Introduction

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₂-Adrenoceptor (AR) autoreceptors are highly expressed in adrenergic neurons (Talley et al., 1996; Nicholas et al., 1997; Lee et al., 1998). These ₂-AR autoreceptors exert a tonic inhibitory control on adrenergic transmission, and both pharmacological approaches and studies of mice displaying null mutations for ₂-AR subtypes (Millan et al., 1994; Lakhlani et al., 1997; Gobert et al., 1998; Mateo and Meana 1999; Trendelenburg et al., 1999; Kable et al., 2000) suggest a principal role of _2A-ARs in this regard. However, locus ceruleus (LC)-localized adrenergic neurons also bear _2C-ARs, which may fulfill a complementary (minor) role in the modulation of norepinephrine (NE) release (Sallinen et al., 1997; Arima et al., 1998; Lee et al., 1998; Trendelenburg et al., 1999).

Actions of ₂-AR ligands at autoreceptors fulfill an important role in their functional properties. First, activation of ₂-AR autoreceptors in the brainstem, in the intermediolateral cell column of the spinal cord and on peripheral, sympathetic terminals underlies hypotensive and bradycardiac effects of ₂-AR agonists, actions in which _2A-ARs are principally involved, although spinal and peripheral (sympathetic) populations of _2C-AR may also be implicated (Nicholas et al., 1997; Trendelenburg et al., 1999; see Kable et al., 2000). Second, although actions integrated by ventral horn motoneurons mediate muscle-relaxant properties of ₂-AR agonists (Coward, 1994; Nicholas et al., 1997), the principle mechanism underlying the sedative/hypnotic (sleep-inducing/anesthetic) actions of ₂-AR agonists is activation of LC-localized ₂-AR autoreceptors (Millan et al., 1994; Hayashi et al., 1995). In line with evidence for a role of _2A-ARs in the modulation of adrenergic transmission, pharmacological, antisense, and gene knockout studies indicate that _2A-ARs are involved in sedative/hypnotic actions of ₂-AR agonists (Millan et al., 1994; Hunter et al., 1997; Lakhlani et al., 1997; Robinson et al., 1999; see Kable et al., 2000). Third, LC-derived adrenergic pathways modulate anxious states (Charney et al., 1995; Cole et al., 1995). Correspondingly, there is evidence for anxiolytic and anxiogenic actions of ₂-AR agonists and antagonists, respectively, although data remain fragmentary (see Cole et al., 1995). Furthermore, the role of ₂-AR subtypes has not, to date, been examined.

₂-ARs also exert an inhibitory influence on ascending serotonergic pathways. This effect partly reflects activation of ₂-AR autoreceptors, thereby suppressing a tonic, excitatory influence of ₁-ARs on serotonergic perikarya in raphe nuclei (Haddjeri et al., 1995; Millan et al., 2000b). In addition, inhibitory ₂-heteroceptors are localized on terminals of serotonergic neurons in corticolimbic structures (Haddjeri et al., 1995; Gobert et al., 1998; see Millan et al., 2000b). A primary role of _2A-ARs is supported by pharmacological (Trendelenburg et al., 1994; Gobert et al., 1998), gene knockout (Sallinen et al., 1997), and neuroanatomical (Talley et al., 1996; Wang et al., 1996; Nicholas et al., 1997) studies. This inhibitory influence of ₂-AR agonists on serotonergic pathways contributes to sedative (Rabin et al., 1996) and potential anxiolytic (see Discussion) actions.

Frontocortical dopaminergic pathways are likewise subject to inhibitory control by ₂-ARs (Gobert et al., 1998). The density of _2A-ARs in ventrotegmental dopaminergic cell bodies projecting to the frontal cortex (FCX) is low, whereas this nucleus possesses a significant density of _2C-ARs in rats (Talley et al., 1996; Lee et al., 1998) but not in mice (Wang et al., 1996). However, ₂-AR-mediated inhibition of mesocortical dopaminergic projections is expressed principally (either directly and/or indirectly) at the level of terminals in the FCX (Grenhoff and Svensson, 1989; Gobert et al., 1998; Matsumoto et al., 1998; Hertel et al., 1999). Therein, both _2A-ARs and (less densely) _2C-ARs are present (Talley et al., 1996; Nicholas et al., 1997), and pharmacological studies suggest a role for the former in modulation of frontocortical dopamine (DA) release (Gobert et al., 1998; see Millan et al., 2000b). Suppression of mesocortical dopaminergic transmission by ₂-AR agonists may be relevant to their modulation of anxious states (Morrow et al., 1999)

The novel spiroimidazoline, S18616 [accompanying article (Millan et al., 2000a)], is an exceptionally potent agonist of native rat ₂-AR (pK_i, 9.8) and cloned human _2A-AR (9.6), _2B-AR (9.6), and _2C-AR (9.5), which display modest affinity at native rat ₁- (7.1), and cloned, human _1A-AR (8.4), _1B-AR (7.7), and _1D-AR (7.6). Furthermore, it is highly selective (>100-fold) versus all other sites (>50) examined. Reflecting engagement of postsynaptic ₂-ARs, S18616 exhibits hypothermic and antinociceptive properties in rodents. In light of these data and the above discussion, the purpose of the studies described herein was 2-fold. First, using an electrophysiological and neurochemical approach, to characterize the influence of S18616 on corticolimbic adrenergic, serotonergic, and (frontocortical) dopaminergic pathways. Second, in parallel, to examine its influence on motor function and its potential anxiolytic properties. Actions of S18616 were compared with those of the potent ₂-AR agonist, dexmedetomidine, and the prototypical partial agonist, clonidine (Bucaffusco, 1992; Aantaa et al., 1993). As in the preceding article (Millan et al., 2000a), it was important to establish the role of ₂- versus ₁-ARs in the functional actions of S18616. To this end, we used several antagonists selective for ₂- versus ₁-ARs: atipamezole, idazoxan, and BRL-44408 (Aantaa et al., 1993; Renouard et al., 1994; Haapalinna et al., 1997). Their actions were compared with those of the selective ₁-AR antagonist, prazosin. The use of these ligands does not permit precise definition of the potential roles of ₂-AR subtypes in the actions of S18616 (itself subtype nonselective). Nevertheless, comparisons between BRL-44408 and prazosin are of interest inasmuch as the former is a preferential antagonist at _2A- versus _2B/2C-ARs, whereas prazosin shows preferential activity at _2B/2C- versus _2A-ARs (Young et al., 1989; Renouard et al., 1994).

	Materials and Methods

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Animals. Unless otherwise specified, these studies used male Wistar rats of 200 to 250 g and Naval Medical Research Institute (NMRI) mice of 22 to 25 g (Iffa Credo, l'Arbresles, France) housed in sawdust-lined cages with unrestricted access to standard chow and water. There was a 12-h/12-h light/dark cycle with lights on at 7:30 AM. Laboratory temperature and humidity were 21 ± 0.5°C and 60 ± 5%, respectively. Animals were adapted to laboratory conditions for at least 1 week before testing. 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.

Influence on the Electrical Activity of Adrenergic, Serotonergic, and Dopaminergic Cell Bodies. The influence of S18616 compared with dexmedetomidine and clonidine on the firing rate of LC-localized adrenergic perikarya, dorsal raphe nucleus (DRN)-localized serotonergic perikarya, and ventral tegmental area (VTA)-localized dopaminergic perikarya was determined using a procedure described in detail previously (Millan et al., 2000b,c). Briefly, after anesthesia with chloral hydrate (400 mg/kg, i.p.), rats were placed in a stereotaxic apparatus, and a tungsten microelectrode was lowered into the LC, DRN, or VTA. Coordinates were as follows. VTA, AP = 5.5 from bregma, L = 0.7 and DV = 7/8.5 from dura. LC, AP = 1.2 from zero, L = 1.2 and DV = 5.5/6.5 from dura. DRN, AP = 7.8 from bregma, L = 0.0 and DV = 5.0/6.5 from dura. As detailed elsewhere (Millan et al., 2000b,c), neurons in each structure are characterized by their distinctive wave form and their discharge rhythm. They are, thus, highly likely to be adrenergic, serotonergic, and dopaminergic cell bodies for the LC, DRN, and VTA, respectively. After baseline recording (5 min), vehicle, S18616, dexmedetomidine, or clonidine were administered i.v. (in a volume of 0.5 ml/kg) in cumulative doses every 2 to 3 min. Subsequent to vehicle or agonist administration, a further injection of idazoxan (63 µg/kg, i.v.) was made. Drug effects were quantified over the 60-s bin corresponding to their time of peak action. Spike2 software (CED, Cambridge, England) was used for data acquisition and analysis. Data are expressed as percentage change from the baseline firing rate (defined as 0%). Data were analyzed by two-way ANOVA followed by Newman-Keuls test for paired data, and the ID₅₀ values [95% confidence limits (CL)] were calculated.

Influence on Extracellular Levels of NE, DA, and 5-HT. Quantification of extracellular levels of NE, serotonin (5-HT), and DA in single dialysate samples of the FCX and hippocampus (NE and 5-HT) was achieved using a protocol extensively described previously (Gobert et al., 1998; Millan et al., 2000b,c). The guide cannula, CMA11, was implanted 1 week before experimentation under pentobarbital anesthesia (60.0 mg/kg, i.p.) at the following coordinates. FCX, AP = +2.2 from bregma, L = ±0.6 and DV = 0.2 from dura. Hippocampus: AP = 3.6 from bregma, L = ±1.2 and DV = 2.3 from dura. A cuprophane CMA/11 probe (4 mm in length for the FCX and 2 mm in length for the hippocampus, and 0.24 mm of outer diameter) was lowered into position, and three basal samples of 20 min each were taken. S18616, dexmedetomidine, clonidine, or vehicle was administered, and samples were taken for a further 3 h. In the FCX antagonism experiments, atipamezole, BRL-44408, prazosin, or vehicle was injected followed, 20 min later, by S18616 (0.0025 mg/kg, s.c.) or vehicle. NE, 5-HT, and DA levels were quantified by HPLC followed by coulometric detection as detailed previously (Gobert et al., 1998). The assay limit of sensitivity was 0.1 to 0.2 pg/sample for NE, 5-HT, and DA in each case. Data were analyzed by ANOVA with sampling time as the repeated within-subject factor. For the decrease in NE levels, because this was 100% (see Results), ID₅₀ values (95% CL) were calculated.

Influence on Cerebral Synthesis of NE, DA, and 5-HT. Using a procedure detailed previously (Millan et al., 2000c), NE synthesis was determined in the hippocampus, and 5-HT synthesis was determined in hippocampus, FCX, striatum, and hypothalamus. The influence of S18616, dexmedetomidine, clonidine, and vehicle was evaluated 60 min after their administration and 30 min after injection of the decarboxylase inhibitor, NSD-1015 (100 mg/kg, s.c.). Tissue levels of L-dihydroxyphenylalanine (L-DOPA) and 5-hydroxytryptophan (5-HTP) were determined by HPLC and electrochemical detection as done previously (Millan et al., 2000c). The influence of drugs on levels of L-DOPA and 5-HTP was expressed relative to those of vehicle values (defined as 0%). Data were analyzed by ANOVA followed by Dunnett's test.

Influence on Ultrasonic Vocalizations in Rats. There were three different experimental phases performed at intervals of 24 h. On day 1 (training), rats were placed in a chamber equipped with a grid floor and were exposed to six randomly distributed electric shocks (800 µA and 8 s) over a 7-min period. On day 2 (selection), they were placed in the chamber for 2 min and received a single shock. They were returned to the chamber 30 min later, and ultrasonic vocalizations were recorded for 10 min. Only rats emitting ultrasonic vocalizations for a total duration of at least 90 s were examined further. On day 3, in dose-response studies, the procedure was identical to day 2, but rats were treated with S18616 or vehicle immediately after the 2-min session. In antagonism studies, 60 min was allowed between the 2-min period and the 10-min test session. Antagonists or vehicle were administrated just after the 2-min period and S18616 or vehicle 30 min later. The total duration of ultrasonic vocalizations was recorded over the 10-min session. The dose effect was analyzed by ANOVA followed by Dunnett's test, and ID₅₀ values (95% CL) were calculated. In the antagonism study, data were analyzed by a two-way ANOVA, followed by Newman-Keuls test.

Action in the Vogel Conflict Test in Rats. The test was conducted in polycarbonate cages (32 × 25 × 30 cm) possessing a grid floor with the spout of a water bottle located 6 cm above the floor. Both the grid and the spout were connected to an Anxiometer (Columbus Instruments, OH) used to record licks and to deliver electrical shocks. During the 3 days preceding testing, rats were housed by four and were restricted to 1 h/day access to tap water (from 9:00 to 10:00 AM). On day 4, just after water delivery, they were isolated in cages with a grid floor. Testing took place on day 5. Each rat was placed in the test cage, and the session was initiated after the animal had made 20 licks and received a first, mild shock (a single, 0.5-s constant current pulse of 0.3-mA intensity) through the spout. Thereafter, a shock was delivered to the animal every 20th lick during a period of 3 min. Animals that did not initiate the session within 5 min were removed. Data were the number of licks emitted by the animal during the 3-min session. Certain control (vehicle) animals did not receive shocks during the session and were used to evaluate free drinking behavior. Drugs were given 30 min before testing. Dose-effects were analyzed using one-way ANOVA followed by Dunnett's test (nonshocked animals were not included in the analysis). The percentage of drug effect was computed as [(drug  vehicle)/(vehicle nonshocked  vehicle)].

Influence on Behavior in the Elevated Plus-Maze Test in Rats. The experiments were performed in a white-mat-painted, plus-maze constructed of wood and elevated to a height of 50 cm. The apparatus comprised two open arms (50 × 10 cm) and two enclosed arms of the same dimensions, with walls 40 cm high. The two open arms were opposite to each other. On the test day, each rat was administered with drug or vehicle and was placed, 30 min later, in the central square of the maze facing one of the enclosed arms. The number of entries and time spent in open and enclosed arms were recorded by an observer situated 2 m from the maze. An entry was counted only when the rat had its four limbs in an individual arm. Data analyzed were the total number of entries, the percentage entries and the percentage time spent in open arms. Drugs were administered 30 min before testing, and their effects were analyzed using ANOVA, followed by Dunnett's test.

Influence on Active Social Interaction in rats. Male Sprague-Dawley rats of 240 to 260 g (Charles River, Saint-Aubin-les-Elbeuf, France) were individually housed for 5 days before testing. On the test day, they were placed in weight-matched pairs (±5 g) in opposite corners of a highly illuminated (300 lux) open-topped arena (57 × 36 × 30 cm) for a 10-min session. A camera was mounted 2 m above the arena and was connected to a monitor and a videotape recorder in an adjacent room. The observer recorded from the screen the duration of active social interaction: i.e., the time spent in grooming, following, sniffing, biting, jumping, or crawling over or under the other animal. If animals remained adjacent to each other without any movement for more than 10 s, scoring was discontinued until active social interaction resumed. At the end of each session, the test arena was carefully cleaned. Animals were administered with drug or vehicle 30 min before testing, with each rat of the same pair receiving the same treatment. Dose-effects were analyzed using ANOVA, followed by Dunnett's test.

Influence on Aggression in Preisolated Mice. Pairs of CD₁ mice, 22 to 25 g (Charles River, Saint-Aubin-les-Elbeuf, France) were isolated in black cages for 1 month. On the test day, one mouse (intruder) was placed into the cage of the other (resident) for 3 min and the total number and duration of fights determined. Both mice were treated 30 min before the test with either drug or vehicle. Data were analyzed by ANOVA followed by Dunnett's test, and ID₅₀ values (95% CL) were calculated.

Influence on Marble Burying Behavior in Mice. Mice were individually placed in transparent, polycarbonate cages (30 × 18 × 19 cm) containing a 5-cm layer of sawdust and 24 glass marbles (1.5 cm in diameter) evenly spaced against the wall of the cage. Thirty min later the animals were removed from the cages and the number of marbles at least two-thirds buried in the sawdust was recorded. The mice were treated 30 min before the test with either drug or vehicle. Data were analyzed using ANOVA, followed by Dunnett's test, and ID₅₀ values (95% CL) were calculated.

Induction of Ataxia in Mice. The latency of mice to fall from an accelerating (from 4 to 40 rpm over 300 s) Rotarod (Ugo Basile, Varese, Italy) was determined. There was a cut-off of 360 s. S18616, dexmedetomidine, clonidine, or vehicle was administered 30 min before the test. Dose-effects were analyzed using ANOVA, followed by Dunnett's test, and ID₅₀ values (95% CL) were calculated.

Induction of a Loss of Righting Reflex in Rats: Sedative/Hypnotic Properties. The loss of righting reflex (LRR) in rats was evaluated according to a scoring system described previously (Millan et al., 1994). Briefly, rats were placed on their backs on a laboratory surface covered with paper wadding, and their ability to right themselves was assessed as follows. Score 0: normal, complete righting reflex. Score 1: attempted righting reflex, turn of at least 90 degrees. Score 2: attempted righting reflex, turn of less than 90 degrees. Score 3: total LRR, no attempt to turn. S18616, dexmedetomidine, clonidine, or vehicle was administered 30 min before determination of the LRR. For antagonist studies, atipamezole, BRL-44408, or prazosin was injected 30 min before S18616 (0.63 mg/kg, s.c.). In one additional antagonist study, a full dose-response to S18616 was performed in the presence of vehicle or prazosin (0.63 mg/kg, s.c.). Data were analyzed nonparametrically. For induction of LRR, as previously (Millan et al., 1994), the percentage of rats displaying a score of 1 or higher was determined: all rats receiving vehicle showed values of 0. For antagonist studies, the percentage of animals displaying a score of 2 or less was determined: 100% (n = 12) of rats receiving S18616 (0.63 mg/kg, s.c.) showed values of 3.0. Differences to vehicle were analyzed by Fisher's exact probability test, and ED₅₀ values (95% CL) were calculated.

Influence on Spontaneous Locomotion in Mice and Rats. Mice were placed for 10 min in individual, white Plexiglas chambers (27 × 27 × 27 cm) equipped with two rows of photocells 2 cm above the floor and 6 cm apart. Rats were individually placed for 12 min in transparent polycarbonate cages (45 × 30 × 20 cm) equipped with two rows of photocells 4 cm above the floor and 24 cm apart. In both cases, a locomotion count corresponds to the consecutive interruption within 2 s of two infrared beams. Drugs or vehicle were given 30 min before testing. Data were analyzed by ANOVA followed by Dunnett's test, and ID₅₀ values (95% CL) were calculated.

Drugs. Drugs were dissolved in sterile water, and if necessary, a few drops of lactic acid were added, and pH was adjusted to as close to normality (>5.0) as possible. Drug salts, structures, and sources were as follows. S18616 HCl {(S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(8'-chloro-1',2',3',4'-tetrahydronaphthalene)]}; atipamezole HCl; dexmedetomidine tartrate; idazoxan HCl; BRL44408 (2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazoline); and WAY100,635 {(N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide} fumarate were synthesized internally (A. Cordi and J.-L. Peglion). Clonidine HCl and prazosin HCl were from Sigma (Chesnes, France).

	Results

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Influence of S18616 Compared with Dexmedetomidine and Clonidine on the Electrical Activity of Neurons of the LC (Figs. 1 and 2). S18616 potently, dose dependently and completely suppressed the electrical activity of perikarya in the LC of anesthetized rats. Similarly, dexmedetomidine reduced the firing rate of these neurons, although it was slightly less potent than S18616. Clonidine was likewise effective although only over a markedly higher dose-range than for S18616. In preliminary experiments (not shown) the actions of the selective ₂-AR antagonist, atipamezole, after i.v. administration proved to be very transient in line with its very short half-life (Aantaa et al., 1993; Scheinin et al., 1998). Thus, for demonstration of a role of ₂-ARs in the actions of S18616 in this model, idazoxan was used. Administration of idazoxan after S18616 completely reversed its inhibitory actions. S18616 (1.25 µg/kg, i.v.)/vehicle = 97 ± 3% versus S18616/idazoxan (63 µg/kg, i.v.) = 2.2 ± 14.0%, P < .001. Upon administration alone, idazoxan enhanced the firing rate, consistent with the existence of a tonic, inhibitory influence of dendritic ₂-AR autoreceptors on the activity of LC-localized neurons. Vehicle = 4.0 ± 0.8% versus idazoxan (63 µg/kg, i.v.) = +42 ± 24%, P < .01. 

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Influence of S18616, dexmedetomidine, and clonidine on the electrical activity of neurons localized in the locus ceruleus, dorsal raphe nucleus, and ventral tegmental area, respectively, of anesthetized rats. Dose-dependent influence of i.v. administration of S18616 (A), dexmedetomidine (B), and clonidine (C) on firing rate expressed as a percentage of preinjection, with basal values defined as 0%. Data are means ± S.E.M. N  5 per value. ID50 (95% CL) in µg/kg, i.v. are as follows. LC: S18616, 0.08 (0.06-0.1); dexmedetomidine, 0.3 (0.1-0.5); and clonidine, 2.0 (1.0-4.0). DRN: S18616, 0.5 (0.1-2.0); dexmedetomidine, 1.5 (1.0-2.0); and clonidine, 3.0 (2.0-4.0). ANOVA results are as follows. A (S18616): LC, F(8,40) = 32.2, P < .01; DRN, F(5,25) = 12.4, P < .01; and VTA, F(6,24) = 4.6, P < .01. B (dexmedetomidine): LC, F(5,20) = 57.0, P < .01; DRN, F(5,20) = 16.1, P < .01; and VTA, F(6,30) = 10.7, P < .01. C (clonidine): LC, F(5,25) = 27.3, P < .01; DRN, F(5,20) = 35.0, P < .01; and VTA, F(6,30) = 13.3, P < .01. Asterisks indicate significance of differences to respective vehicle (VEH) values in Newman-Keuls test. *P < .05.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Influence of S18616 on representative neurons localized in the locus ceruleus, dorsal raphe nucleus, and ventral tegmental area, respectively, of anesthetized rats. Recordings were acquired in three independent experiments and show the influence of cumulative, incremental doses of S18616 (0.02-1.25 µg/kg, i.v.), followed by a single dose of the 2-AR antagonist, idazoxan (63 µg/kg, i.v.), which reversed its actions.

Influence of S18616 Compared with Dexmedetomidine and Clonidine on the Electrical Activity of Neurons of the DRN (Figs. 1 and 2). S18616 dose dependently and completely blocked the firing rate of DRN-localized perikarya with a potency ca. 5-fold lower than that suppressing the activity of LC-localized neurons. Dexmedetomidine and clonidine mimicked the action of S18616 at 3- and 6-fold higher doses, respectively. Idazoxan significantly reversed the action of S18616. S18616 (2.5 µg/kg, i.v.)/vehicle = 99 ± 1% versus S18616/idazoxan (63 µg/kg, i.v.) = 11.0 ± 8.1%, P < .001. Upon administration alone, idazoxan partially reduced the DRN firing rate. Vehicle = +2.5 ± 2.6% versus idazoxan (63 µg/kg, i.v.) = 42 ± 5%, P < .001. This action, which likely reflects its 5-HT_1A agonist properties, was reversed by WAY100,635 (31 µg/kg, i.v.): idazoxan/WAY100,635 = +25 ± 18%, P < .001. WAY100,635 was inactive alone (not shown).

Influence of S18616 Compared with Dexmedetomidine and Clonidine on the Electrical Activity of Neurons in the VTA (Figs. 1 and 2). S18616 did not decrease the firing rate of VTA-localized neurons at doses that suppressed the electrical activity of LC-localized cell bodies (vide supra). Furthermore, it likewise did not modify their firing pattern: bursting or regular (not shown). Over a (25-fold) higher dose range, however, S18616 elicited a slight and dose-dependent increase in their firing rate, an action reversed by idazoxan. S18616 (10 µg/kg, i.v.)/vehicle = +41 ± 11% versus S18616/idazoxan (63 µg/kg, i.v.) = +8.1 ± 8.3%, P < .01. Similarly, dexmedetomidine and clonidine both slightly increased the firing rate of VTA-localized cells. Upon administration alone, idazoxan, did not modify firing rate. Vehicle = 1.5 ± 0.7 versus idazoxan (63 µg/kg, i.v.) = +4.9 ± 3.9%, P > .05.

Influence of S18616 on Extracellular Levels of NE, 5-HT, and DA in the FCX and Hippocampus (Fig. 3 and Table 1). S18616 potently and dose dependently (0.00004-0.16 mg/kg, s.c.) decreased levels of NE, 5-HT, and DA simultaneously quantified in single dialysis samples of the FCX of freely moving rats. Levels of NE were maximally decreased by the highest dose (0.16) of S18616 examined. Administered at a single dose (0.16 mg/kg, s.c.), S18616 similarly elicited a marked reduction in dialysate levels of NE and 5-HT in the dorsal hippocampus. (DA was undetectable in this structure.) All these effects were sustained, being fully expressed throughout the 3-h sampling period.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Influence of S18616 on extracellular levels of NE, 5-HT, and DA in the frontal cortex and dorsal hippocampus of freely moving rats. A to C, dose-dependent inhibitory influence of S18616 on dialysate levels of NE, 5-HT, and DA in the FCX. D and E, inhibitory influence of S18616 on dialysate levels of NE and 5-HT in dorsal hippocampus. Levels are expressed as a percentage of basal, preinjection values, which were defined as 0%. These were as follows (pg/20 µl of dialysate). NE, FCX = 1.3 ± 0.2; 5-HT, FCX = 0.68 ± 0.06; DA, FCX = 1.1 ± 0.1; NE, hippocampus = 0.9 ± 0.1 and 5-HT, hippocampus = 0.37 ± 0.02. Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. A: F(5,40) = 51.3, P < .01. B: F(5,40) = 16.9, P < .01. C: F(5,41) = 22.0, P < .01. D: F(1,17) = 82.4, P < .01. E: F(1,17) = 27.6, P < .01. Asterisks indicated significance of differences to respective vehicle values in Dunnett's test. *P < .05.

Influence of Dexmedetomidine and Clonidine on Extracellular Levels of NE, 5-HT, and DA in the FCX (Fig. 4 and Table 1). In analogy to S18616, dexmedetomidine (0.00063-0.16 mg/kg, s.c.) dose dependently reduced extracellular levels of NE, 5-HT, and DA in the FCX. Clonidine (0.00063-0.16 mg/kg, s.c.) behaved similarly. In analogy to S18616, levels of NE were maximally decreased at 0.16 mg/kg, s.c. of dexmedetomidine and clonidine.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Influence of dexmedetomidine and clonidine on extracellular levels of NE, 5-HT, and DA in the frontal cortex and hippocampus of freely moving rats. A to C, dose-dependent inhibitory influence of dexmedetomidine on dialysate levels of NE, 5-HT, and DA levels in the FCX. D to F, dose-dependent inhibitory influence of clonidine on dialysate levels of NE, 5-HT, and DA levels in FCX. NE, 5-HT, and DA levels are expressed as a percentage basal, preinjection of values, which were defined as 0% (see legend to Fig. 3). Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. A: F(3,22) = 36.9, P < .01. B: F(3,24) = 25.6, P < .01. C: F(3,24) = 25.6, P < .01. D: F(3,21) = 32.3, P < .01. E: F(3,23) = 23.0, P < .01. F: F(3,22) = 36.2, P < .01. Asterisks indicated significance of differences to respective vehicle values in Dunnett's test. *P < .05.

Influence of Atipamezole on Extracellular Levels of NE, 5-HT, and DA in the FCX and Their Modulation by S18616 (Fig. 5). Atipamezole (0.01-0.63 mg/kg, s.c.) elicited a dose-dependent increase in extracellular levels of NE and DA, but not 5-HT, in FCX, in line with a tonic inhibitory influence of ₂-ARs on NE and DA release in this structure (Millan et al., 2000b,c). When S18616 (0.0025 mg/kg, s.c.) was administered after atipamezole (0.16 mg/kg, s.c.), its suppressive influence on extracellular levels of NE, 5-HT, and DA was abolished.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Influence of atipamezole on extracellular levels of NE, 5-HT, and DA in the frontal cortex of freely moving rats and their modulation by S18616. A to C, dose-dependent influence of atipamezole alone. D to F, influence of S18616 (0.0025 mg/kg, s.c.) in the presence of atipamezole (0.16 mg/kg, s.c.). Levels are expressed as a percentage of values, which were defined as 0% (see legend to Fig. 3). Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. A: F(4,27) = 32.9, P < .01. B: F(4,26) = 0.1, P > .05. C: F(4,27) = 21.9, P < .01. D: influence of atipamezole, F(1,16) = 40.4, P < .01; influence of S18616, F(1,15) = 36.9, P < .01; and interaction, F(1,12) = 67.1, P < .01. E: influence of atipamezole, F(1,17) = 0.1, P > .05; influence of S18616, F(1,16) = 25.9, P < .01; and interaction, F(1,11) = 21.3, P < .01. F: influence of atipamezole, F(1,15) = 22.7, P < .01; influence of S18616, F(1,16) = 18.9, P < .01; and interaction, F(1,12) = 36.9, P < .01. Asterisks indicate significance of differences of atipamezole versus vehicle and of atipamezole/S18616 versus vehicle/S18616 values in Dunnett's test. *P < .05.

Influence of BRL-44408 Compared with Prazosin on Extracellular Levels of NE, 5-HT, and DA in the FCX and Their Modulation by S18616 (Figs. 6 and 7). BRL-44408 dose dependently (0.63-40.0 mg/kg, s.c.) elevated FCX levels of NE and DA but not 5-HT. In its presence (10.0 mg/kg, s.c.), the influence of S18616 (0.0025 mg/kg, s.c.) on levels of NE, DA, or 5-HT was blocked. In distinction to BRL-44408, prazosin (0.04-0.63 mg/kg, s.c.), administered alone, dose dependently reduced levels of 5-HT while not affecting those of NE and DA. In contrast to BRL-44408, prazosin (0.63 mg/kg, s.c.) failed to modify the influence of S18616 on levels of NE, DA, or 5-HT.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Influence of BRL-44408 on extracellular levels of NE, 5-HT, and DA in the frontal cortex of freely moving rats and their modulation by S18616. A to C, dose-dependent influence of BRL-44408 alone. D to F, influence of S18616 (0.0025 mg/kg, s.c.) in the presence of BRL-44408 (10.0 mg/kg, s.c.). Levels are expressed as a percentage of values, which were defined as 0% (see legend to Fig. 3). Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. A: F(4,25) = 23.4, P < .01. B: F(4,27) = 0.3, P > .05. C: F(4,26) = 15.1, P < .01. D: influence of BRL-44408, F(1,14) = 27.4, P < .01; influence of S18616, F(1,15) = 36.9, P < .01; and interaction, F(1,11) = 82.9, P < .01. E: influence of BRL-44408, F(1,16) = 0.1, P > .05; influence of S18616, F(1,16) = 25.9, P < .01; and interaction, F(1,11) = 24.7, P < .01. F: influence of BRL-44408, F(1,15) = 56.7, P < .01; influence of S18616, F(1,16) = 18.9, P < .01; and interaction, F(1,11) = 76.4, P < .01. Asterisks indicate significance of differences of BRL-44408 versus vehicle and of BRL-44408/S18616 versus vehicle/S18616 values in Dunnett's test. *P < .05.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Influence of prazosin on extracellular levels of NE, 5-HT, and DA in the frontal cortex of freely moving rats and their modulation by S18616. A to C, dose-dependent influence of prazosin alone. D to F, influence of S18616 (0.0025 mg/kg, s.c.) in the presence of prazosin (0.63 mg/kg, s.c.). Levels are expressed as a percentage of values, which were defined as 0% (see legend to Fig. 3). Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. A: F(3,22) = 0.4, P > .05. B: F(3,24) = 30.9, P < .01. C: F(3,23) = 0.6, P > .05. D: influence of prazosin, F(1,15) = 1.8, P > .05; influence of S18616, F(1,15) = 36.9, P < .01; and interaction, F(1,12) = 1.7, P > .05. E: influence of prazosin, F(1,16) = 43.7, P < .01; influence of S18616, F(1,16) = 25.9, P < .01; and interaction, F(1,12) = 0.6, P > .05. F: influence of prazosin, F(1,15) = 0.2, P > .05; influence of S18616, F(1,16) = 18.9, P < .01; and interaction, F(1,12) = 1.0, P > .05. Asterisks indicate significance of differences of prazosin versus vehicle values in Dunnett's test. *P < .05.

Influence of S18616 Compared with Dexmedetomidine and Clonidine on Hippocampal Synthesis of NE (Fig. 8). S18616 dose dependently reduced hippocampal synthesis of NE, as determined by accumulation of its precursor, L-DOPA. This action was mimicked by dexmedetomidine and clonidine. In distinction to S18616, atipamezole (0.0025-0.63 mg/kg, s.c.) and BRL-44408 (0.16-40.0 mg/kg, s.c.), but not prazosin (0.63-40.0 mg/kg, s.c.), dose dependently and significantly (P < .01) elevated hippocampal NE turnover (atipamezole, not shown; for BRL-44408 and prazosin, see Millan et al., 1994).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Influence of S18616 compared with dexmedetomidine and clonidine on cerebral synthesis of NE and 5-HT. A, NE, hippocampus; B, 5-HT, hippocampus; C, 5-HT, FCX; D, 5-HT, striatum; and E, 5-HT, hypothalamus. Levels are expressed as a percentage of vehicle (VEH) values, which were defined as 0%. These were as follows (ng/mg of protein): hippocampus, L-DOPA = 0.5 ± 0.02 and 5-HTP = 1.1 ± 0.06; FCX, 5-HTP = 0.7 ± 0.02; striatum, 5-HTP = 1.1 ± 0.04 and hypothalamus, 5-HTP = 2.7 ± 0.1. Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. Hippocampus, L-DOPA: S18616, F(6,47) = 11.2, P < .01; dexmedetomidine, F(4,31) = 11.0, P < .01; and clonidine, F(6,55) = 6.0, P < .01. Hippocampus, 5-HTP: S18616, F(6,48) = 9.6, P < .01; dexmedetomidine, F(4,32) = 24.8, P < .01; and clonidine, F(6,56) = 12.2, P < .01; FCX, 5-HTP: S18616, F(5,32) = 18.2, P < .01; dexmedetomidine, F(3,14) = 9.5, P < .01; and clonidine, F(5,25) = 5.6, P < .01. Striatum, 5-HTP: S18616, F(5,31) = 21.3, P < .01; dexmedetomidine, F(3,15) = 14.7, P < .01; and clonidine, F(6,35) = 3.0, P < .05. Hypothalamus, 5-HTP: S18616, F(3,15) = 37.9, P < .01; dexmedetomidine, F(3,15) = 22.3, P < .01; and clonidine, F(6,35) = 11.2, P < .01. Asterisks indicate significance of differences to vehicle values in Dunnett's test. *P < .05.

Influence of S18616 Compared with Dexmedetomidine and Clonidine on Cerebral Synthesis of 5-HT (Fig. 8). In the hippocampus, FCX, striatum, and hypothalamus, S18616 dose dependently reduced 5-HT synthesis, as determined by accumulation of its precursor, 5-HTP. Dexmedetomidine and clonidine behaved similarly. Administered at doses indicated above for modulation of NE synthesis, atipamezole and BRL4408 did not affect 5-HT synthesis, whereas prazosin decreased it (not shown), likely due to its ₁-AR antagonist properties (Haddjeri et al., 1995; Gobert et al., 1998).

Influence of S18616 Compared with Dexmedetomidine and Clonidine on Motor Behavior (Fig. 9 and Table 1). In mice, S18616 dose dependently elicited ataxia in the Rotarod procedure. It also decreased spontaneous locomotion in both mice and rats. Dexmedetomidine displayed similar actions over a comparable dose range, although clonidine elicited these effects only at higher doses. In rats, S18616 induced a LRR, consistent with hypnotic-sedative properties. Dexmedetomidine proved to be more potent than S18616 in evoking LRR. In contrast to S18616 and dexmedetomidine, clonidine showed only partial activity in this protocol, eliciting a submaximal LRR even at high doses.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Induction of a LRR by S18616 compared with dexmedetomidine and clonidine in rats. A, induction of LRR by S18616 compared with dexmedetomidine and clonidine; B, inhibition of the action of S18616 (0.63 mg/kg, s.c.) by atipamezole and BRL-44408 compared with prazosin; and C, potentiation by prazosin (0.63 mg/kg, s.c.) of the actions of S18616: displacement of its dose-response curve to the left. Data are mean percentage of rats showing a score of >1. N  6 per value. Asterisks indicate significance of differences to respective vehicle (VEH) values in Fisher's exact probability test. *P < .05.

Influence of Antagonists on the Sedative/Hypnotic Properties of S18616 (Fig. 9). The LRR elicited by S18616 was dose dependently abolished by atipamezole and BRL-44408 with ED₅₀ values (95% CL) of 0.11 (0.04-0.26) and 1.18 (0.89-1.55) mg/kg, s.c., respectively. Prazosin was ineffective. No antagonist significantly elicited a LRR on administration alone (not shown). However, prazosin dose dependently facilitated the induction of LRR by S18616, shifting its dose-response curve to the left. In the presence of vehicle, ED₅₀ (95% CL) = 0.02 (0.01-0.03) and, in the presence of prazosin, ED₅₀ (95% CL) = 0.003 (0.001-0.03).

Influence of S18616 Compared with Dexmedetomidine and Clonidine on Ultrasonic Vocalizations in Rats, and on Marble-Burying and Aggressive Behavior in Mice (Fig. 10 and Table 2). S18616 potently, dose dependently and completely blocked ultrasonic vocalizations emitted by rats placed in an environment in which they had previously been exposed to a mild aversive stimulus. Dexmedetomidine and clonidine similarly reduced ultrasonic vocalizations. S18616 similarly elicited a potent and dose-dependent reduction in marble-burying and aggressive behaviors in mice. Dexmedetomidine and, at somewhat higher doses, clonidine, mimicked these actions of S18616.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Influence of S18616 compared with dexmedetomidine and clonidine on ultrasonic vocalizations in rats, on aggressive behavior in isolated mice and on marble-burying behavior in mice. A, influence on ultrasonic vocalizations emitted by rats placed in an aversive environment; B, influence on marble-burying in mice; C, influence on aggressive behavior in resident to intruder mice (number of attacks); and D, influence on aggressive behavior of resident to intruder mice (duration of attacks). Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. A: S18616, F(4,20) = 17.4, P < .01; dexmedetomidine, F(3,26) = 11.7, P < .01; and clonidine, F(3,21) = 8.3, P < .01. B: S18616, F(4,30) = 16.3, P < .01; dexmedetomidine, F(4,26) = 26.5, P < .01; and clonidine, F(3,23) = 22.6, P < .01. C: S18616, F(4,32) = 10.1, P < .01; dexmedetomidine, F(4,34) = 6.7, P < .01; and clonidine, F(4,28) = 4.1, P < .01. D: S18616, F(4,32) = 5.4, P < .01; dexmedetomidine, F(4,34) = 6.0, P < .01; and clonidine, F(4,28) = 3.6, P < .05. Asterisks indicate significance of drug versus vehicle (VEH) values in Dunnett's test. *P < .05.

Influence of ₂-AR Antagonists on the Suppression of Ultrasonic Vocalizations by S18616 (Fig. 11). Atipamezole (0.16 mg/kg, s.c.) and BRL-44408 (10.0 mg/kg, s.c.) significantly attenuated the inhibitory influence of S18616 (0.01 mg/kg, s.c.) on ultrasonic vocalizations, although they themselves tended to decrease ultrasonic vocalizations alone. Prazosin (0.16 mg/kg, s.c.) did not significantly modify the action of S18616.

View larger version (18K): [in this window] [in a new window]   Fig. 11.   Influence of atipamezole, BRL-44408, and prazosin on the actions of S18616 in the ultrasonic vocalizations procedure. A, atipamezole; B, BRL-44408; and C, prazosin. Data means ± S.E.M. N > 6 per point. Two-way ANOVA results are as follows. A: influence of S18616, F(1,30) = 5.0, P < .05; influence of atipamezole, F(1,30) = 0.4, P > .05; and interaction, F(1,30) = 9.8, P < .01. B: influence of S18616, F(1,23) = 6.7, P < .05; influence of BRL-44408, F(1,23) = 0.7, P > .05; and interaction, F(1,23) = 14.1, P < .01. C: influence of S18616, F(1,60) = 44.3, P < .001; influence of prazosin, F(1,60) = 16.1, P < .001; and interaction, F(1,60) = 2.4, P > .05. Closed asterisks indicate significance of the difference of vehicle/S18616 to vehicle/vehicle values, and open asterisks of antagonist/S18616 to vehicle/S18616 values in Newman-Keuls test after ANOVA. *P < .05.

Influence of S18616 Compared with Dexmedetomidine and Clonidine on Behavior in the Elevated Plus-Maze in Rats (Fig. 12). S18616 did not significantly elevate arm entries or the percentage of time spent in open arms. With increasing doses, it significantly suppressed total arm entries. Dexmedetomidine and clonidine showed similar patterns of action.

View larger version (19K): [in this window] [in a new window]   Fig. 12.   Influence of S18616 compared with dexmedetomidine and clonidine on behavior in the elevated plus-maze test in rats. A, influence on percentage of entries in open arms; B, influence on percentage time in open arms; and C, influence on total arm entries. Data are means ± S.E.M. N  6 per value. ANOVA results are as follows. Percentage entries in open arms, S18616, F(5,62) = 2.5, P < .05; dexmedetomidine, F(3,26) = 0.6, P > .05; and clonidine F(3,30) = 1.3, P > .05. Percentage time in open arms: S18616, F(5,62) = 1.3, P > .05; dexmedetomidine, F(3,26) = 1.7, P > .05; and clonidine, F(3,30) = 1.3, P > .05. Total entries: S18616, F(5,62) = 14.3, P < .01; dexmedetomidine, F(3,26) = 15.5, P < .01; and clonidine, F(3,30) = 16.8, P < .01. Asterisks indicate significance of drug versus vehicle (VEH) values in Dunnett's test. *P < .05.

Influence of S18616 Compared with Dexmedetomidine and Clonidine on Behavior in the Vogel and Social Interaction Tests in Rats (Fig. 13 and Table 2). At a low dose, S18616 elicited a mild and significant increase in punished responses in the Vogel procedure. Dexmedetomidine and clonidine showed a similar elevation in responses restricted to low doses. Higher doses of S18616, dexmedetomidine, and clonidine suppressed responding. In the social interaction model, at low doses, S18616, but not dexmedetomidine and clonidine, elicited a mild and significant increase in active social interaction. For S18616, dexmedetomidine, and clonidine, higher doses led to a marked reduction in social interaction in each case.

View larger version (16K): [in this window] [in a new window]   Fig. 13.   Influence of S18616 compared with dexmedetomidine and clonidine on behavior in the Vogel conflict and social interaction tests in rats. A to C, influence on responding in the Vogel procedure. D to F, influence on active social interaction. At a higher dose of dexmedetomidine (0.01), responding was completely suppressed (not shown). Data are means ± S.E.M. N  5 per value. ANOVA results are as follows. Vogel procedure: S18616, F(3,46) = 2.9, P < .05; dexmedetomidine, F(3,61) = 4.5, P < .01; and clonidine, F(4,58) = 3.0, P < .05. Social interaction: S18616, F(4,32) = 18.5, P < .01; dexmedetomidine, F(4,22) = 34.6, P < .01; and clonidine, F(4,22) = 12.3, P < .01. Asterisks indicate significance of drug versus vehicle (VEH) values in Dunnett's test. *P < .05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Adrenergic Transmission. In amplification of previous work, dexmedetomidine and clonidine abolished the firing rate of LC-localized adrenergic perikarya, suppressed extracellular levels of NE in the FCX, and decreased hippocampal synthesis of NE (Aantaa et al., 1993; Chiu et al., 1995; Dalley and Stanford, 1995; Gobert et al., 1998; Mateo and Meana, 1999; see Millan et al., 2000b). These actions were mimicked by S18616, which likewise attenuated dialysate levels of NE in hippocampus, demonstrating engagement of ₂-AR autoreceptors in vivo. ₂-AR autoreceptors possess marked tonic activity, as revealed by the opposite, facilitatory influence of atipamezole and idazoxan, observations extending previous studies of these and other ₂-AR antagonists (Millan et al., 1994, 2000b,c; Haapalinna et al., 1997; Gobert et al., 1998). It has been speculated that imidazoline I_1/2 receptors, via actions integrated in the LC, modulate central adrenergic transmission (Piletz and Halaris, 1994; Dalley and Stanford, 1995; Nutt et al., 1997). However, recent studies suggest that actions of imidazoline compounds are mediated either via an, as yet, poorly characterized interaction with K⁺ channels (Ugedo et al., 1998) or, principally, via ₂-ARs themselves (Szabo et al., 1996). Indeed, atipamezole, which is devoid of affinity at I_1/2 receptors, accelerates adrenergic transmission (Fig. 4) (vide supra) and blocks the inhibitory influence of S18616, which shows >100-fold higher affinity for ₂-ARs versus I_1/2 sites. Studies of dendritic ₂-autoreceptors in the LC (Chiu et al., 1995; Callado and Stamford, 1999; Mateo and Meana, 1999) and their terminal counterparts in the FCX (Trendelenburg et al., 1999) support a key role of _2A-ARs, an interpretation underpinned by gene knockout and neuroanatomical studies (see the Introduction). Indeed, in analogy to their influence on other ₂-AR agonists (Gobert et al., 1998), BRL-44408 (a preferential _2A- versus _2B/2C-AR antagonist) potentiated frontocortical NE release and suppressed the inhibitory action of S18616, whereas prazosin (a preferential antagonist of _2B/2C- versus _2A-ARs) was ineffective. Nevertheless, S18616 is not selective for _2A-ARs so the precise contribution of ₂-AR subtypes to its influence on adrenergic pathways requires further examination.

Serotonergic Transmission. Dexmedetomidine and clonidine reduced frontocortical 5-HT release and suppressed 5-HT synthesis (Aantaa et al., 1993; Sallinen et al., 1997; Gobert et al., 1998), actions potently mimicked by S18616. One underlying mechanism may comprise activation of ₂-heteroceptors on terminals of serotonergic fibers (Trendelenburg et al., 1994; Haddjeri et al., 1995; Millan et al., 2000b). However, S18616, dexmedetomidine, and clonidine (idazoxan, reversibly) reduced the electrical activity of DRN serotonergic perikarya. This action likely involves stimulation of ₂-AR autoreceptors on adrenergic neurons innervating the DRN, thereby eliminating a tonic excitatory influence of ₁-ARs (Haddjeri et al., 1995). Indeed, ₁-AR antagonist properties of prazosin underlie blockade of the firing rate of DRN-serotonergic perikarya (Haddjeri et al., 1995; see Millan et al., 2000b) and release of 5-HT in postsynaptic structures (Gobert et al., 1998). Interestingly, however, certain studies observed an excitatory influence of ₂-AR antagonists on 5-HT release (Haddjeri et al., 1995), whereas others reported no effect (Haapalinna et al., 1997; Millan et al., 2000b,c), as herein. One reason underlying divergent data is that tonic control of serotonergic transmission by ₂-AR heteroceptors is expressed less consistently than that of adrenergic pathways by ₂-AR autoreceptors (Millan et al., 2000b,c). Attenuation of frontocortical release of 5-HT by S18616 was blocked by atipamezole and BRL-44408, but not prazosin, supporting a role of ₂- versus ₁-ARs in inhibitory control of serotonergic transmission and in line with anatomical, pharmacological, and gene knockout studies suggesting participation of _2A-ARs (see the Introduction).

Frontocortical Dopaminergic Transmission. Clonidine failed to inhibit VTA-dopaminergic perikarya (Grenhoff and Svensson, 1989), and dexmedetomidine and S18616 were similarly ineffective. Indeed, they all increased the firing rate. Although activation of ₁-ARs might be invoked (Grenhoff and Svensson, 1989), idazoxan reversed this excitatory influence of S18616 implicating of ₂-ARs. These might be localized on terminals of serotonergic pathways inhibitory to dopaminergic perikarya (Millan et al., 2000b): their engagement would, thus, disinhibit mesocortical dopaminergic pathways. However, doses of S18616, dexmedetomidine, and clonidine exciting VTA neurons were rather high, and the mechanisms underlying this effect would benefit from further evaluation. Furthermore, S18616, dexmedetomidine, and clonidine suppressed DA levels in FCX, reflecting engagement of ₂-ARs localized on terminals of frontocortical dopaminergic pathways (Matsumoto et al., 1998; Hertel et al., 1999; Millan et al., 2000b). Correspondingly, atipamezole and BRL-44408, but not prazosin, dose dependently elevated dialysate levels of DA and attenuated their suppression by S18616. Inasmuch as VTA-dopaminergic neurons express low levels of mRNA encoding _2A-ARs, other neuronal pathways likely intervene in the influence of ₂-AR agonists on frontocortical DA (Matsumoto et al., 1998), although the presence of _2C-ARs should not be neglected (Lee et al., 1998). Furthermore, NE transporters on adrenergic terminals contribute to clearance of extracellular DA in the FCX (Yamamoto and Novotney, 1998; Millan et al., 2000b). Thus, changes in frontocortical levels of DA may, at least partially, be secondary to alterations in levels of NE.

Motor-Suppressive Properties. The suppressive influence of ₂-AR agonists on motor behavior reflects multiple actions, including engagement of ventral horn-localized ₂-ARs (Coward, 1994; Nicholas et al., 1997) and, most prominently, as concerns sedative/hypnotic properties, activation of _2A-ARs in the LC (Aantaa and Scheinin, 1993; Hayashi and Maze, 1993; Hayashi et al., 1995). Inhibition of serotonergic activity may also be involved (Rabin et al., 1996). Although the respective role of these mechanisms requires clarification, S18616, dexmedetomidine, and clonidine all manifested motor-suppressive properties. Notably, doses disrupting motor function were close to those reducing frontocortical levels of NE (Table 1) suggesting that the principal underlying mechanism is suppression of adrenergic transmission via activation of ₂-AR autoreceptors. In line with extensive experimental and clinical observations of ₂-AR agonists, there was little separation between these motor-disruptive versus other behavioral (including anxiolytic) actions of S18616 [Hayashi an