered serotonin
transport or turnover is also implicated in the patho-
genesis of the central nervous system complications of
acute liver failure in humans.
The mechanism responsible for the ®nding of
increased extracellular serotonin in brain in acute liver
failure is currently unknown. However, there is evi-
dence to suggest that ammonia may be involved. Brain
ammonia concentrations in acute (ischemic) liver fail-
ure attain millimolar levels at severe stages of encepha-
lopathy (Swain et al., 1992) and millimolar
concentrations of ammonia have been shown to stimu-
late serotonin release from rat brain synaptosomes in a
dose-dependent manner (Erecinska et al., 1987). It was
postulated that this e€ect was due to ammonia-
induced alkalinization of the intracellular storage ves-
icles causing extrusion of the amine into the synapse
through a reversal of the plasma membrane transpor-
ter. Ammonia administration to rats with chronic liver
failure results in increased extracellular brain concen-
trations of serotonin and 5-HIAA (Bergqvist et al.,
1995), a ®nding which is consistent with an e€ect of
ammonia on serotonin release or uptake.
Increased plasma and brain concentrations of aro-
matic amino acids including L-tryptophan, the seroto-
nin precursor amino acid, have been consistently
described in association with hyperammonemia result-
ing from urease administration to rats (Bachmann and
Colombo, 1983) as well as from acute (ischemic) liver
failure in both experimental animals (Mans et al., 1994
and the present study) and humans (Record et al.,
1976). Results of the microdialysis experiments in the
present study reveal a signi®cant increase in extracellu-
lar brain concentrations of L-tryptophan as well as
phenylalanine and tyrosine suggesting that these
increases could result from increased blood±brain bar-
rier transport via the large neutral amino acid carrier.
An additional contributing factor, therefore, to the ob-
servation of increased brain concentrations of 5-HIAA
could relate to increased availability of L-tryptophan
resulting in increased serotonin turnover.
Direct evidence for a role for serotoninergic mechan-
isms in the pathogenesis of the neuropsychiatric symp-
toms of acute liver failure is lacking. However, a
signi®cant positive correlation was reported between
brain concentrations of L-tryptophan and grade of
encephalopathy in acute (ischemic) liver failure (Mans
et al., 1994). A previous study showed downregulation
of postsynaptic serotonin2A receptors in brain in acute
liver failure (Kaneko et al., 1998) and a brief com-
munication described improvement of encephalopathy
grade following treatment of rats with thioacetamide-
induced acute liver failure with the serotonin receptor

Fig. 2. Regional distribution of binding sites for the serotonin transporter ligand [3H]-citalopram in the brain of rats with acute liver failure at
coma stages of hepatic encephalopathy and control rats. SM: sham operation; SH: portacaval shunt; HAL: hepatic artery ligation. Data rep-
resent mean2SEM …n ˆ 8). Values signi®cantly di€erent from control groups indicated by p < 0:05 by ANOVA, Tukey±Kramer multiple com-
parisons test. Fr: frontal cortex; FrPar: frontoparietal cortex; Hipp: hippocampus; Th: thalamus; Hyp: hypothalamus; SuG: superior colliculus
(super®cial grey layer); CG: central grey; SN: substantia nigra; DT: dorsal tegmental nucleus; LC: locus coeruleus; DR: dorsal raphe nucleus.
168 A. Michalak et al. / Neurochemistry International 38 (2001) 163±168
antagonist methysergide (Yurdaydin et al., 1996).
Further studies of the e€ects of pharmacological ma-
nipulation of the serotonin system in relation to the
central nervous system complications of acute liver
failure are clearly warranted.
Several neurotransmitter pathways appear to be dis-
turbed in brain in acute liver failure including the glu-
tamatergic (Michalak et al., 1996; Knecht et al., 1997;
Michalak and Butterworth, 1997), noradrenergic
(Michalak et al., 1998) and serotoninergic (present
study) systems. However, it is unclear whether these
alterations are interdependent or form part of a cas-
cade of transmitter changes resulting from a primary
de®cit. One factor in common with these systems is the
loss of transporter sites in experimental acute liver fail-
ure. Whether this generalized loss of transporter sites
is the consequence of altered membrane function as a
result of a phenomenon such as cell swelling due to ex-
posure to ammonia awaits further studies.
Acknowledgements
The authors would like to thank Ms Dominique D.
Roy for her secretarial assistance. This project was
funded by The Medical Research Council of Canada.
AM was a recipient of a postdoctoral fellowship from
the Canadian Association for the Study of the Liver.
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