You are on page 1of 9

Psychopharmacology (1997) 134:378386

Springer-Verlag 1997

O R I G I N A L I N V E S T I G AT I O N

&roles:F. Borsini R. Cesana J. Kelly B. E. Leonard M. McNamara J. Richards L. Seiden

BIMT 17: a putative antidepressant with a fast onset of action?

&misc:Received: 10 March 1997 / Final version: 9 July 1997

&p.1:Abstract BIMT 17, the only compound reported to be a full 5-HT1A agonist and a 5-HT2A antagonist at the frontal cortex, was assessed in three animal paradigms sensitive to antidepressants in rats: olfactory bulbectomy (OB), differential-reinforcement-of-low rate 72-s (DRL 72-s) and learned helplessness (LH). In the OB rats, BIMT 17, given once daily for 14 consecutive days at an IP dose of 10 mg/kg, but not of 20 mg/kg, reduced the increase in ambulation of OB rats, 24 h after the last administration. In the DRL 72-s test, BIMT 17 had a different profile than imipramine. A single IP injection of 5, 10, 15 or 20 mg/kg BIMT 17, in contrast to the same doses of imipramine, did not affect response and reinforcement rate in DRL 72-s 1 h after the administration. On the other hand, BIMT 17 slightly shifted the peak of the interresponse time (IRT) distribution towards shorter IRT duration, while imipramine shifted the peak of the IRT distribution towards longer IRT duration. In the LH test, acute oral doses (36, 48 or 60 mg/kg) of BIMT 17, given 30 min before testing, reduced the number of escape failures in LH without altering the intertrial crossings. This effect was also induced by a repeated, but not single, administration with 8 or 16 mg/kg imipramine. The plasma levels following IP 10 or oral 48 mg/kg BIMT 17 were in the same range. These results indicate that BIMT 17 does not behave like imipramine in all the tests, and suggest that BIMT 17 acts through different mechanisms of action than imipramine. Only clinical trials will tell whether these mechanisms will be relevant, but if so, BIMT 17 might induce a faster onset of therapeutic activity.

&kwd:Key words BIMT 17 Animal models of depression Serotonin&bdy:

Introduction
BIMT 17 (2H-benzimidazol-2-one, 1,3-dihydro-1-[2-[4[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl]) (Fig. 1) has recently been described as the first compound capable of inhibiting forskolin-stimulated cAMP (Borsini et al. 1995b) and electrical activity (Borsini et al. 1995a) in the rat frontal cortex, by activating postsynaptic serotonin (5HT) 5-HT1A receptors and antagonizing 5-HT2A receptors (Borsini et al. 1995a,b). This mechanism of action in the frontal cortex has also been reported for antidepressants following chronic treatment (Borsini 1994). Thus, BIMT 17 may represent a prototype of a novel class of antidepressants with a potential faster onset of action. Interestingly, in the chronic mild stress model in mice, an animal model sensitive to antidepressants, a single administration of BIMT 17 had an effect similar to that following repeated treatment with fluoxetine (Willner 1995). An antidepressant-like effect of BIMT 17 has also been reported in the forced swimming test in mice (Cesana et al. 1995). The purpose of the present study was two-fold: a) to evaluate further the antidepressant potential of BIMT 17 in the rat and b) to confirm the faster onset of activity of BIMT 17. The effect of repeated treatment with BIMT 17 was evaluated in the olfactory bulbectomy (OB) model and the effect of a single administration of BIMT 17 in the

F. Borsini (u) R. Cesana Boehringer Ingelheim Italia, Via Lorenzini 8, I-20139 Milano, Italy J. Kelly B.E. Leonard M. McNamara Pharmacology Department, University College Galway, Ireland J. Richards L. Seiden Department of Pharmacological and Physiological Sciences, University of Chicago, 947 East 58th Street, Chicago, IL 60637, USA&/fn-block:

Fig. 1 Chemical structure of BIMT 17ig.c:&/f

379

differential-reinforcement-of-low rate 72-s (DRL 72-s) paradigm. In addition, to evaluate further the possibility of a faster onset of action of BIMT 17, the learned helplessness (LH) model was used, and the effect of a single administration of BIMT 17 was evaluated in comparison to single and repeated treatments with imipramine.

0600 and 0800 hours. Each animal was placed in the centre of the open field apparatus and the following parameters were measured over a 3-min period: ambulation (number of squares crossed), rearing (number of times the rat simultaneously raised both forepaws off the floor of the apparatus), grooming (number of times the rat stopped and groomed itself) and defaecation (the number of faecal boli deposited). Data analysis. &p.2:Two-way analysis of variance was performed to evaluate body weights. If any statistically significant change was found, the data was further analysed using the Student Newman Keuls test. These results are expressed as group mean and standard error of the mean. Kruskal-Wallis test was performed on the data relative to the open field test. If any statistically significant changes were found, these data were then tested using a MannWhitney U-test. These results are expressed as group median and inter-quartile range. DRL 72-s This experiment was carried out by J. R. and L. S. Animals. &p.2:Fifteen male Sprague-Dawley rats (Hotzman, Madison, Wisc., USA) weighing between 350 and 500 g at the time of drug administration were used. The rats were housed two per cage in hanging stainless steel wire cages. Lights were on in the colony room from 0700 to 1900 hours. Food (4% Tek Lab rat chow) was available ad lib. Access to water was restricted to 20 min per day. On training days the rats received 20 min access to water at the end of their training session. On non-training days (weekends), the rats were given 20 min access to water between 1000 and 1400 hours. Apparatus. &p.2:This was according to Richards et al. (1994). The operant chambers were 20.5 cm wide, 20.5 cm deep and 23.5 cm long. The operant chambers had grid floors, aluminium front and back walls and Plexiglas sides. A lever was mounted 3 cm above the grid floor 4.5 cm from the nearest side. A downward force of approximately 0.15 N was required for a lever press to be detected. A solenoid operated dipper was located 10 cm to the left of the lever. Access to the dipper was through a round 4.5 cm diameter hole in the front panel. Reinforcement consisted of lifting the dipper (0.025 ml) from a water trough to within reach of the rats tongue for a period of 4 s. A stimulus light mounted 15 cm above the floor on the back wall of the chamber provided the only illumination within the chamber. The stimulus light was turned on when a training session began, and off when the training session ended. The operant chambers were enclosed in 80 quart Coleman ice chests to attenuate external stimuli. Fans mounted on the ice chests provided ventilation and masking noise. The operant chambers were connected to a PDP-11/73 micro computer via a Coulbourn Lablinc interface. The schedule contingencies were programmed using the SKED-11 software system. The timing resolution of the system was 0.01 s. Training. &p.2:Upon arrival in the colony the rats were adapted to the 20 min per day access to water regimen for 1 week. The rats were then trained to bar press in over-night training sessions using an alternative FR1, FT 1-min schedule. Rats which did not acquire the lever press response after five overnight training sessions were manually trained to press it. The rats were then shifted to a DRL-72-s training regimen. DRL 72-s overnight training consisted of six 1-h sessions with a 30-min time-out (house light off) between each session. The rats were trained overnight on the DRL 72-s schedule for 10 nights. After completion of overnight DRL 72-s training the rats were trained during daily (5 days a week) 1-h sessions on the DRL 72-s schedule. The rats had received daily 1-h DRL 72-s schedule sessions for 10 weeks before drug testing was initiated. Drug administration. &p.2:Both BIMT 17 chloride and imipramine chloride (Ciba) were injected IP 1 h before the session in doses of 0, 5, 10, 15 and 20 mg/kg, calculated as a salt. The compounds

Material and methods


Procedures involving animals and their care were conducted in conformity with the institutional guidelines, in compliance with national and international laws and policies (EEC Council Directive 86/609, = J L 358,1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals. NIH publication no. 8523, 1985). Bulbectomized rats This experiment was carried out by M. M., J. K. and B. E. L. Animals. &p.2:Male Sprague-Dawley rats were obtained from Harlan Olac, UK (weight on arrival: 230250 g). The animals were housed four per cage in plastic bottomed cages and were given a 1-week acclimatisation period prior to surgery. They were allowed free access to food and water throughout the course of the experiment. Lighting was controlled on a 12-h light: dark cycle (light period: 08002000 hours) and temperature was maintaned between 20 and 22C. At the time of surgery, rats were individually marked with an ear notch for identification purposes. Olfactory bulbectomy (OB). &p.2:Bilateral olfactory bulbectomy was performed in rats anaesthetised with 2.5% w/v 2-22 tribromoethanol (10 ml/kg IP) essentially as described by Cairncross at al. (1977). The head was shaved and a midline sagittal incision was made extending at least 1 cm rostral to the bregma. Sufficient pressure was applied to ensure that the periostium on the underlying bone had been penetrated. Two drill holes of 2 mm diameter were made in the skull 5 mm rostral to the bregma and 2 mm lateral to the midline. For sham animals, the dura was carefully pierced and the wound closed. For OB animals, the olfactory bulbs were aspirated using a water suction pump. Care was taken not to damage the frontal cortex. After the operation, bleeding was controlled by plugging the holes with haemostatic sponge (Haemofibrine, Specialities Septodont, France). Oxytetracycline dusting powder was sprinkled on the wound prior to closure. The animals were allowed to recover for 14 days following surgery; they were handled daily throughout the recovery period to eliminate any aggressiveness that would otherwise arise (Leonard and Tuite 1981). Drug administration. &p.2:After surgery, the rats were allocated to six groups (nine animals in each group): sham + vehicle; sham + 10 mg/kg BIMT 17; sham + 20 mg/kg BIMT 17; OB + vehicle; OB + 10 mg/kg BIMT 17; OB + 20 mg/kg BIMT 17. The doses of BIMT 17 refer to its salt form. Body weights of each animal in all the groups were measured at days 1 and 15. BIMT 17 chloride was mixed with Tween 80 until a milky, homogenous dispersion was obtained. Saline was then added, to give a concentration of either 10 or 20 mg/ml. This was administered IP at 1 ml/kg given a single daily dose between 0800 and 1000 hours of 10 or 20 mg/kg for 14 consecutive days. Controls received injections of vehicle alone. Open field. &p.2:The rats were tested on day 15 of experiment. This apparatus is essentially as described by Gray and Lalljee (1974). The open field consisted of a circular base, 90 cm in diameter which was divided into 10 cm squares by faint yellow lines. The wall surrounding the base consisted of a 75 cm high aluminium sheet. Illumination was provided by a 60-W bulb, positioned 90 cm above the floor of the apparatus. All measurements were carried out in a darkened room on the morning of day 15 between

380 were dissolved in vehicle (V) to form a solution of 1 ml/kg for injection. BIMT 17 was combined with Tween 80 in a ratio of 1 mg BIMT 17 to 4 mg Tween 80. A glass vial and a small magnetic stir rod were used to obtain a homogeneous dispersion. Water was then incrementally added to obtain the appropriate drug dose. This procedure resulted in a clear fluid with no visible particles. Tween 80, 80 mg/ml water, was used for the BIMT 17 vehicle injection. The vehicle for imipramine was saline. Drug doses were prepared immediately before administration. The doses of BIMT 17 and imipramine were given in ascending order. Drugs were administered on Tuesday and Friday of each week. Control performance was the average of the Thursdays which occurred during each drugs dose-response determination. BIMT 17 and imipramine were tested using a cross-over procedure. Eight of the rats received the BIMT 17 dose determination first, followed by the imipramine dose determination. The remaining seven rats received the imipramine dose determination first, followed by the BIMT 17 dose determination. Data analysis. &p.2:Response rate, reinforcement rate, peak area (PkA), and peak location (PkL) measures were determined for each rat. The PkA and PkL metrics quantitatively characterize the profile of the interresponse time (IRT) distributions generated by responding on the DRL 72-s schedule (Richards and Seiden 1991; Richards et al. 1993a). Rats well trained on the DRL 72-s schedule generate IRT distributions which have well defined peaks. PkA indicates the area of the IRT distribution peak and PkL indicates the location of the IRT distribution peak. In Fig. 4, the BIMT 17 vehicle histogram (top left histogram) illustrates how the profile of the IRT distributions was quantitatively characterized by PkA and PkL. The single shaded histogram bar on the left indicates the burst component of the IRT distribution (IRT < 6 s). The bars to the right of the burst component indicate the pause component of the IRT distribution (IRTs 6s). The connected dots indicate the expected appearance of the pause component of the IRT distribution if the rat emitted the same number of responses, but randomly in time with respect to the preceding response. This expected curve is called the corresponding negative exponential. PkA is indicated by the shaded region of the obtained IRT histogram above the corresponding negative exponential. The peak location (PkL) is the median IRT duration which bisects the shaded region above the corresponding negative exponential. Decreases in the value of PkA indicate that the rats are responding in a less coherent fashion and that the distribution of waiting times (IRTs) is more dispersed. Increases in the value of PkA indicate that the rats are responding in a more coherent fashion and that the IRT distribution is less dispersed. PkL indicates the central location of the peak. A decrease in the value of PkL indicates the rats are not waiting as long between responses. An increase in PkL indicates the rats are waiting longer. A two-factor ANOVA, with treatment order as a betweengroups factor and drug dose as a within-groups factor, was done for both BIMT 17 and imipramine to determine if treatment order affected the results. The results of this analysis showed no significant between group differences. This result indicated that there was no effect of treatment order. After determining that there was no effect of treatment order, the data were combined and analyzed using within group t-tests. Control values (average of the Thursdays which occurred during each drugs dose response determination) for each measure were compared to each drug dose (including vehicle) using within group t-tests. A Bonferroni correction was used to guard against a type 1 error due to multiple comparisons. The overall level of significance was set at P<0.05, twotailed. All four dependent variables (response rate, reinforcement rate, peak area and peak location) were examined in this fashion. Learned helplessness This experiment was carried out by R. C. Animals. &p.2:Naive male Crl:(WI)BR Wistar rats (Charles River Italia S.p.A., Calco, Italy) weighing 200250 g were used. The animals were kept in the experimental room with a regulated environment (21 1C, 5055% humidity and 12-h light-dark cycle, light on at 7.00 a.m.). The animals were housed two per cage in makrolon cages (422615 h cm) with free access to water and food (4RF21 pellets Mucedola Srl, Settimo Milanese, Italy). Helplessness (LH) induction. &p.2:Rats were randomly assigned to groups and subjected to one session of inescapable footshock (IS+). Electric footshock was delivered to the animals by mean of eight chambers (222227 h cm) with grey Plexiglas walls and covers and with the floor consisting of stainless-steel grids (Ugo Basile). A constant-current shocker delivered 60 scrambled, randomized inescapable shocks (15 s duration, 1.2 mA, every 30 24 s) to the grid floor. Control rats were placed in identical chambers for 45 min but shock was not given (IS). All the helplessness induction trials were performed on day 1 in the morning. Shuttle-box test. &p.2:The shuttle-box test was carried out according to Geoffroy et al. (1990). The test took place on day 4 between 08.30 and 12.30 a.m. Four two-way shuttle-boxes (Ugo Basile, mod. 7502) were used. After an initial 5-min habituation, a total of 25 unsignalled escape trials were presented to each animal (each trial started with the simultaneous onset of a light and a 0.8-mA scrambled electric shock). In the first ten trials the rats were required to change side once (FR1) and in the following 15 trials the rats had to change side twice (FR2) in order to terminate both shock and light. The shock duration was 15 s max with an intertrial time of 25 s. The number of escape failures (i.e., the failure to respond during the 15-s shock on) and the number of intertrial crossings (i.e., the number of side-to-side crossing during the 25-s shock off) was recorded by mean of a computerized data acquisition system (Basilink, Ugo Basile). For the evaluation of rats performances, only the FR2 escape responses were analysed. It has already been noted, in fact, that rats exposed to inescapable shock often do not fail to learn FR1 shuttle-box response, whereas the FR2 response is always reduced (Maier and Seligman 1976). Drug administration. &p.2:Three different experiments were conducted: one with BIMT 17 base given acutely and the others with imipramine given acutely or chronically. In the experiment with BIMT 17, 96 rats were divided into four experimental blocks of 24 rats each (four rats for each group). The experimental groups were 6 (IS; IS+ 0; IS+ 24; IS+ 36; IS+ 48 and IS+ 60 mg/kg BIMT 17 base) of 16 rats each. Two experimental blocks per week were carried out. BIMT 17 was given orally, in a volume of 5 ml/kg, 30 min before testing. The compound was solubilized in the following vehicle: 25% v/v polyethylene glycol-400 acidified (1.5% citric) distilled water. Imipramine hydrochloride (Sigma) was dissolved in saline and given IP at doses of 4, 8 and 16 mg/kg on a volume of 2 ml/kg. Doses of imipramine refer to the base. Imipramine was given either acutely or repeatedly. The repeated treatment with imipramine was performed as follows: the first injection was done 2 h after the session of inescapable shock (day 1); injections were given twice daily (0800 and 1600 hours) on day 2 and 3. On day 4, the animals were injected 30 min before the shuttle-box test. Acute treated rats received vehicle on day 1, 2 and 3 and the test compound in the last injection (day 4). Control groups (IS and IS+) received the corresponding vehicle treatment. Data analysis. &p.2:The behavioural data (FR2 escape failures and intertrial crossings) were expressed as medians and interquartile range. The Wilcoxon two-sample test was used to compare the results relative to the control groups (IS+ versus IS). The effect of treatment versus IS+ group on the number of escape failures was analysed by the Kruskal-Wallis test followed by the Dunn test. The effect of treatment versus IS- group on the number of intertrial crossings was analysed by the Kruskal-Wallis test. The aim of the latter comparison was to verify if the motility of the drug-treated animals was greater than the motility of unshocked rats. The statistical evaluations were carried out with the program system SAS (SAS Institute Inc., Cary, North Carolina), Version 6.07 on a DEC Computer, o.s. VMS.

381 Plasma levels of BIMT 17 Plasma samples (0.5 ml) were addedd with 100 l of internal standard (1 g/ml BIMT 31), adjusted to 3 ml with 0.5 M KH2PO4, vortexed and added to solid-phase extraction cartridges, conditioned with 2.5 ml methanol followed by 2.5 ml water. The cartridges were washed twice with 1 ml of a mixture acetonitrile/water (1:1, v/v) and dried under vacuum for about 30 min. The analytes were eluted twice with 1 ml ammonia solution in methanol (33%) and taken to dryness under nitrogen stream at about 45C. The dried samples were taken up in mobile phase and injected into the HPLC system (Perkin-Elmer), consisting of a Series LC 250 pump, ISS 200 autosampler, UV/Vis detector mod LC 95 and Turbochrom data collection system. Mobile phase consisted of 28% (v/v) of acetonitrile and 72% of the following acqueous solution: 10 mM KH2PO4 and triethylamine adjusted at pH 3 with 2 M H3PO4. BIMT 17 and internal standard were separated with a reverse phase ODS Hypersyl column (1002.1 mm i.d.; HewlettPackard). Column temperature was set at 40C and flow rate was 0.26 ml/min. Detection was UV at = 210 nm; under these chromatographic conditions, approximate retention time for BIMT 17 and internal standard were 10 and 13 min, respectively. Three different groups of animals (six rats/group) were administered IP with 10 or 20 mg/kg or orally with 48 mg/kg. Plasma levels were collected 60 min after the IP injections or 30 min after the oral administration.

Fig. 2 Effect of BIMT 17 in the open-field in sham and OB rats. Results are expressed as median, and interquartile range (in brackets), of nine animals. Rats were placed in the open field after 2 weeks of treatment with BIMT 17 (IP, once daily). The test was carried out on day 15, i.e. 24 h after the last injection. # P<0.05 versus Sham-vehicle; * P<0.05 versus OB-vehicleig.c:&/f

Results
Bulbectomized rats Prior to drug treatment, the mean body weights (g SEM) of the control OB group were reduced when compared to its sham group prior to treatment (344 6 and 370 9, respectively), as demonstrated by a significant lesion effect [F(1, 48) = 5.98; P = 0.018], but no drug effect [F(2, 48) = 0.14; P = 0.873], or lesion*drug interaction [F(2, 48) = 0.37; P = 0.695]. On day 15, the mean body weights for OB and sham control groups were 361 13 and 417 9, respectively (P<0.01). At this time, the lesion effect was still evident [F(1, 48) = 22.14; P = 0.000], but there was no drug effect [F(2, 48) = 0.03; P = 0.970], or lesion*drug interaction [F(2, 48) = 1.34; P = 0.272]. A typical hyperactivity was observed in OB rats when placed in the open field apparatus when compared to shams (P < 0.05) (Fig. 2); in contrast, no significant differences were found in rearing, grooming or defecation (data not shown). Chronic 10 mg/kg BIMT 17 (P<0.05) but not 20 mg/kg BIMT 17 treatment significantly reduced the increase in ambulation of OB rats (Fig. 2), without affecting rearing, grooming or defecation in sham or OB rats (data not shown). DRL 72-s There was no significant effect of acute BIMT 17 treatment on response [F(5, 70) = 1.18] and reinforcement [F(5, 70) = 1.61] rate up to a dose of 20 mg/kg (top panel on Fig. 3), whereas imipramine decreased [F(5, 70) = 27.65; P < 0.0001] and increased [F(5, 70) = 12.64; P < 0.0001] these parameters, respectively (top panel on The acute oral administration of BIMT 17 caused a reduction (Kruskal-Wallis test P = 0.041) in the number of escape failures of helpless rats during the shuttle-box test session (Fig. 5). The effect was statistically significant after administration of 48 mg/kg BIMT 17. The reversal of the escape deficit was obtained without affecting the number of intertrial crossings (Table 1). The single adFig. 3). A higher dose of BIMT 17, 40 mg/kg, was used but produced an almost catatonic state and failures in nine of 15 rats to respond in the operant chamber 1 h after administration. Examination of the histograms (Fig. 4) shows that the BIMT 17 and imipramine had very different effects on the IRT distribution profiles. BIMT 17 decreased PkA [F(5, 70) = 15.04; P < 0.0001] at 15 and 20 mg/kg: such a decrease is reflected in the IRT histograms presented on the left side of Fig. 3. BIMT 17 increased the dispersion of the waiting time distribution (Fig. 4). In contrast, imipramine had no effect on PkA [F(5, 70) = 0.62] (Fig. 3). Thus, BIMT 17 shifted the peak of the IRT distribution [F(5, 70) = 3.7; P < 0.01] toward shorter IRT durations (bottom right panel of Fig. 3). This effect was significant at the 15 mg/kg dose. The BIMT 17-induced shift of PkL toward shorter waiting times is graphically shown in the IRT distributions presented in Fig. 4. In contrast, imipramine shifted the peak of the IRT distribution [F(5, 70) = 7.56; P < 0.0001] toward longer IRT durations in a dose-dependent fashion (Fig. 3). The imipramine induced shift of PkL toward longer waiting times is graphically shown in the IRT distribution presented on the right side of Fig. 4. Learned helplessness

382 Fig. 3 Effects of BIMT 17 and imipramine on response rate, reinforcement rate, peak area, and peak location. Peak area and location are metrics designed to measure the IRT distribution profile (see text and Fig. 4). BIMT 17, imipramine and vehicle were given IP 1 h before the session. * P < 0.05 versus respective V-groupig.c:&/f

Table 1 Effect of acute BIMT 17 and imipramine, and repeated imipramine on the intertrial crossings in the LH test in rats&/tbl.c:

Group Acute bimt 17 IS IS+ IS+ IS+ IS+ IS+ Acute imipramine ISIS+ IS+ IS+ IS+ Repeated imipramine IS IS+ IS+ IS+ IS+

Treatment

Dose mg/kg

No. of intertrial crossings median (interquartile range)

BIMT 17 was given orally 30 min before testing. Imipramine was given as a single or a repeated administration. Imipramine was given as a single dose 30 min before testing. Repeated administered imipramine was given i.p. 2 h after inescapable shock on day 1, twice daily on days 2 and 3, 30 min before testing on day 4. Group sizes were constituted by 16 rats for BIMT 17 experiment and by 8 rats for the imipramine experiments. KruskalWallis test: P >0.05 &/tbl.:

Vehicle Vehicle BIMT 17 BIMT 17 BIMT 17 BIMT 17 Vehicle Vehicle Imipramine Imipramine Imipramine Vehicle Vehicle Imipramine Imipramine Imipramine

0 0 24 36 48 60 0 0 4 8 16 0 0 4 8 16

0.5 2.0 0.5 2.0 2.0 2.0 0 0.5 1.5 0 0 0 0.5 0.5 0 0.5

(01) (03) (01) (14.5) (14.5) (0.54) (00.5) (01.5) (0.52) (01) (00) (00) (01) (02.5) (00.5) (01)

ministration of 4, 8 and 16 mg/kg imipramine (Fig. 5) was completely devoid of effects (Kruskal-Wallis test P = 0.88). The repeated administration of 4, 8 and 16 mg/kg imipramine decreased the number of escape failures of helpless rats during the shuttle-box test session (Kruskal-Wallis test P = 0.001), without affecting the number of intertrial crossings (Table 1), 8 and 16 mg/kg being the active doses.

Plasma levels The plasma levels (g/ml) of BIMT 17 60 min after its IP administration with 10 or 20 mg/kg were (mean SEM) 1.24 0.05 and 1.59 0.18, respectively; whereas 30 min after its oral administration with 48 mg/kg they were (mean SEM) 1.27 0.36.

383 Fig. 4 Interresponse time (IRT) histograms visualizing how BIMT 17 and imipramine effected the DRL 72-s IRT distribution profiles. The IRT distributions are bimodal with one mode at short IRT durations (IRT < 6 s; shaded histogram bar on the left) indicating bursting and a second mode at longer IRT durations (IRT 6 s; open histogram bars) indicating pausing. This second mode shows that the rats have learned to wait between responses (although not quite long enough). The BIMT 17 vehicle histogram (top left histogram) illustrates how the IRT distribution profile is quantitatively characterized by the peak area (PkA) and peak location (PkL) metrics (see text for explanation). The triangle in the burst category indicates the relative frequency of burst responses predicted by extrapolation of the corresponding negative exponential into the burst component. The single dot at the far right of the histogram indicates the relative frequency of IRTs > 144 s predicted to occur in the tail of the corresponding negative exponential. Similarly, the single histogram bar at the far right indicates the relative frequency of IRTs > 144 s in the tail of the obtained IRT distribution. The dashed vertical line indicates the 72-s IRT duration requirement for reinforcement. The histograms represent the averaged relative frequencies of 15 ratsig.c:&/f

Discussion
Given orally, BIMT 17 is less potent than when given IP (Cesana et al. 1995), and this finding has been confirmed in the present study where IP 10 mg/kg and oral 48 mg/kg gave similar plasma levels. Both the OB and LH models only permit the observation of very strong effects, due to either the small window for drugs to restore a normal behaviour or to the variability of data. In fact, in both tests, the data were expressed as median and interquartiles because the distribution of values was not Gaussian. This might explain why the effect of BIMT 17 in the OB model apparently appears similar at both doses, 10 and 20 mg/kg, but only the lowest dose induced a statistically significant effect. Similar consideration holds with the LH model, where the highest dose exerted an effect which was smaller than that observed at a lower dose. Alternatively, it may be that doses greater than 10 mg/kg IP (or 48 mg/kg PO, which produces similar plasma levels of 10 mg/kg IP) interfere with rat performance, as also evidenced by DRL 72-s.

Bulbectomized rats The ablation of the olfactory bulbs induces many behavioural changes in the rat (Leonard 1984; Jesberger and Richardson 1986). The primary index of potential antidepressant activity is reversal of increased ambulation scores (Jancsar and Leonard 1983; Van Riezen and Leonard 1990). Repeated administration of BIMT 17 at a dose of 10 mg/kg significantly reversed the increased ambulation associated with the OB rat in the open field test. Reversal of increase in ambulation in OB rats has also been reported for antidepressants, both typical and atypical including selective serotonin reuptake inhibitors, but not for centrally acting drugs which lack antidepressant activity (Jancsar and Leonard 1983; Leonard 1984; Song and Leonard 1994): repeated treatment with these antidepressants seems to be necessary to observe an effect (Van Riezen et al. 1977; Leonard 1982, 1984; Song and Leonard 1994). Interestingly, OB rats have an altered 5-HT metabolism in the frontal cortex (Lumia et al. 1992), and in oth-

384

Fig. 5 Effect of BIMT 17 and imipramine on the escape failures in the LH test. Results are expressed as median, and interquartile range (in brackets). BIMT 17 (top panel) was given orally 30 min before testing. Imipramine was given as a single ( middle panel) or a repeated (bottom panel) administration. Imipramine was given as a single dose 30 min before testing. Repeated administered imipramine was given IP 2 h after inescapable shock on day 1, twice daily on days 2 and 3, 30 min before testing on day 4. Group sizes were constituted by 16 rats for BIMT 17 experiment and by eight rats for the imipramine experiments. # P < 0.01 versus IS+ (Wilcoxon two-sample test); * P < 0.01 versus vehicle-treated group (Dunn test)ig.c:&/f

er brain regions. In addition, OB mice showed an increase in 5-HT2 receptors only in the frontal cortex (Gurevich et al. 1993) and, in these animals, repeated administration with amitriptyline and trazodone had the common effect of reducing the number of 5-HT1A receptors in the frontal cortex but not in other areas (Gurevich et al. 1993). The OB rodent model has been suggested as a model for agitated hyposerotonergic depression (Lumia et al. 1992). Thus, BIMT 17, an agonist at 5-HT1A receptors and an antagonist upon 5-HT2A receptors in the frontal cortex (Borsini et al. 1995a,b), showed an antidepressant-like effect in the OB model following chronic administration. DRL 72-s The profiles of the acute effects of BIMT 17 and the antidepressant imipramine on DRL 72-s performance are very different. BIMT 17 had no effect on response and reinforcement rate while imipramine decreased response rate and increased reinforcement rate. BIMT 17, but not imipramine, induced decreases in PkA. BIMT 17 shifted the PkL toward shorter IRT durations while imipramine shifted PkL toward longer IRT durations. Previous re-

search (Seiden et al. 1985; Marek and Seiden 1988a; Richard et al. 1993b) has shown that a variety of antidepressant compounds shift the peak of the IRT distribution toward longer IRT durations without decreasing PkA. This shift in the IRT distribution toward longer waiting times results in an increased reinforcement rate. By these criteria, BIMT 17 does not resemble the acute effects of antidepressants on the DRL 72-s screen. Although BIMT 17 did not affect response and reinforcement rate at the doses tested, it did significantly affect the IRT distribution profile. The shift in the peak toward shorter waiting times and the increased dispersion of the waiting times indicates that BIMT 17 disrupted performance at doses greater than 10 mg/kg. It has been suggested that antidepressants may act in DRL 72-s by blocking 5-HT2 receptors and activating 5HT1A receptors (Marek and Seiden 1988b; Marek et al. 1989a,b). Even if not all 5-HT1A agonists exert an antidepressant-like effect in this test (Richards et al. 1994), it is surprising that BIMT 17, a 5-HT2A antagonist and 5HT1A agonist in the cortex, did not affect DRL 72-s behaviour. The fact that BIMT 17 is the first full postsynaptic agonist (Borsini et al. 1995a,b) differentiates it from the other agonists. In fact, it should be pointed out that 8-OH-DPAT and buspirone do not behave as agonists in the frontal cortex (Borsini et al. 1995b). Single doses of 8-OH-DPAT and buspirone (Borsini et al. 1995a), imipramine (Ceci et al. 1992) or fluoxetine (Ceci et al. 1993) increase the firing rate of frontocortical neurons, whereas BIMT 17 decreases the firing rate by activating 5-HT1A receptors (Borsini et al. 1995a). In addition, BIMT 17 has a direct action on postsynaptic 5-HT receptors (Borsini et al. 1995a; Cesana et al. 1995), whereas all the other compounds referred to seem to activate presynaptic 5-HT mechanisms. It may be that this difference in the mechanism of action can explain the different effect of BIMT 17 in comparison with antidepressants in this animal model. Learned helplessness Among the animal models of depression, the LH possesses an interesting degree of face validity and predictive validity (Willner 1991). In fact, reversal of LH is obtained after repeated, but not after acute, administration of tricyclic antidepressants, monoamine oxidase inhibitors, selective 5-HT reuptake inhibitors and electroconvulsive shock. Reversal of LH is also obtained after acute administration of psychostimulant compounds (e.g. amphetamine) but in contrast to antidepressants, they also affect locomotor activity, increasing the number of intertrial crossings (Christensen 1993). The reversal of LH observed after the single administration of BIMT 17 did not seem to depend on modification of the number of intertrial crossings. Thus, BIMT 17 induced an antidepressant-like behavioural effect in the LH paradigm after a single administration whilst antidepressants must be given repeatedly.

385

It is worth noting that a supersensitivity of 5-HT2 receptors possibly related to LH behaviour has been suggested and that the repeated administration of antidepressants induces a down-regulation of these receptors which parallels the reversal of LH behaviour (Barone et al. 1990; Nakai et al. 1995). In addition, it has been reported that fluoxetine, after repeated treatment, prevents the escape deficit in this animal model also by activating 5HT1A receptors, although a different experimental protocol of the test was used (Gambarana et al. 1995). Interestingly, the helpless behavior is reversed in the LH model by repeated treatment with putative 5-HT1A agonists (Giral et al. 1988) and it has been shown that their effect is due to the activation of postsynaptic 5-HT1A receptors (Martin et al. 1990, 1991). Moreover, it has been reported that LH induction modifies in vivo cortical 5HT release (Petty and Sherman 1983; Petty et al. 1994) and that tricyclic antidepressants normalize this change (Sherman and Petty 1982); such effects correlate with antidepressant concentrations in the anterior neocortex (Petty et al. 1982). The prefrontal cortex was the only brain region in which microinjection of 5-HT reversed learned helplessness 1 h after injection (Petty and Sherman 1980). Due to the serotonergic activity of BIMT 17, it is not surprising that BIMT 17 can reverse the behavioural deficit of rats in the LH after a single administration. General discussion The purpose of this study was two-fold. Firstly, to confirm the potential antidepressant activity of BIMT 17 as previously shown in two animal paradigms sensitive to antidepressants, i.e. the forced swimming test (Cesana et al. 1995) and the chronic mild stress procedure (Willner 1995). Thus, BIMT 17 was tested in OB rats, in the LH procedure and in the DRL 72-s model and induced antidepressant-like effects in the former two paradigms but not in the latter one. Secondly, to confirm the faster onset of action of BIMT 17, as has already been seen in the chronic mild stress procedure (Willner 1995), by using the LH paradigm where it was found that BIMT 17 induced antidepressant-like effect after a single administration. It is worth noting that BIMT 17 and not imipramine induced an effect in the LH, whereas imipramine and not BIMT 17 induced an effect on DRL 72-s. These differences are not surprising, as these two compounds may exert different effects on the 5-HT-containing neurons. 5HT uptake inhibitors, after a single administration, are more effective on the dendro-somatic region than on the postsynaptic 5-HT receptors (Adell and Artigas 1991; Artigas 1993; Bel and Artigas 1993). Thus, the different effect of BIMT 17 and the 5-HT uptake inhibitors may depend on this different location of action. This may be also substantiated by the fact that the 5-HT1A agonist, 8OH-DPAT, produces an effect when microinjected at postsynaptic receptors in the LH paradigm whereas the

action of a low dose of fluoxetine in the DRL 72-s paradigm is reduced after destruction of 5-HT-containing neurons by a selective 5-HT neurotoxin (Marek et al. 1989a). Similar results with fuoxetine and the 5-HT neurotoxin were also obtained in the LH test (Martin et al. 1990). Thus, the different activities of acute BIMT 17 and imipramine may depend on their postsynaptic (BIMT 17) and presynaptic (imipramine) component. Interestingly, when the postsynaptic component of imipramine is unmasked after repeated treatment, an effect common to antidepressants (see Borsini 1994), imipramine was also active in the LH. A study to investigate the exact mechanism of action of BIMT 17 in the LH is currently under investigation. In conclusion, taken as a whole, these results indicate that BIMT 17 does not behave like imipramine in all the tests and suggest that BIMT 17 acts through different mechanisms of action than imipramine. Only clinical trials will tell whether these mechanisms will be relevant, but if so, BIMT 17 might induce a faster onset of therapeutic activity.
&p.2:Acknowledgements This study was mostly supported by IMI contract no. 55312/46.

References
Adell A, Artigas F (1991) Differential effects of clomipramine given locally or systemically on extracellular 5-hydroxytryptamine in raphe nuclei and frontal cortex. Naunyn-Schmiedebergs Arch Pharmacol 343: 237244 Artigas F (1993) 5-HT and antidepressants: new views from microdialysis studies. Trends Pharmacol Sci 14: 262 Bel N, Artigas F (1993) Chronic treatment with fluvoxamine increases extracellular serotonin in frontal cortex but not in raphe nuclei. Synapse 15: 243245 Barone P, Atger F, Martin P, Puech A, Fillion G (1990) Increased number of 5HT2 receptors in the learned helplessness rat. Eur J Pharmacol 183: 19001901 Borsini F (1994) Balance between cortical 5-HT 1A and 5-HT2 receptor function: hypothesis for a faster antidepressant action. Pharmacol Res 30: 111 Borsini F, Ceci A, Bietti G, Donetti A (1995a) BIMT 17 a 5-HT 1A agonist/5-HT2A antagonist, directly activates postsynaptic 5HT inhibitory responses in the cortex. Naunyn-Schmiedebergs Arch Pharmacol 352: 283290 Borsini F, Giraldo E, Monferini E, Antonini G, Parenti M, Bietti G, Donetti A (1995b) BIMT 17, a 5-HT 2A antagonist and 5HT1A receptor full agonist, in rat cerebral cortex. NaunynSchmiedebergs Arch Pharmacol 352: 276282 Cairncross KD, Wren AF, Cox B, Schnieden H (1977) Effects of olfactory bulbectomy and domicile on stress induced corticosterone release in the rat. Physiol Behav 19: 485487 Ceci A, Baschirotto A, Borsini F (1992) Effect of 5HT monoamine uptake blockers and 5HT1A agonists on spontaneous firing rate of prefrontal cortical neurons. Soc Neurosci Abstr 18: 317.1 Ceci A, Baschirotto A, Borsini F (1993) Effect of fluoxetine on the spontaneous electrical activity of fronto-cortical neurons. Eur J Pharmacol 250: 461464 Cesana R, Ciprandi C, Borsini F (1995) The effect of BIMT 17, a new potential antidepressant, in the forced swimming test in mice. Behav Pharmacol 6: 688694 Christensen AV (1993) Learned helplessness: an animal model of depression? Neuropsychopharmacology 9: 167S Gambarana C, Ghieri O, Taddei A, Tagliamonte, de Montis MG (1995) Imipramine and fluoxetine prevent the stress-induced

386 escape deficits in rats through a distinct mechanism of action. Behav Pharmacol 6: 6673 Geoffroy M, Scheel-Kruger J, Christensen AV (1990) Effect of imipramine in the learned helplessness model of depression in rats is not mimicked by combination of specific reuptake inhibitors and scopolamine. Psychopharmacology 101: 371375 Giral P, Martin P, Soubrie P, Simon P (1988) Reversal of helpless behavior in rats by putative 5-HT1A agonists. Biol Psychiatry 23: 237242 Gray JA, Lalljee B (1974) Sex differences in emotional behaviour in the rat: correlation between the open field defecation and active avoidance. Anim Behav 22: 856861 Gurevich EV, Aleksandrova IA, Otmakhova NA, Katkov YA, Nesterova IV, Bobkova NV (1993) Effects of bulbectomy and subsequent antidepressant treatment on brain 5-HT2 and 5HT1A receptors in mice. Pharmacol Biochem Behav 45: 6570 Jancsar S, Leonard BE (1983) The olfactory bulbectomized rat as a model of depression. In: Usdin E, Goldstein M, Friedhoff AJ, Georgotas A (eds) Frontiers in neuropsychiatric research. MacMillan, New York, pp 357372 Jesberger JA, Richardson JS (1986) Effects of antidepressant drugs on the behavior of olfactory bulbectomized and shamoperated rats. Behav Neurosci 100: 256274 Leonard BE (1982) On the mode of action of mianserin. Adv Biochem Psychopharmacol 31: 301320 Leonard BE (1984) The olfactory bulbectomized rat as a model of depression. Pol J Pharmacol Pharm 36: 561569 Leonard BE, Tuite M (1981) Anatomical, physiological and behavioural aspect of olfactory bulbectomy in the rat. Int Rev Neurobiol 22: 251286 Lumia AR, Teicher MH, Salchli F, Ayers E, Possidente B (1992) Olfactory bulbectomy as a model for agitated hyposerotonergic depression. Brain Res 587: 181185 Maier SF, Seligman MEP (1976) Learned helplessness: theory and evidence. J Exp Psychol Gen 105: 346 Marek GJ, Seiden LS (1988a) Selective inhibition of MAO-A, not MAO-B, results in antidepressant-like effects on DRL 72-second behavior. Psychopharmacology 96: 153160 Marek GJ, Seiden LS (1988b) Effects of selective 5-hydroxytryptamine-2 and nonselective 5-hydroxytryptamine antagonists on the differential-reinforcement-of-low-rate 72-second schedule. J Pharmacol Exp Ther 244: 650658 Marek GJ, Li AA, Seiden LS (1989a) Evidence for involvement of 5-hydroxytryptamine-1 receptors in antidepressant-like drug effects on differential-reinforcement-of-low-rate 72-second behavior. J Pharmacol Exp Ther 250: 6071 Marek GJ, Li AA, Seiden LS (1989b) Selective 5-hydroxytryptamine-2 antagonists have antidepressant-like drug effects on differential-reinforcement-of-low-rate 72-second schedule. J Pharmacol Exp Ther 250: 5259 Martin P, Beninger RJ, Hamon M, Puech AJ (1990) Antidepressant-like action of 8-OH-DPAT, a 5-HT1A agonist, in the learned helplessness paradigm: evidence for a postsynaptic mechanism. Behav Brain Res 38: 135144 Martin P, Tissier MH, Adrien J, Puech AJ (1991) Antidepressantlike effects of buspirone mediated by the 5-HT1A post-synaptic receptors in the learned helplessness paradigm. Life Sci 48: 25052511 Nakai M, Yamada S, Muneoka K, Tiru M (1995) Increased 5-HT2 receptor-mediated behavior 11 days after shock in learned helplessness rats. Eur J Pharmacol 281: 123130 Petty F, Sherman AD (1983) Learned helplessness induction decreases in vivo cortical serotonin release. Pharmacol Biochem Behav 18: 649650 Petty F, Sherman AD (1980) Regional aspects of the prevention of learned helplessness by desipramine. Life Sci 26: 14471452 Petty F, Sacquitne JL, Sherman AD (1982) Tricyclic antidepressant drug action correlates with its tissue levels in anterior neocortex. Neuropharmacology 21: 475477 Petty F, Kramer G, Wilson, Jordan S (1994) In vivo serotonin release and learned helplessness. Psychiatry Res 52: 285293 Richards JB, Seiden LS (1991) A quantitative interresponse-time analysis of DRL performance differentiates similar effects of the antidepressant desipramine and the novel anxiolytic gepirone. J Exp Anal Behav 56: 173192 Richards JB, Sabol KE, Seiden LS (1993a) DRL interresponsetime distributions: quantification by peak deviation analysis. J Exp Anal Behav 60: 361385 Richards JB, Sabol KE, Seiden LS (1993b) Fluoxetine prevents the disruptive effects of fenfluramine on differential-reinforcement-of-low-rate 72-second schedule performance. J Pharmacol Exp Ther 267: 12561263 Richards JB, Sabol KE, Hand TH, Jolly DC, Marek GJ, Seiden LS (1994) Buspirone, gepirone, ipsapirone, and zalospirone have distinct effects on the differential-reinforcement-of-low-rat 72second schedule when compared with 5-HTP and diazepam. Psychopharmacology 114: 3946 Seiden LS, Dahms JL, Shaughnessy RA (1985) Behavioral screen for antidepressants: the effect of drugs and electroconvulsive shock on performance under a differential-reinforcement-oflow-rate schedule. Psychopharmacology 86: 5560 Sherman AD, Petty F (1982) Additivity of neurochemical changes in learned helplessness and imipramine. Behav Neural Biol 35: 344353 Song C, Leonard BE (1994) Serotonin reuptake inhibitors reverse the impairments in behaviour, neurotransmitter and immune functions in the olfactory bulbectomized rat. Hum Psychopharmacol 9: 135146 Van Riezen H, Leonard BE (1990) Effects of psychotropic drugs on the behaviour and neurochemestry of olfactory bulbectomized rats. Pharmacol Ther 47: 2143 Van Riezen H, Schneiden H, Wren AF (1977) Olfactory bulb ablation in the rat: behavioural changes and their reversal by antidepressive drugs. Br J Pharmacol 60: 521346 Willner P (1991) Animal models of depression. In: Willner P (ed) Behavioural models in psychopharmacology. University Press, Cambridge, pp 91125 Willner P (1995) Animal models of depression: validity and applications. In: Gessa G, Fratta W, Pani L, Serra G (eds) Depression and mania: from neurobiology to treatment. Raven Press, New York, pp 1941