Department of Psychopharmacology, Institut de Recherches Servier,
Paris, France
In a dialysis procedure not requiring perfusate addition of
acetylcholinesterase inhibitors to "boost" basal levels of
acetylcholine (ACh), the influence of the antiparkinson agent piribedil
upon levels of ACh in frontal cortex and dorsal hippocampus of freely moving rats was compared with those of other antiparkinson drugs and
selective ligands at 
2-adrenoceptors (ARs). Suggesting a tonic, inhibitory influence of 2A-ARs upon cholinergic
transmission, the 2-AR agonist
5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline tartrate
(UK14,304), and the preferential 2A-AR agonist
guanabenz reduced levels of ACh. They were elevated by the antagonists
2(2-methoxy-1,4 benzodioxan-2-yl)-2-imidazoline HCl (RX821002) and
atipamezole and by the preferential 2A-AR antagonist
2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole (BRL44008). In contrast,
trans-2,3,9,13b-tetrahydro-1,2-dimethyl-1H-dibenz[c,f]imidazo[1,5-a]azepine (BRL41992) and prazosin, preferential 2B/2C-AR
antagonists, were inactive. The dopaminergic agonist and antiparkinson
agent piribedil, which behaves as an antagonist at
2-ARs, dose dependently increased extracellular levels
of ACh. This action was absent upon pretreatment with a maximally
effective dose of RX821002. On the other hand, a further dopaminergic
agonist and antiparkinson agent, talipexole, which possesses agonist
properties at 2-ARs, dose dependently reduced levels of
ACh. This action was also blocked by RX821002. In contrast to piribedil
and talipexole, quinelorane, which interacts with dopaminergic
receptors but not 2-ARs, failed to affect ACh levels.
Finally, in analogy to the frontal cortex, piribedil likewise elicited
a dose-dependent increase in extracellular levels of ACh in the dorsal
hippocampus. In conclusion, in distinction to talipexole and
quinelorane, and reflecting its antagonist properties at
2A-ARs, piribedil reinforces cholinergic transmission in
the frontal cortex and dorsal hippocampus of freely moving rats. These actions may be related to its facilitatory influence upon cognitive function.
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Introduction |
In
Parkinson's disease (PD), progressive degeneration of nigrostriatal
dopaminergic pathways results in a profound disruption of motor
function, including such cardinal features as rigidity, bradykinesia,
and an inability to initiate movement (Jenner, 1995
). In addition,
patients frequently reveal sensory deficits, depressed mood, and a
perturbation of cognitive function. Although the dopamine (DA)
precursor L-dihydroxyphenylalanine (L-DOPA) is
universally used in the treatment of PD, certain motor symptoms, as
well as the accompanying mnemic, sensory, and emotional deficits, are little improved (Jenner, 1995). Furthermore, L-DOPA may
elicit pronounced dyskinesias (Jenner, 1995). Most disturbingly, its actions eventually become variable with abrupt transitions between "on" (effective) and "off" (ineffective) phases. These
observations underpin interest in dopaminergic agents for the
management of PD. Although they elicit their own spectrum of side
effects (hallucinations, sleep-attacks, and sedation), their low
dyskinetic potential and potential neuroprotective properties render
them attractive as alternatives (or adjuncts) to L-DOPA, in
particular in younger patients (Jenner, 1995; Rascol et al., 2000). The
improvement of motor function may primarily be attributed to activation
of postsynaptic D2 receptors in the basal ganglia
(Jenner, 1995; Wang et al., 2000). Although D4
receptors are not of major significance, it remains unclear whether
engagement of their D3 counterparts is
advantageous or deleterious in the management of PD (Newman-Tancredi et
al., 2002a).
In fact, antiparkinson agents do not exclusively interact with
dopaminergic receptors (Millan et al., 2002; Newman-Tancredi et al.,
2002a,b). Notably, several recognize 2-ARs.
For example, talipexole is an agonist both at
D2/D3 receptors and at
2-ARs (Meltzer et al., 1989; Millan et al.,
2002; Newman-Tancredi et al., 2002a), whereas piribedil (Jenner, 1995;
Smith et al., 2002) behaves as an agonist at
D2/D3 receptors yet as an
antagonist at 2A- and
2C-ARs (Millan et al., 2001, 2002;
Newman-Tancredi et al., 2002a,b). Furthermore, in distinction to most
other antiparkinson agents, both piribedil and talipexole show
negligible affinity for serotonergic receptors (Newman-Tancredi et al.,
2002b). The distinctive profile of piribedil is of considerable
interest inasmuch as 2-AR antagonists enhance
antiparkinson actions of dopaminergic agonists and L-DOPA
in rodent and primate models of PD, and suppress the induction of
dyskinesias (Brefel-Courbon et al., 1998; Bezard et al., 2001). These
actions may reflect blockade of 2C-ARs, which
are enriched in the striatum (Rosin et al., 1996; Bücheler et
al., 2002). They also likely reflect blockade of tonically active,
inhibitory 2A-AR autoreceptors on ascending
adrenergic neurons, which play an important role in the control of
motor behavior, cognition and mood (Kable et al., 2000; Millan et al., 2000; Chopin et al., 2002). Indeed, degeneration of adrenergic pathways
aggravates PD (Sandyk and Iacono, 1990) and, in experimental models,
renders subjects more sensitive to dopaminergic neurotoxins (Bing et
al., 1994). In line with these observations, like selective 2-AR antagonists, piribedil reinforces
ascending adrenergic transmission (Millan et al., 2001).
The significance of 2-AR antagonist properties
may not, however, be restricted to an enhancement of adrenergic
transmission. The frontal cortex (FCX) receives an intense input from
ascending cholinergic projections originating in the nucleus basalis
magnocellularis (Amassiri-Teule et al., 1993; Descarries and Umbriaco,
1995). Together with cholinergic pathways innervating the hippocampus, frontocortical cholinergic projections exert a facilitatory influence upon mnemic processes by the engagement of postsynaptic muscarinic and
nicotinic receptors (Broersen et al., 1995; Hironaka et al., 2001). By
analogy to Alzheimer's disease, reduced activity of ascending
cholinergic projections contributes to cognitive deficits and the
perturbation of mood in parkinsonian patients (Dubois et al., 1986;
Sarter and Bruno, 1998; Perry et al., 1999; Reading et al., 2001).
There is ultrastructural evidence that adrenergic and cholinergic
projections interact at the terminal level in the cortex and limbic
regions (Descarries and Umbriaco, 1995; Li et al., 2001), whereas
adrenergic neurons derived from the locus coeruleus also target
cholinergic perikarya (Smiley et al., 1999; Hajszan and Zaborszky,
2002). Correspondingly, 2-ARs are localized in
the FCX, hippocampus, and cerebral regions containing cholinergic
perikarya (Rosin et al., 1996; Talley et al., 1996). These observations
provide an anatomical substrate for functional interactions among
cholinergic and adrenergic pathways (Cuadra and Giacobini, 1995;
Niitykoski et al., 1997) and for neurochemical evidence that
2-ARs inhibit release of ACh both in the FCX
(Moroni et al., 1983; Tellez et al., 1997) and, according to a recent study (Shirazi-Southall et al., 2002), the hippocampus. However, the
identity of the 2-AR subtype(s) involved has
not been determined and, with few exceptions (Cuadra and Giacobini,
1995; DeBoer and Abercrombie, 1996; Ichikawa et al., 2000, 2002),
dialysis studies have resorted to AChE inhibitors to "boost"
otherwise undetectable basal levels of ACh (Toide and Arima, 1989; Liu
and Kato, 1994; Sarter and Bruno, 1998; Shirazi-Southall et al., 2002).
In light of the above-mentioned observations, we hypothesized that, in
analogy to 2-AR antagonists, piribedil should
reinforce corticolimbic release of ACh in rats. The objectives of this
study were, thus, as follows. First, by use of a procedure not
requiring the use of AChE inhibitors (Ichikawa et al., 2000, 2002), we
characterized the influence of agonists and antagonists possessing
contrasting affinities at 2-AR subtypes (Table
1) upon extracellular levels of ACh in
the FCX of conscious rats. Second, the influence of piribedil upon ACh
levels in FCX was compared with the effects of talipexole and of
quinelorane, the latter a potent dopaminergic agonist lacking affinity
at 2-ARs (Table 1; Millan et al., 2002; Newman-Tancredi et al., 2002a,b). In a parallel experiment, their influence upon extracellular levels of DA in this structure was also
examined. Finally, the influence of piribedil, compared with 2-AR ligands, upon levels of ACh in the dorsal
hippocampus was evaluated.
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Materials and Methods |
Animals.
Male Wistar rats (Iffa Credo, l'Arbresle, France)
of 225 to 250 g were allowed free access to food and water and
housed singly. Laboratory temperature was 21 ± 1°C and humidity
60 ± 5%. There was a 12-h light/dark cycle (lights on at 7:30
AM). All animal use procedures conformed to international European
ethical standards (86/609-EEC) and the French National Committee
(décret 87/848) for the care and use of laboratory animals.
Dialysis Procedure.
Surgery was performed under
pentobarbital anesthesia (60 mg/kg i.p.). As described previously
(Millan et al., 2001), rats were mounted in a Kopf stereotaxic frame
and a single guide cannulae (CMA/11) implanted in the FCX or dorsal
hippocampus with coordinates as follow: AP, +2.2; L, 0.6; DV, 
0.2; or
AP, 3.8; L, 2.0; DV, 2.0, respectively. Rats were single-housed and
allowed to recover for 5 days before dialysis. On the day of dialysis,
a cuprophan CMA/11 probe (4 mm in length for the FCX and 2 mm in length
for the dorsal hippocampus, 0.24 mm o.d.) was slowly lowered into position. It was perfused at 1 µl/min with a phosphate-buffered solution of 147.2 mM NaCl, 4 mM KCl, and 2.3 mM
CaCl2, pH 7.3. Two hours after implantation,
20-min dialysate samples were collected for 3 h. Three basal
samples were collected before drug administration. In the antagonist
studies, RX821002 was injected 20 min before piribedil or
talipexole. The influence of drugs and vehicle was expressed relative
to basal values (defined as 0%).
Chromatographic Procedures.
ACh was quantified in the
absence of AChE inhibitors, essentially as described by Ichikawa et al.
(2000). Twenty-microliter dialysate samples were collected on 10 µl
of 0.01% acetic acid. Twenty-microliter aliquots were then analyzed by
high-performance liquid chromatography. The mobile phase was
composed of 50 mM Na2HPO4
and 0.5% proclin (BAS, Congleton, UK), adjusted to pH 8.2 with
H3PO4. The stationary phase
was comprised of a cation ion exchanger (Sepstik, 530 × 1.0 mm,
particle size 10 µm; BAS), a precolumn (preimmobilized enzyme
reactor, 55 × 1 mm) of choline oxydase/catalase (BAS), and a
postcolumn (postimmobilized enzyme reactor, 50 × 1 mm) of choline
oxydase/AChE (BAS) maintained at 35°C. An amperometric detector
(LC-4B; BAS) was used for quantification. The electrode was set at +100
mV versus Ag/AgCl. The glassy carbon electrode (MF2098, BAS) was coated
with the peroxidase-redox polymer. The mobile phase was delivered at a
flow rate of 0.14 ml/min. The sensitivity of the essay for ACh was 0.1 pg (0.55 fmol) (injected in a volume of 20 µl). DA levels were
quantified by high-performance liquid chromatography followed by
coulometric detection as described previously (Millan et al.,
2001). The assay limit of sensitivity was 0.1 pg/sample. Data were
analyzed by ANOVA with sampling time as the repeated within-subject factor.
Chemicals and Drugs.
All drugs were injected s.c. in a
volume of 1.0 ml/kg. Drugs were dissolved in sterile water plus a few
drops of lactic acid if necessary and the pH adjusted to >5.0.
Guanabenz base, quinelorane 2HCl, 2(2-methoxy-1,4
benzodioxan-2-yl)-2-imidazoline HCl (RX821002), prazosin HCl and
talipexole 2HCl were purchased from Sigma Chemie (Chesnes, France).
Atipamezole HCl, piribedil monomethane sulfonate (Trivastal),
5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline tartrate (UK14,304),
trans-2,3,9,13b-tetrahydro-1,2-dimethyl-1H-dibenz[c,f]imidazo[1,5-a]azepine (BRL41992 maleate), and
2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole (BRL44408 base) were synthesized by Servier (Institut de Recherches Servier, Paris, France) chemists.
| |
Results |
Influence of 2-AR Agonists and Antagonists upon
Dialysis Levels of ACh in the FCX of Freely Moving Rats.
In the
absence of AChE inhibitors, basal dialysate levels of ACh were
2.18 ± 0.38 pg/20 µl (12 ± 2 fmol/20 µl) (Fig.
1). As shown in Fig.
2, the injection of vehicle (1 ml/kg)
induced a significant, although modest and transient (20 min), increase in extracellular levels of ACh in the FCX. The
2-AR receptor agonist UK14,304 induced a
pronounced and dose-dependent (0.16-2.5 mg/kg s.c.) decrease (maximal
effect, 82 ± 3% versus basal values) in ACh levels (Fig. 2),
an action mimicked by the preferential 2A-AR
agonist guanabenz (0.16-10.0 mg/kg s.c.) (maximal effect, 69 ± 3% versus basal values), although with a less sustained duration of
action (Fig. 3). In contrast, the
selective 2-AR antagonists atipamezole
(0.63-630 µg/kg s.c.) and RX821002 (0.01-2.5 mg/kg s.c.), dose
dependently elevated levels of ACh, with peak effects of +168 ± 26 and +130 ± 40%, respectively (Fig. 2). Likewise, the
selective 2A-AR antagonist BRL44408 (2.5-40.0
mg/kg s.c.) markedly elevated levels of ACh (maximal effect, +115 ± 15% versus basal values). In contrast, BRL41992 (10.0 mg/kg s.c.),
a preferential 2B/2C-AR antagonist, and
prazosin (10.0 mg/kg s.c.), a preferential antagonist at
2B/2C-ARs (and a potent
1-AR antagonist), were inactive (Fig. 3).
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Fig. 1.
Chromatogram showing identification and
quantification of acetylcholine. A, 20 µl of standards (1, 5, and 10 pg) of ACh were injected onto the column using chromatographic
conditions as described under Materials and Methods. The
retention time of ACh was 7.8 min. B, 20 µl of a 20-µl basal
microdialysis sample plus 10 µl of acetic acid 0.01% was injected
onto the column. In a representative, basal, frontocortical dialysate
sample, the quantity of ACh was 1.9 pg. The administration of piribedil
(10.0 mg/kg s.c.) increased FCX dialysate levels of ACh to a peak of
ACh of 5.9 pg.
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Fig. 2.
Influence of the 2-AR agonist UK14,304
and of the 2-AR antagonists atipamezole and RX821002
upon dialysis levels of acetylcholine in the frontal cortex of freely
moving rats. A, UK14,304. B, atipamezole. and C, RX821002. Data are
means ± S.E.M. In the frontal cortex, basal levels of ACh were
2.18 ± 0.38 pg/20 µl. ANOVA data are as follows: UK14,304
(0.16; n = 5) F(1,9) = 2.9, P > 0.05; UK14,304 (0.63; n = 5) F(1,9) = 5.2, P < 0.05;
UK14,304 (0.63; n = 5) F(1,9) = 59.4, P < 0.01; atipamezole (0.00063;
n = 5) F(1,9) = 0.3, P > 0.05; atipamezole (0.01; n = 6) F(1,10) = 2.7, P > 0.05;
atipamezole (0.16; n = 6)
F(1,10) = 19.8, P < 0.01;
atipamezole (0.63; n = 6)
F(1,10) = 27.6, P < 0.01;
RX821002 (0.01; n = 5) F(1,9) = 0.1, P > 0.05; RX821002 (0.04;
n = 5) F(1,9) = 6.4, P < 0.05; RX821002 (0.16; n = 6) F(1,10) = 45.9, P < 0.01;
and RX821002 (2.5; n = 6)
F(1,10) = 5.2, P < 0.05. Asterisks indicate significance of drug-treated versus vehicle-treated
(n = 6) values. *, P < 0.05.
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Fig. 3.
Influence of the 2A-AR agonist
guanabenz, the 2A-AR antagonist BRL44408, and the
preferential 2B/2C-AR antagonists BRL41992 and prazosin
upon dialysis levels of acetylcholine in the frontal cortex of freely
moving rats. A, guanabenz. B, BRL44408. C, prazosin. Data are
means ± S.E.M. ANOVA data are as follows: guanabenz (0.16;
n = 7) F(1,11) = 0.4, P > 0.05; guanabenz (2.5; n = 6) F(1,10) = 11.9, P < 0.01;
guanabenz (10.0; n = 5) F(1,9) = 43.4, P < 0.01; BRL44408 (2.5;
n = 5) F(1,9) = 0.6, P > 0.05; BRL44408 (10.0; n = 6) F(1,10) = 5.7, P < 0.05;
BRL44408 (40.0; n = 7) F(1,11) = 28.3, P < 0.01; prazosin (10.0;
n = 6) F(1,10) = 0.3, P > 0.05; and BRL41992 (10.0;
n = 5) F(1,9) = 0.1, P > 0.05. Asterisks indicate significance of
drug-treated versus vehicle-treated (n = 6) values.
*, P < 0.05.
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Influence of Single Doses of Piribedil, Talipexole, and Quinelorane
upon Dialysis Levels of DA Compared with ACh in the FCX of Freely
Moving Rats.
In an initial study, we examined the influence
of single, equieffective doses of piribedil, talipexole, and
quinelorane upon dialysis levels of DA in FCX. Reflecting their agonist
properties at D2/D3
autoreceptors (Millan et al., 2000), they all elicited marked and
significant decreases in frontocortical levels of DA with comparable
maximal effects of 49 ± 9, 55 ± 10, and 50 ± 8% versus basal values, respectively (Fig.
4). At these equivalent doses, it can be
seen from Fig. 4 that piribedil elicited a pronounced and significant
elevation in ACh levels in FCX, whereas talipexole, in an opposite
manner, reduced levels of ACh; quinelorane did not significantly
modify ACh levels. Thus, despite a common, suppressive influence upon
DA levels, piribedil, talipexole, and quinelorane differentially
modified extracellular levels of ACh in FCX.
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Fig. 4.
Influence of piribedil, talipexole, and quinelorane
upon dialysis levels of dopamine compared with acetylcholine levels in
the frontal cortex of freely moving rats. A and D, piribedil. B and E,
talipexole. C and F, quinelorane. Frontocortical basal levels of
dopamine were 1.1 ± 0.3 pg/20 µl (right). Data are means ± S.E.M. For DA, ANOVA data are as follows: piribedil (5.0;
n = 5) F(1,8) = 34.4, P < 0.01; talipexole (2.5; n = 6) F(1,9) = 32.7, P < 0.01;
and quinelorane (0.16; n = 6)
F(1,9) = 70.8, P < 0.01. Asterisks indicate significance of drug-treated versus vehicle-treated
(n = 5) values. *, P < 0.05. For ACh, piribedil (5.0; n = 5)
F(1,9) = 9.2, P < 0.05;
talipexole (2.5; n = 6) F(1,10) = 24.0, P < 0.01; and quinelorane (0.16;
n = 6) F(1,10) = 0.8, P > 0.05. Asterisks indicate significance of
drug-treated versus vehicle-treated (n = 6) values.
*, P < 0.05.
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Dose-Dependent Influence of Piribedil Compared with
Talipexole upon Dialysis Levels of ACh in the FCX of Freely Moving
Rats.
In subsequent studies, it was found that piribedil elicited
a dose-dependent (0.63-40.0 mg/kg s.c.), pronounced, and sustained increase in dialysis levels of ACh (maximal effect, +219 ± 24% versus basal values) (Fig. 5). In
distinction, talipexole provoked a dose-dependent (0.63-10.0 mg/kg
s.c.) reduction in extracellular levels of ACh (maximal effect,
79 ± 5% versus basal values) (Fig. 5). After pretreatment with
a maximally effective dose of RX821002 (2.5 mg/kg s.c.), piribedil
(10.0 mg/kg s.c.) failed to significantly modify levels of ACh. This
lack of "additive" or "synergistic" effects indicates that they
act at a common site. The inhibitory influence of talipexole (10.0 mg/kg s.c.) upon ACh levels was further "canceled out" by
pretreatment with RX821002 (Fig. 5).
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Fig. 5.
Influence of piribedil compared with talipexole upon
dialysis levels of acetylcholine in the frontal cortex of freely moving
rats: dose-response relationships and influence of pretreatment with
RX821002. A, piribedil. B, talipexole. C, piribedil after pretreatment
with RX821002. D, talipexole after pretreatment with RX821002. Data are
means ± S.E.M. ANOVA data are as follows: A and B, piribedil
(0.63; n = 5) F(1,9) = 0.1, P > 0.05; piribedil (2.5; n = 5) F(1,9) = 10.7, P < 0.01;
piribedil (10.0; n = 7) F(1,11) = 6.6, P < 0.05; piribedil (40.0;
n = 5) F(1,9) = 86.8, P < 0.01; talipexole (0.63; n = 5) F(1,9) = 2.0, P > 0.05;
and talipexole (10.0; n = 6)
F(1,10) = 90.6, P < 0.01. Asterisks indicate significance of drug-treated versus vehicle-treated
(n = 6) values. *, P < 0.05. C and D, influence of RX821002 (n = 8)
F(1,12) = 9.9, P < 0.01;
influence of piribedil (n = 6)
F(1,10) = 32.8, P < 0.01; and
interaction (n = 8) F(1,12) = 0.1, P > 0.05. Influence of RX821002
(n = 8) F(1,12) = 9.9, P < 0.01; influence of talipexole
(n = 6) F(1,10) = 73.7, P < 0.01; and interaction (n = 5) F(1,9) = 31.7, P < 0.01. Asterisks indicate significance of drug-treated versus
vehicle/vehicle-treated (n = 6) values. *,
P < 0.05.
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Influence of Piribedil Compared with RX821002 and UK14,304 upon
Dialysis Levels of ACh in the Dorsal Hippocampus of Freely Moving
Rats.
Whereas the 2-AR agonist UK14,304
(2.5 mg/kg s.c.) markedly suppressed dialysis levels of ACh in dorsal
hippocampus, they were elevated by the 2-AR
antagonist RX821002 (2.5 mg/kg s.c.) (maximal effects, 71.9 ± 6.7 and + 122.0 ± 27.0% versus basal values, respectively) (Fig.
6). In analogy to the FCX, piribedil elicited a dose-dependent (2.5-40.0 mg/kg s.c.) and sustained increase
in dialysis levels of ACh (maximal effect, +126.7 ± 33.0% versus
basal values) (Fig. 6).
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Fig. 6.
Influence of piribedil compared with RX821002 and
UK14,304 upon dialysis levels of acetylcholine in the dorsal
hippocampus of freely moving rats. A, piribedil. B, RX821002. C,
UK14,304. Data are means ± S.E.M. In the dorsal hippocampus,
basal levels of ACh were 1.24 ± 0.14 pg/20 µl. ANOVA data are
as follows: piribedil (2.5; n = 5)
F(1,11) = 2.9, P > 0.05;
piribedil (5.0; n = 6) F(1,12) = 5.1, P < 0.05; piribedil (10.0;
n = 6) F(1,12) = 22.7, P < 0.01; piribedil (40.0; n = 5) F(1,11) = 21.7, P < 0.01;
RX821002 (2.5; n = 5) F(1,11) = 34.0, P < 0.01; and UK14,304 (2.5;
n = 6) F(1,12) = 21.7, P < 0.01. Asterisks indicate significance of
drug-treated versus vehicle-treated (n = 8) values.
*, P < 0.05.
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Discussion |
Technical Considerations: Muscarinic Modulation of Frontocortical
Release of ACh.
Owing to the high capacity and rapid kinetics of
AChE, extracellular levels of ACh are greatly (~1000-fold) exceeded
by those of its metabolite, choline. This renders detection of
extracellular levels of ACh difficult and has necessitated addition of
AChE inhibitors to dialysis perfusates. In contrast, corroborating the
work of Ichikawa et al. (2000, 2002), introduction of a supplementary, choline oxydase-loaded, "enzyme-immobilized" column before the "analytical" column eliminated choline from the chromatogram; thus,
the fidelity and sensitivity of ACh detection was substantially improved. Accordingly, even "resting" levels of ACh could be
reproducibly quantified and values of 2.18 ± 0.38 pg/20 µl
(15.0 ± 2.6 fmol/20 µl) correspond well to those of Ichikawa et
al. (2000, 2002) (19.5 ± 0.7 fmol/20 µl). They are considerably
(>20-fold) lower than "basal" levels generated in the presence of
AChE inhibitors (Cuadra and Giacobini, 1995; Tellez et al., 1997).
Furthermore, the AChE inhibitor, eserine, increased ACh levels by
~7-fold (A. Gobert and M. J. Millan, unpublished observation)
in line with its pronounced increase in ACh levels upon local perfusion
(Ichikawa et al., 2002). In an extension of the work of Ichikawa et al.
(2002), we demonstrate herein that this technique also permits the
reliable detection and quantitation of ACh levels in dorsal
hippocampus. In this structure, basal levels of ACh herein, 1.30 ± 0.16 pg/20 µl (9.0 ± 1.0 fmol/20 µl), were substantially
lower than those documented using AChE inhibitors (e.g., 860 fmol/36
µl with 0.3 µM neostigmine; Shirazi-Southall et al., 2002).
Quantification of ACh levels in the absence of AChE inhibitors avoids
potentially misleading effects due to pharmacological or metabolic
interactions with the drug under study (DeBoer and Abercrombie, 1996;
Ichikawa et al., 2000, 2002). Furthermore, inasmuch as ACh exerts a
tonic, inhibitory feedback upon its own release via muscarinic
autoreceptors (Zhang et al., 2002), an elevation in its levels by
inhibition of AChE directly modifies actions of agonists and
antagonists at these sites (Toide and Arima, 1989; Liu and Kato, 1994;
Ichikawa et al., 2002). In addition, for all drug classes, the apparent
magnitude of their actions relative to basal values will be
distorted by the use of AChE inhibitors.
By analogy to Ichikawa et al. (2000), in vehicle-treated rats, levels
of ACh in FCX were transiently increased relative to basal values.
Similarly, levels of ACh in dorsal hippocampus displayed a short-lived
increase upon vehicle injection (Shirazi-Southall et al., 2002). These
responses reflect arousal and cognitive-attentional factors associated
with handling and motor activity (Sarter and Bruno, 2000; Giovannini et
al., 2001; Hironaka et al., 2001).
2-AR Modulation of Frontocortical Release of
ACh.
The finding that the 2-AR agonist
UK14,304 and the 2-AR antagonists atipamezole
and RX821002, respectively, suppressed and enhanced frontocortical ACh
release demonstrates that 2-ARs exert a tonic,
inhibitory influence upon ACh release in the FCX of conscious rats.
This observation amplifies findings of in vitro studies (Williams and
Reiner, 1993) and in vivo studies using AChE inhibitors (Moroni et al.,
1983; Tellez et al., 1997). Furthermore, ACh release was reduced by the
preferential 2A-AR agonist guanabenz and
accelerated by the selective 2A-AR antagonist
BRL44408 (Young et al., 1989; Renouard et al., 1994), suggesting a role
for the 2A-AR subtype in this effect. Indeed,
prazosin, which displays higher affinity at
2B/2C- versus 2A-ARs
(Renouard et al., 1994), did not modify ACh levels, in line with a
study of Acquas et al. (1998). This observation was underpinned by the
lack of effect of a further preferential antagonist at
2B/2C- versus 2A-ARs,
BRL41992 (Young et al., 1989), upon ACh levels. Notably, RX821002 does
not interact with imidazoline receptors, which cannot, therefore, be
implicated in its induction of ACh release. This pattern of effects
resembles studies of frontocortical release of noradrenaline and DA and suggests that 2A-ARs are inhibitory to ACh
release (Kable et al., 2000; Millan et al., 2000), consistent with
their high density in the FCX and localization on cholinergic cell
bodies (Zaborszky et al., 1995; Talley et al., 1996). The doses of
BRL44408 used herein were shown to block
2A-ARs in previous investigations including,
for example, the modulation of frontocortical release of DA and
noradrenaline under conditions analogous to the present study of ACh
release (Millan et al., 1994; Gobert et al., 1998). Furthermore, the
preferential 2B/2C-AR antagonists BRL41992 and prazosin were used herein at doses previously demonstrated to not block
2A-ARs (Millan et al., 1994; Gobert et al.,
1998). However, there is no currently well defined functional model of the role of cerebral 2B- and/or
2C-ARs appropriate to the precise definition
of their active dose ranges at these sites. Thus, it is necessary to be
cautious as regards the apparent exclusion of a role of
2B- and/or 2C-ARs in
the modulation of ACh release. Indeed, in would be of interest to
undertake complementary studies in genetically transformed mice lacking
(or overexpressing) specific subtypes of 2-AR
to corroborate the present observations. Such an approach indicated
that 2C-ARs also, albeit to a minor degree relative to their 2A-AR counterparts, modulate
cerebral monoaminergic transmission (Kable et al., 2000; Bücheler
et al., 2002).
Facilitatory Influence of Piribedil upon Frontocortical Levels of
ACh.
Piribedil, which displays marked antagonist properties at
2A- and 2C-ARs
(Millan et al., 2001, 2002; Newman-Tancredi et al., 2002a), provoked a
rapid, dose-dependent and sustained increase in extracellular levels of
ACh in FCX. There are several possible explanations for this finding.
First, piribedil might interact directly with muscarinic mechanisms.
However, it shows negligible affinity for cloned human M2 receptors, other (M1,
M3, and M4) muscarinic
sites, and for AChE (M. J. Millan, unpublished observation). On
structural grounds, it is unlikely that metabolites of piribedil would
interact with muscarinic mechanisms: in line with this contention,
piribedil does not modify muscarinic responses in vivo (M. J. Millan, unpublished observation). Second, a role of
D2 and/or D3 receptors
might be evoked. However,
D2/D3 agonists, such as
quinpirole, did not increase ACh release in FCX in a previous study
(Day and Fibiger, 1993). Accordingly, the potent
D2/D3 agonist quinelorane,
which is devoid of affinity for 2-ARs, failed
to modify dialysis levels of ACh and several other selective
D2/D3 agonists also do not
enhance ACh levels (A. Gobert, unpublished observation). Furthermore, this hypothesis cannot accommodate the opposite facilitatory and inhibitory influence of piribedil and talipexole upon ACh levels, respectively, despite their mutual agonist properties at
D2/D3 receptors. Indeed, at
doses that elicited an equivalent reduction in FCX release of DA
(reflecting activation of
D2/D3 autoreceptors), piribedil, talipexole, and quinelorane exerted contrasting influences (increase, decrease, and no change, respectively) upon dialysis levels
of ACh (Fig. 4). Third, the weak antagonist properties of piribedil at
1-ARs (Millan et al., 2002; Newman-Tancredi et al., 2002a) are unlikely to be implicated because they are shared by
talipexole, whereas prazosin (a potent 1-AR
antagonist) did not enhance ACh release in FCX.
Thus, in line with above-discussed evidence for a tonic, inhibitory
influence of 2-AR heteroceptors upon
frontocortical cholinergic transmission, the induction of ACh release
in FCX by piribedil likely reflects its antagonist properties at
2-ARs. This interpretation accounts for the
opposite suppressive influence of talipexole, an agonist at
2-ARs (Millan et al., 2002; Newman-Tancredi et al., 2002a), upon ACh levels. Moreover, in the presence of a maximally effective dose of RX821002, piribedil failed to elevate ACh levels, indicating a common site of action, whereas the inhibitory influence of
talipexole was canceled out by pretreatment with RX821002. Further
supporting a role of 2-ARs, the dose range of
piribedil that elevated FCX levels of ACh was identical to that which
augments frontocortical levels of noradrenaline by blockade of
2A-ARs (Millan et al., 2001). Although
blockade of the 2A-AR subtype likely
participates in the influence of piribedil upon ACh release (vide
supra), this issue remains to be directly addressed. Moreover, inasmuch
as 2-ARs inhibit ACh release at both the
cortical and dendritic level (Moroni et al., 1983; Bertorelli et al.,
1991), the precise locus(i) of action of piribedil will require future evaluation.
Facilitatory Influence of Piribedil upon Dorsal Hippocampus Levels
of ACh.
Although the 2-AR antagonist
yohimbine increased extracellular levels of ACh in the ventral
hippocampus of rats, it is poorly selective for
2-ARs (Millan et al., 2000) and that study
used AChE inhibitors in the dialysate perfusate (Shirazi-Southall et al., 2002). It is thus of interest that using the present procedure, UK14,304 and RX821002, respectively, decreased and enhanced
extracellular levels of ACh in the dorsal hippocampus. This observation
provides further evidence for a tonic, inhibitory influence of
2-ARs upon ACh release in the dorsal
hippocampus, a structure in which their density is particularly high
(Talley et al., 1996). Correspondingly, reflecting its antagonist
properties at 2-ARs, piribedil dose dependently elevated dialysis levels of ACh in the dorsal hippocampus, a finding paralleling its actions in the FCX.
General Considerations.
First, the present study exploited a
technique developed by Ichikawa et al. (2000, 2002) in freely moving
rats that does not require systemic or local administration of drugs to
artificially elevate basal values of ACh. This strategy, analogous to
that used for evaluation of extracellular levels of monoamines (Gobert et al., 1998; Millan et al., 2000), should prove invaluable in the
characterization of the modulation of cerebral cholinergic transmission
by psychotropic agents.
Second, piribedil, via its distinctive antagonist properties at
2-ARs (Millan et al., 2001, 2002), reinforced
frontocortical and hippocampal cholinergic transmission. This action
may well contribute to its enhancement of cognitive-attentional
function (Maurin et al., 2001; Nagaraja and Jayashree, 2001; Smith et
al., 2002). Indeed, although behavioral studies are required to
underpin this contention, there is preliminary evidence that AChE
inhibitors exert a favorable influence upon cognitive function in
parkinsonian patients (Reading et al., 2001). Inasmuch as piribedil
(like other 2-AR antagonists) also enhances
noradrenaline release in FCX (Millan et al., 2001), the relative
contribution of cholinergic versus adrenergic mechanisms to its
influence upon cognitive-attentional function will be of interest to evaluate.
Third, frontocortical cholinergic pathways also influence motor
function, anxiety, sleep and mood (Perry et al., 1999; Sarter and
Bruno, 2000; Giovannini et al., 2001; Ichikawa et al., 2002). Thus, a
broader exploration of the functional significance of an increase in
FCX release of ACh to the management of PD would be justified. Notably,
deficits in cholinergic (frontocortical and pedunculopontine)
transmission are implicated in the perturbation of sleep and
hallucinations experienced by parkinsonian patients (Perry et al.,
1999; Sarter and Bruno, 2000). Furthermore, AChE inhibitors have
been reported to ameliorate psychotic symptoms in patients in PD
(Reading et al., 2001; Bergman and Lerner, 2002).
Finally, although their pronounced side effects (including disruption
of sleep and induction of psychosis and cognitive deficits; c.f.,
paragraphs above) greatly limit their use, muscarinic antagonists have
been used in the treatment of PD, principally in the management of
refractory tremor and severe L-DOPA-induced dyskinesias
(Hurtig, 1997; Wilms et al., 1999; Jenner, 2000; Singer, 2002). These
actions do not reflect their blockade of autoreceptors (thereby
enhancing ACh release), rather antagonism of postsynaptic sites in the
striatum. Furthermore, D2 receptors exert an
inhibitory influence upon ACh release in the striatum (Di Chiara et
al., 1994; DeBoer and Abercrombie, 1996). In the light of these
comments, an interesting question concerns the influence of piribedil
compared with other agents upon the striatal release of ACh. In fact,
there is no evidence for a role of 2-ARs in
the control of striatal cholinergic transmission, so its influence upon
ACh release therein should not, in principle, differ from those of
talipexole, quinelorane, or other agents. This remains to be directly
demonstrated. In any case, notwithstanding possible benefits of
increased corticolimbic release of ACh in the control of
cognitive-attentional function (vide supra), such actions would not be
expected to markedly modify the motor symptoms of PD per se.
Conclusions.
Using an innovative dialysis approach not
requiring use of AChE inhibitors, the present study demonstrates that
the antiparkinson agent piribedil, which possesses marked antagonist
properties at 2-ARs, markedly enhances release
of ACh in the FCX and dorsal hippocampus of freely moving rats. These
actions may be distinguished to the inhibitory influence of talipexole,
which acts as an agonist at 2-ARs, and to the
lack of effect of quinelorane, which does not interact with
2-ARs. A reinforcement of frontocortical
cholinergic transmission may contribute to the facilitatory influence
of piribedil upon cognitive-attentional function, which is compromised
in PD, although it would not be expected to modify motor performance per se. Thus, the present data encourage additional neurochemical, behavioral, and clinical studies of the functional significance of
cholinergic transmission and its modulation by
2-ARs to the etiology and management of PD.
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2A-Adrenoceptors: A Dialysis Comparison to Talipexole
and Quinelorane in the Absence of Acetylcholinesterase Inhibitors
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Abstract
Introduction
-aminobutyric acid release in freely moving guinea pigs: effects of clonidine and other adrenergic drugs.
J Pharmacol Exp Ther
277:
435-440.
