Neuropharmacological profile of a novel and selective ligand of the sigma site: SR 31742A

Newophwmacology Vol. 32,No. 6, pp. 60-15, 1993 Print& in Great Britain. All rights ramed

Copyright 0

0028.3908/93 $6.00+ 0.00 1993 Pergamon PressLtd

NEUROPHARMACOLOGICAL PROFILE OF A NOVEL AND SELECTIVE LIGAND OF THE SIGMA SITE: SR 31742A M. PONCELET, V. SANTLXXI, R. PAUL, C. G-ET,

S. LAVASTRF,,J. GUITARD, R. STEINBERG,

J. P. TERRANOVA, J. C. BRELR%E, P. SOUBRIB and

G. LE FUR Sanofi Recherche, Neuropsychiatry Department, 371 rue du Professeur J. Blayac, 34184 Montpellier Cedex 4, France (Accepted 29 October1992) Summary-The biochemical, electrophysiological and behavioural effects of SR 31742% a novel and selective ligand of sigma sites in brain, labelled with (+)-[3H]3PPP (Ki = 5.3 f 0.3 nM), were investigated in rodents and compared with those of DA antagonists having (halo~ridol) or not (spiro~~dol) a high affinity for sigma sites. Like haloperidol but unlike spiroperidol, SR 31742A, (ED, = 0.065 mg/kg, i.p., and 0.21 mg/kg, p.o.) antagonized sigma-dependent turning behaviour in mice and inhibited (0.5 mg/kg, i.v.) the spontaneous firing of hippocampal (CA,) neurones in urethane-anaesthetized rats. In chloral hydrate-anaesthetized rats, like classical antipsychotic compounds, SR 31742A (0.625-5 mg/kg, i.p.) increased the number of spontaneously active 4 and A,,, DA cells after single administration and produced an opposite effect after repeated injections. The drug SR 31742A reduced (2.5, 5, lOmg/kg, i.p.) the hyperactivity elicited by various drugs including that produced by injection of (+)-amphetamine into the nucleus accumbens and impaired avoidance responses at doses (5, 10 mg/kg, i.p.), sparing escape behaviour. SR 31742A lacked affinity for DA receptors and neither did the compound induce catalepsy nor antagonize such effects elicited by apomorphine as climbing, hypothermia, stereotypy or the inhibition of firing of DA neurones. SR 317429 did not affect the basal metabolism of DA but at 10 mg/kg (i.p.) it significantly reduced the amphetamine-induced rise in levels of 3-MT in the striatum of mice. Together, these results indicate a modulatory role for sigma sites upon the activity of hippocampal and DA systems and that sigma ligands exert effects, which suggest antipsychotic potential. Key words-SR

31742A, sigma sites, DA neurones, hippocampus, antipsychotic drugs, rodents.

Radioligand studies have revealed the existence in brain of sigma binding sites, distinct from dopamine (DA), phencyclidine and classical opiate receptors (Quirion, Bowen, Itzhak, Junien, Musacchio, Rothman, Su, Tam and Taylor, 1992). These binding sites are particularly abundant in cortical and limbic areas, as well as in DA-rich regions of the mammalian (including human) brain ~eissman, Casanova, Kleinman, London and De Souza, 1991). Investigation of the functional significance of sigma sites in brain has been hampered by the relative lack of specificity of drugs used as tools and by the lack of a coherent classification into postulated agonists and antagonists (Itzhak and Stein, 1990; Quirion et al., 1992). Thus, although some hypotheses have been proposed in terms of second messenger transduction systems (Itzkak and Stein, 1990) or of ion channels (Bobker, Shen, Surprenant and Williams, 1989), the sigma site-coupled events remain to be elucidated. Two lines of evidence however formed the basis for the hypothesis that sigma ligands might have antipsychotic activity. Sigma ligands, such as benzomorphan derivatives, are known to produce hallucinations, dysphoria and de~~nalisation although the involvement of sigma

sites in such phenomena has recently been challenged (Musacchio, Klein and Canoll, 1989). On the other hand, prototype neuroleptic drugs (e.g. haloperidol) display a nanomolar affinity for sigma sites and these sites are reportedly involved in the regulation of neuronal activity of DA neurones in the midbrain (Iyengar, Dilworth, Mick, Contreras, Monahan, Rao and Wood, 1990; Steinfels and Tam, 1989; Engberg and WikstrGm, 1991). In addition, Weissman er al. (1991) reported alterations in sigma binding in schizophrenic patients. On this account, an original compound (Fig. 1), SR 3 1742A [cis-3-(hexahydroazepin-1 -yl)l-(3-chloro4-cyclohexylphenyl)propene- 1, hydrochloride], displaying a high and selective affinity for central sigma sites, was investigated on animal models selected in order to assess the potential antipsychotic activity of this compound and its propensity to produce the side effects associated with conventional antipsychotics. For this purpose, two series of experiments have been performed. The first was aimed at uncovering tests able to discriminate pure antipsychotic DA blockers (e.g. spiroperidol) from antipsychotics having additional high affinity for sigma receptors (e.g. halo~~dol) and at studying SR 31742A on

605

M. PONCELETet al.

606

these tests. The second was devoted to the investigation of the effects of SR 31742A on various laboratory screens for neuroleptic drugs.

‘I’

(IQ\

Cl

METHODS

Animals

Female CD, (20-25 and 25-30g) and OF, mice (22-26 g) were used for in vivo binding assays, turning behaviour and drug-induced hyperactivity, respectively. Male Wistar rats (150-200 g) were used for methylphenidate-induced hyperactivity and conditioned avoidance responding. Male SpragueDawley rats (180 -410 g) were used for in vitro binding assays, amphetamine intra-accumbens-induced hyperactivity and electrophysiological studies. Animals were from Charles River or Iffa Credo, France. One week before the experiments, mice were caged in groups of 10 and rats in groups of 3 (except for chronically implanted rats, which were caged singly) and housed in a room at constant temperature (21 + 1’C) on a automatic dark/light schedule (light 7a.m.-7p.m.) with food and tap water freely available.

N

3

Fig. 1. Chemical structure of SR 31742A [cis-3-(hexahydroazepin-l-yl)l-(3-chloro-4-cyclohexylphenyl)pro~ne-1 hydrochloride]. vitro binding to [3H]DTG receptors was performed

ively.

according to Weber, Sonders, Quarum, MacLean, Pou and Keana (1986) in membranes from rat brain (6OOpg protein per assay), incubated for 90 min at 25°C in the presence of [3H]DTG (2 x 10m9M) and 10-‘0-10-5M of SR 31742A (triplicate determinations). Non-specific binding was determined in the presence of IO-’ M DTG. In vitro binding to (+)-[ ‘Hlpentazocine receptors was performed according to de Costa, Bowen, Hellewell, Walker, Thurkauf, Jacobson and Rice (1989) in membranes from rat brain (750 pg protein per assay), incubated for 120 min at 25°C in the presence of 3.10V9M (+)-[3H]pentazocine and IO-” to 10m6M of SR 31742A (triplicate determinations). Non-specific binding was determined in the presence of pentazocine 10m5M. Proteins were assayed according to Lowry, Rosebrough, Farr and Randall (1951). In vivo binding of ( +)-[3H]3PPP was performed in mice according to Koe, Burkhart and Lebel (1989). Briefly, (+)-[ ‘HJ3PPP was injected intravenously (370 kBq/mouse) 10 min before sacrifice. Brains (without cerebellum) were homogenized in 20ml Tris-HCl 50mM buffer, pH 8.0 and aliquots of 1 ml were filtered on to GF/B, previously soaked with polyethylenimine (PEl). The SR 31742A (0.5-10 mg/kg) was administered 20 (i.p.) or 60 min (p.0.) before sacrifice. In the time-course study, SR 31742A was administered 1, 2, 4, 8, 16 and 24 hr before sacrifice. For control, the in vivo affinity of SR 31742A for DA receptors was investigated. For this purpose [ ‘Hlspiroperidol (74 kBq/mouse) was injected 30 min before sacrifice, SR 3 1742A being given intraperitoneally (10 mg/kg) 60 min before sacrifice. Nonspecific binding was determined with haloperidol (5 mg/kg, i.p.). Groups of 5 mice were used per dose or time.

Binding assays

Behavioural studies

In vitro binding to ( +)-[3H]3PPP receptors was performed according to Largent, Gundlach and Snyder (1986) in membranes from rat brain (750 pg protein per assay) incubated for 90 min at 4°C in the presence of (+)-[3H]3PPP (3 x 10e9 M) and 10-‘1-10-6M of SR 31742A (triplicate determinations). Non-specific binding was determined in the presence of haloperidol lo-* M. In saturation experiments, SR 31742A (0 and 8 x 10e9 M) was added to (+)-[‘H]3PPP (2 x lo-“-2 x lo-‘M). In

Turning behaviour in mice. Intrastriatal injection was performed according to Worms, Gueudet and Bizilre (1986). Briefly, the injection was made freehand in a volume of 1~1, in the right striatum of conscious, non-restrained mice, by means of a 5 ~1 Hamilton microsyringe and a 10 mm calibrated needle (final length below the skin: 3.5 mm). The descending point of the needle was lightly internal and caudal to the right orbitus. The duration of the injection was 2-3 sec. Control mice received 1~1 of

Drugs (+)-[ 3H]3PPP {( +)-[ ‘HI-3-(3-hydroxyphenyl)-N(I-propyl) piperidine, 90 Ci/mmol}, [ ‘HJDTG ([‘HI1,3-Di-o-tolylguanidine, 50 Ci/mmol}, {[3H] spiroperidol, 30 Ci/mmol} and (+)-[ 3H]pentazocine, 42Ci/mmol} were from (N.E.N.). The SR 31742A HCl (Sanofi Recherche, Montpellier, France), (+)amphetamine sulphate, cocaine HCl (Cooperative Pharmaceutique Francaise), methylphenidate HCl (Ciba-Geigy), morphine HCl (Sanofi Recherche), apomorphine HCl (Sigma) and spiroperidol (RBI) were dissolved in sterile distilled water. Haloperidol (Sigma) was dissolved in tartaric acid (0.1%) and distilled water, (+)-3PPP HCl (RBI) was dissolved in phosphate buffer (Merck, pH = 6.9) and DTG (Interchim) was dissolved in 5% DMSO. The injection volume for the intraperitoneal and oral routes was 0.4ml/20 g and 0.5 ml/l00 g body weight for mice and rats, respectively; the injection volume for the intravenous route was 0.1 ml/20 g and 0.1 ml/l00 g body weight for mice and rats, respect-

Neurophannacology of SR 31742A vehicle. After the injection, the animals were placed individually in Plexiglas cages (10 x 10 x 15 cm). The number of complete contralateral rotations (e.g. away from the injected side) was recorded visually and cumulated over 3 periods of 2 min (2-4, 5-7, 8 -10 min) after the intrastrial injection. Whatever the vehicle used, the control injection usually produced no behavioural effect, at most 2-3 ipsiiateral rotations (e.g. towards the injected side). After preliminary experiments, the dose close to the ED*, of (+>3PPP and DTG to be injected in one striatum was fixed at 0.05kg delivered in 1 pl. SR 31742A was given intraperitoneally and orally 30 and 6Omin before the intrastriatal injection, respectively. For comparative purposes, SR 3 1742A was also assessed under the same conditions against apomorphine (0.5 pg/pl, into one striatum)-induced turning, the 3 observation periods being 2-4, 8-10, 13 -15 min after injection of apomorphine. Depending on the test and the route of a~inistration, the injected doses of SR 31742A ranged from 0.015 to 20mgfkg. For selectivity analysis, haloperidol and spiroperidol were also investigated in both tests of turning behaviour. Haloperidol (dose range 0.3-lOOpg/kg) and spiroperidol (2pg/kg to 5 mg/kg) were injected intraperitoneally, 30 min before the intrastriatal injection. Ten animals were used per dose group. Statistical analysis was performed using analysis of variance (ANOVA), followed by Dunnett’s t-test. Models of hyperactively in mice and rats. Hyperactivity was induced by the intra~~toneal injection in mice of either (+)-amphet~ine (2.5 mgjkg), cocaine (16 mg/kg) or morphine (64 mg/kg), doses selected according to previous work (Simon, Sultan, Chermat and Boissier, 1972; Soubrie, Simon and Boissier 1975). The SR 31742A (0, 1.25, 2.5, 5 and 10 mg/kg) was given intraperitoneally 30 min before injection of drug and the 1 hr placement in the translucent boxes (20 x 20 x 30 cm) of a Digiscan actimeter (Omnitech Electronics Inc.). Locomotion was evaluated as the number of automatically recorded photocell impulses, cumulated over 30 min (last 30 min of the recording period). Twelve or multiples of 12 animals were used per dose group. Statistical analysis was performed using ANOVA, followed by Dunnett’s t-test. To further investigate the profile of action of SR 31742A, two models of hyperactivity were performed in rats. In the first, hyperactivity was produced by methylphenidate, a releaser of the reserpine-sensitive DA pool (16 mg/kg, i.p.). Locomotion was measured, as described previously for mice in translucent boxes (40 x 40 x 30 cm) of a Digiscan actimeter. In the second, hyperactivity was elicited by a bilateral injection of (+)-amphetamine (10 pg) in each nucleus accumbens of chronically implanted rats [A + 9.9, H - 3.9, L rt: 1.6 from Paxinos and Watson (198611 according to Costall, Domeney, Naylor and Tyers (1987). Locomotor ac-

607

tivity was evaluated over 1 hr as the cumulated number of photocell impulses in activity cages (39X39Xl5cm, Apelex, France). Eight (Experiment I) or 6 (Experiment II) animals were used per dose group. One week before the test, rats were matched according to their reactivity to injection of amphetamine in the nucleus accumbens. On ~ompIetion of the experiments, the rats were anaesthetized and decapitated and the brains were removed and fixed in 3.7% formalin in 0.9% NaCl. The brains were frozen and sectioned on a freezing microtome and the site of deposition of drug (or vehicle) identified. The locations were found to be correct in all the rats for which intra-accumbal injections of (+)-amphetamine produced hyperactivity. Results were expressed as the mean ( + SEM) of the locomotor scores for each group. Differences between treated and corresponding control (vehicle) groups were assessed by Student’s t-test. Conditioned ~oidance response in rats. The procedure used was from Arnt (1982). Briefly, during a period of 3 days, rats were trained once daily (30 min session) to avoid a scrambled electric shock, delivered through the grid floor of an automatically controlled shuttle-box (Campden Instruments, Phymep, France), monitored by an Apple 2 computer. A daily session involved about 40 trials, each consisting of a 3-set warning “light only”, followed by 6-set warning “light plus shock” (0.8 mA) and a 45-set intertrial interval. Moving from one compartment of the shuttle-box to the other imm~iately terminated the stimuli being presented and prolonged the intertrial interval. After this training phase, rats (n = 11) with stable avoidance performance were subjected to 3 experimental sessions, identical to the training ones, with at least 3 days recovery between test sessions. The SR 31742A or distilled water was administered intraperitoneally, 30 min before placing the rats in the shuttle-box. Statistical analysis on the number of avoidance responses was performed using ANOVA, followed by Dunnett’s f-test.

Standard tests for the study of DA-blockers climbing, apomorphine(apomo~hine-indu~ induced stereotypy, catalepsy and prochlorperazineinduced catalepsy), were performed as previously described (see Puech, Simon and Boissier, 1978; Boissier and Simon, 1963; Timsit, 1966; Worms and Lloyd, 1980). Neurochemistry Levels of DA, DOPAC (3, 4-dihydroxyphenylacetic acid) and 3-MT (3-methoxytyramine) were measured with high pressure liquid chromatography (HPLC, Waters instruments, 715 ultra Wisp, 510 solvent delivery System and 460 el~tro-chemica1 detector). A p Bondapack phenyl column (Water Ass.) was used for the separation. The mobile phase

608

M. PONCELETet al.

consisted of a 3% methanol in 0.1 M Na-phosphate buffer pH 2.5 and 1 mM l-octane sulphonic acid (PIC B-8 Waters). Frozen samples were homogenized in 0.1 N HClO,,, containing 4 mM Na-metabisulphite and 1 mM EDTA. Rats were killed by microwave irradiation (Sacron 8000, Sairem, Villeurbanne, France; 3.4 KW/cm2/1.6 set). Electrophysiological studies A,-Alo DA cells. Rats were anaesthetized with chloral hydrate (4OOmg/kg, i.p.) and maintained throughout the experiment under a low-speed infusion of the anaesthetic (120 mg/kg/hr, iv.). After appropriate surgery, glass micropipettes (1.5 mm o.d., pulled to 2.5pm at the tip) filled with 0.9% saline, were stereotaxically aimed at the vicinity of & or Alo DA cells (coordinates according to Paxinos and Watson (1986): A,: A(bregma) 5.4, L 2.0, H 7.5-8.5 mm; and A,,: A 5.4, L0.9, H 7.5-8.5 mm). The spikes recorded from the micropipette were amplified, filtered (100 Hz to 2 kHz) and fed into a window discriminator linked to a PC AT, through an intelligent interface (Cambridge Electronic Design, Cambridge, U.K.). The signals were also recorded on magnetic tape for further analysis. Two main types of response were studied in A, and A,, areas: the firing rate of DA cells (spontaneous or reduced by a small dose of apomorphine, 5 pg/kg, i.v.) and the number of spontaneously active DA cells. For this latter purpose, 12 electrode tracks (downwards direction only) were defined on a 1Zbox grid, according to Chiodo and Bunney (1983). The DA cells were identified on-line according to usual criteria of firing rate and signal morphology (see Chiodo, 1988). For the firing experiments, after the rate was stabilized (5-10min) SR 31742A (0.5 mg/kg) or saline was injected intravenously, followed or not (5 min later) by an intravenous injection of apomorphine (5 pg/kg), the total recording period being 15-20 min. One neurone was recorded per animal and, except otherwise noted, 7-15 rats were used per group. The effects of SR 31742A on the number of active DA cells were investigated according to two treatment schedules (acute vs repeated administration). In the acute experiment, SR 31742A (0, 0.625, 1.25, 2.5 and 5 mg/kg) was compared to haloperidol (0.125 and 0.25 mg/kg), both compounds being given intraperitoneally 1 hr before recording. Four to nine animals were used per dose, the total number of controls being 25 (A,) and 19 (A,,). In the subchronic experiment, SR 31742A (5 mg/kg, i.p.) was given once a day for a maximum of 4 weeks, separate groups of rats (n = 4-6) being tested after varying durations of treatment. As in the acute experiments, the last injection was performed 1 hr before recording. Statistical analysis was performed using Student’s t-test or ANOVA, followed by Dunnett’s t-test.

Hippocampal CA3 neurones. Rats were anaesthetized with urethane (1.25 g/kg, i.p.) and maintained throughout the experiment under low-speed infusion of the anaesthetic (60 mg/kg/hr, iv.). Extracellular recordings were performed in the CA3 subfield [A (bregma) - 3.8, L 3.5, H 3.4-3.7 mm, Paxinos and Watson (1986)], using micropipettes and processing systems identical to those described above. The effect of SR 31742A (0.5 mg/kg, i.v.) were compared to those of haloperidol (0.05 mg/kg, i.v.) and spiroperidol (0.05 mg/kg, i.v.), 4-6 rats being used per group (one cell per rat). Injections were performed 5-10 min after signal stabilization and the firing rate was recorded for 30 min or until the cell was lost. Statistical analysis was performed using ANOVA, followed by Dunnett’s t-test, For all these electrophysiological studies, body temperature was maintained at 37 f 0.5”C by means of a homeothermic blanket. RESULTS

Binding assays

In 3 independent experiments, SR 31742A was found to inhibit the binding of (+)-[‘H]3PPP with a K, of 5.3 + 0.3 nM and a Hill coefficient of 1.16 + 0.6 (Fig. 2). The corresponding values were 16.8 + 2.1 and 0.89 _+0.09 nM vs binding of [‘HIDTG (not shown). Under identical experimental conditions, the Ki values for haloperidol were 28.2 f 2.3 nM vs (+)-[3H]3PPP and 31.3 _+3.9 nM vs [3H]DTG. In 3 independent experiments, the IC, of SR 31742A vs (+)-[ ‘Hlpentazocine was 2.0 + 0.1 nM (not shown). Scatchard analysis (Fig. 2) of saturation curves of (+)-[ 3H]3PPP, in the absence or presence of SR 3 1742A (8 nM), revealed an apparent competitive interaction, Kd values being shifted from 37 + 2 to 72 f 5 nM with no significant change in the B,,,,, values: 1073 * 31 vs 1003 + 55 fmol/mg protein.

log [St? 31742A], M

SR 31742A 8 nM 0

400 Specific

bound

Ed0 (fmollmg

1200 ptotein)

Fig. 2. Interaction between SR 3 1742A and sigma sites in rat brain. (A) Scatchard analysis of (+)-[‘H]3PPP (0.2-200 nM) saturation curves in absence (0) or presence (a) of 8 nM SR 31742A. (B) Competition curve between SR 31742A and (+)-[3H]3PPP (3 nM).

Neuropharmacology of SR 31742A

609

Table I. Radioligand binding protile of SR 31742A Receptor/site

Concentration (nM)

Linand

Membranes (rat tissue)

Opiates brain brain brain Brain

* I

IwfCP

3.0 1.0 1.0 1.0

Whole Whok Whole Whole

[‘H]SCH 23390 [‘HjSpiropcridol

0.3 0.4

striatum St&urn

[‘)ilPiren7.epine [sH]N-Methyl-Scopolamine

1.0 0.3

Brain cortex Heart

6.3 f 0.9 2.9 f 0.5

[‘H]WB 4101 [sH]Idaxoxan

0.3 0.5

Brain cortex Brain cortex

. 3.6 f 0.5

(‘HJS-OH-DPAT [‘HIS-HT [‘HjKetanserine [‘*‘I]substance P [ ‘2rl]Neurotensine [ ‘qcCK8

1.0 2.0 1.0 0.04 0.05 0.05

Hippocampus Striatum Brain cortex Brain cortex Striatum Pancreas

[‘WilDAGO [‘HIDADLE [‘H]Ethylketccychuwine

:: K Phencyclidine Dopamine Dl D2 Muscarinic Ml M2 Adrenergic Crl a2 Serotonergic 5-HT,, 5-HT,, S-HT, Substance P Neurotensine CCKA

IC w OcM)

l

1 l l

l

. l

. * .

*Less than 25% displacement at IOm5M. ‘Mean + SEM calculated from 3 independent experiments. DAGD = [o-Ala’, NMe-Phe’, glyoll_cnkephalin; DADLE = [gala’, n-Leu’]-enkephalin; TCP = I-[l-(2-thienyl)cyclohexyl]piperidine; SCH 23390 = R( +)-7chloro-8-hydroxy-3-methyl-l -phenyl-2,3,4,5-tetrahydro-I H3-betuazepine; WB 4101 = 2-(2,6dimethoxyphenoxyethyl-aminoethyl-l,4-benxodioxane; I-OH-DPAT = 8hydroxydipropylaminotetralin; CCK = chokcystokinin.

Under standard conditions for binding to opiate (p, S, K), phencyclidine (PCP), DA and many other receptors for neurotransmitters, no significant displacement activity was observed, at concentrations of SR 31742A less than 10e5 M (Table 1). In uiuo binding assays in brain using (+)-[ ‘H]3PPP (Fig. 3) showed that the IDm of SR 31742A was 0.4 f 0.1 and 1.6 h 0.5 mg/kg after intraperitoneal and oral administration, respectively. Doses close to 5 mg/kg (i.p.) or lOmg/kg @.o.), were found to produce a complete inhibition (saturation of sigma sites) of the binding of ( + )-[‘H]3PPP. The time-course study indicated that displacement

-0.i70.25

0.5

1

’

2

4

6

16 24 72

(Br)

Fig. 3. Interaction between SR 31742A and sigma sites in brain in uivo: dose-effect relationship (insert) and time-course study. Mice were injected intravenously with (+)-[)H]3PPP, 10min befort sacrifice. Data are the mean f SEM percentage inhibition of binding of (+)-[)H]3PPP. *P < 0.05 (Dunnett’s r-test).

of (+)-[‘H]3PPP was complete from 1 to 6 hr after SR 3 1742A (5 mg/kg) and then declined progressively to reach nonsignificant values after 24 hr. In contrast, SR 3 1742A (10 mg/kg) did not exhibit significant displacement activity against [ 3H]-spiroperidol (data not shown). Behavioural studies Turning behaviour in mice. The mean number of contralateral rotations induced by (+)-3PPP and apomorphine in control mice in the intraperitoneal study was 13.6 + 0.7 and 13.3 f 0.7, respectively. As shown in Fig. 4, SR 31742A given intraperitoneally 30min before the test, dose-dependently reduced (+)-3PPP-induced turning (linear regression: F(1,72) = 114.4, P < O.OOl), a significant antagonism being observed from 30 pg/kg onwards, with an EDSo of 65pg/kg (62-67, 95% confidence limits). SR 3 1742A did not affect apomorphine-induced turning, until the dose of 5mg/kg was reached. Under the same conditions, haloperidol dose-dependently reduced (+)-3PPP- and apomorphine-induced tuming (linear regression: F(l, 63) = 94.6, P < 0.001; F(l,36) = 54.6, P < 0.001, respectively) the first significant effects being obtained at 3 and 30pg/kg, respectively (Fig. 3). In contrast, spiroperidol did not affect (+)-3PPP-induced rotations at 62.5 pg/kg, whereas it dose-dependently reduced apomorphineinduced turning (linear regression: F(l,72) = 210.7, P < O.OOOl),the first significantly active dose being 4pg/kg (Fig. 3). The mean number of rotations induced by (+)-3PPP and DTG in control mice in the oral study was 11.9 f 1.0 and 10.3 f 0.9, respectively. SR 31742A, given orally, dose-dependently

M.P~NCELET

0.3

3

30

300

pglkg, i.p.-30

in

hyperactivity

mice

1.25

SR 31742A

min

antagonized (+)-3PPP- and DTG-induced turning [EDSo= 0.21 mg/kg (0.19- 0.25, 95% confidence limits) and 0.9 mg/kg (0.78-1.06, 95% limits of confidence), respectively (not shown)]. The time-course study, performed with SR 31742A at 0.625 mg/kg (p.0.) against (+)-3PPP-induced turning, revealed that the compound produced a significant effect (-65%) 15 min after injection, a maximum antagonism (80-85%) being observed between 0.5 and 2 hr after the injection. No significant antagonism of rotations was observed at post-injection times exceeding 8 hr (not shown). of

b

5000

Fig. 4. Effect of SR 31742A, haloperidol and spiroperidol on (+)-3PPP- and apomorphine-induced turning in mice (mean + SEM). Turning was induced by a unilateral intrastriatal injection of 0.05 pg of (+)-3PPP and 0.5 pg of apomorphine and visually recorded over 6 min. *P d 0.05 (Dunnett’s r-test).

Models

et al.

rats.

and

SR 31742A dose-dependently counteracted each type of drug-induced hyperactivity in mice, a significant antagonism being observed at the dose of 2.5 mg/kg in all three models (linear regression: F(1,112)=22.12, P < 0.001; F(l, 56) = 32.13, P c 0.001; F(l, 90) = 54.08, P c 0.0001 for (+)amphetamine-, cocaineand morphine-induced hyperactivity, respectively) (Fig. 5).

2.5

5

mglkg, i.p.-30

SR 3174214 (Table 2) also reduced drug-induced hyperactivity in rats. At the dose of 5 mg/kg, it significantly attenuated the IO-fold increase in motility caused by methylphenidate. Similarly, in two independent experiments, SR 31742A attenuated the 2.5-fold stimulation of locomotor activity, elicited by intra-accumbens injection of amphetamine. In the range of doses studied, SR 31742A did not affect spontaneous locomotor activity in mice and rats. Conditioned avoidance response in rats. SR 3 1742A dose-dependently reduced the number of avoidance responses in the CAR paradigm (linear regression: F( 1,20) = 3 1.97, P < O.OOOl),this effect being associated with a significant compensatory increase in the number of escape responses at the larger dose tested (Table 2). Miscellaneous

SR 31742A did not induce catalepsy nor did it significantly affect responses associated with direct stimulation or blockade of DA-receptors.

Hyperactivity

SR 31742A (mg/kg. i.p.) 0 2.5 5 10

’ 16052 f 2301

8850 f 1557.

:

-

Shuttle-box behaviour

Amphetamine (Nucleus accumbens) Experiment 1

Experiment 2

1655 f 81 1381 f 206 850 + 204.

1746 f 198 1465 f 265 927 + 182’

-

min

Fig. 5. Antagonism by SR 31742A of (+)-amphetamine (2.5 mg/kg), cocaine (16 mg/kg)- and morphine (64 mg/kg)induced hyperactivity in mice. Locomotor activity was measured in a D&scan actimeter and the number of photocell impulses (mean k SEM) was automatically recorded and evaluated during the last 30 min of the recording period. *P d 0.05 (Dunnett’s r-test).

Table 2. Effects of SR 31742A on drug-induced hyperactivity and conditioned avoidance responses in rats

Methylphenidate

10

-

Avoidance 27.8 f 2.7 18.8 + 4.7’ 12.9 + 3.3’

Escape 13.0 f 2.6 19.4 + 4.6 21.8 & 3.1’

Methylphenidate (16 mg/kg)-induced hyperactivity was measured in a Dig&an actimeter and the number of photocell impulses was automatically recorded during the last 30 min of the recording period; hyperactivity induced by (+)-amphetamine (2 x IOpg) injected into the nucleus accumbens was measured in an Apelex actimeter and the number of photocell impulses was evaluated over 1hr (mean + SEM). Avoidance and escape responses (mean + SEM) were measured in a CAR paradigm during 30 min (about 40 trials) shuttle-box session. lP C 0.05 (Dunnett’s I-test or Student’s r-test).

611

Neuropharmacology of SR 3 1742A Table 3. Effects of SR 31742A (0.5 mg/kg, i.v.) on the spontaneous firing rate (in spikes/set) of A, DA neurones and on the inhibition of Bring rate induced by apomorphine (0.005 mg/kg, i.v.)

Neurochemistry The levels of DA and DOPAC were not altered in the striatum and nucleus accumbens of rats, 1 and 2 hr after intra~~toneal a~inistration of 10 mgikg of SR 3174214 (not shown). In mice, in two independent experiments, SR 3 1742A 10 mg/kg given 40 min before (+)-amphetamine (4mg/kg, i.p.) reduced by 20 f 5%(n = 10; P < 0.01) and 21 f 7% (n = 10; P -C0.05), the amphetamine-induced rise in striatal levels of 3-MT [3-MT in ng/mg wet tissue (n = 20), controls, 57 t_ 4, (+)-amphetamine, 143 f IO]. At this dose, SR 31742A did not alter basal levels of 3-MT.

Firing rate (mean f SEM) (n = 5)

saline 3.12 & 0.5

3.68 f 0.5

Slow (n = 7) Fast (n = 8)

Saline 1.75 f 0.2 5.19 + 0.4

SR 31742A 2.15 * 0.2” 5.38 f 0.4

Soline

Firing rate inhibition (mean difference i SEM) Saline + APO (rz = 7) SR 31742A+AF’O (rt =7)

-0.83 + 0.2 - 1.33 + 0.4b

“Significantly greater than controls (paired r-test) “Non-significantly different from apomorphine alone (Student’s r-test).

Electrophysiological studies

Injected intravenously, SR 3 174214 produced inconsistent effects on the firing rate of DA cells (Table 3). However, after distinction between fast (>4 Hz) and slow (~2 Hz) (n = 8, II = 7, respectively) A, neurones, SR 31742A was found not to affect the former type, whereas it slightly (+ 23%) but significantly increased the number of discharges of the latter type. An identical trend was observed in A,, cells but the number of slow cells encountered (n = 3) precluded any statistical analysis. This acceleration of rate was in fact associated with a significant increase in the number of interspike intervals in the burst domain [< 160 msec; see Grace and Bunney (1983)]: controls: 9.6 & 4.4~s SR 31742A; 29.4 _t 8.5, P < 0.01 for the seven A, neurones. Tested on A,, neurones (n = 7 cells/group), SR 31742A did not prevent the suppressant effect of apomorphine on rate (Table 3). The single administration of SR 31742A resulted in a dose-dependent and significant increase in the number of spontaneously active A, and A,, DA ceils, as compared to baseline saline controls (Fig. 6).

Upon repeated injection of SR 3 1742A, the number of spontaneously active cells diminished rapidly in A,, and more progressively in AP (Fig. 6). Values significantly lower than saline baseline, were reached from the third and the fourth week of treatment onwards in A,, and A, cells, respectively. Mathematical analysis revealed that these experimental values could be fitted by exponential functions, whose tl,2 were 4.4 and 10.2 injections in A,, and Ag, respectively. This indicated a lag time (about 6 days) in the effect of SR 31742A in A, vs Aa, DA neurones. Injected intravenously, SR 31742A (0.5 mg/kg) produced a pronounced time-dependent inhibition of the spontaneous firing activity of CA, neurones, peak effect (- 85%) occurring 15 min after the injection (n = 6). For cells which could be recorded longer (n = 4), a tendency to recovery was observed (Fig. 7). Haloperidol (0.05 mg/kg) showed an identical profile of action, whereas spiropetidol (0.05 mg/kg) was ineffective, although the dose used was pha~acoiogically

active in other respects.

r-l aA

OJ , 0.625

I

1.25

mglkg,

2.5

i.p.

5

1

4 7

14

21

28

Daily injections Fig. 6. Effects of SR 3174244on the mean ( f SEM) number of spontaneously active b’(O) and A,, (V) DA neurones after single (left) and repeated (right) administration.(-) and (---) represent the mean & SEM number of DA cells in control conditions (& = 1.05 rf:0.11; A,, = 0.96 f 0.04 for single and As = 0.92 + 0.03; A,, = 1.02 f 0.02 for repeated ~ministration). *P Q 0.05 (Dunnett’s r-test).

612

M. PONCELETet al. .

0

SR 31742A

Haloperidol

Time

V Spiroperldol

(min)

25

t 0

*

1

I

I

I

I

I

C

5

10

15

20

25

Time

I 30

(min)

Fig. 7. Effects of intravenous administration of SR 31742A (O.Smg/kg), haloperidol (0.05 mg/kg) firing rate of CA, hippocampal neurones. Top and spiroperidol (0.05 mg/kg) on the spontaneous diagrams illustrate typical samples with arrows indicating the time of injection. Bottom curves reflect the time-course (consecutive 5-min periods) of mean (*SEM) changes in rate as a percentage of corresponding controls (c). Symbols refer to top diagrams. *P < 0.05 (Dunnett’s r-test).

DISCUSSION In vitro binding assays using ( +)-[3H]3PPP and [ 3H]DTG as ligands indicated that SR 3174214 behaved as a competitive sigma ligand on membranes from the brain of the rat. SR 31742A also displayed a similar or slightly higher affinity for (+)-[ 3H]pentazocine-labelled sites. In comparison, no significant binding could be detected to PCP, opiate or DA receptors or to other neurotransmitter binding sites. In vivo binding studies performed in mice with (+)-[ ‘H]3PPP and [ ‘Hlspiperidol, further support the idea that SR 31742A is a potent sigma ligand devoid of affinity for DA receptors. Indeed, the in vivo binding data related to the range of active doses, the intraperitoneal/oral ratio and the time-course of the displacement of ( +)-[3H]3PPP fitted with the functional data obtained. Although, blockade of turning behaviour occurred at doses producing less than 25% inhibition of the binding of

(+)-[ 3H]3PPP, most of the effects were observed at doses occupying 60-90% of the binding sites. The first objective was to identify test procedures able to reveal actions of SR 3 1742A and antipsychotic drugs on systems in brain that may occur through sigma sites, rather than DA receptors. Sigma sites densely populate the nigrostriatal system, and biochemical (Iyengar et al., 1990), electrophysiological (Engberg and Wikstriim, 1991) as well as behavioural (Goldstein, Matsumoto, Thompson, Patrick, Bowen and Walker, 1989) experiments, suggest a modulatory role of these sites on DA systems. Circling behaviour can be elicited by microinjection of sigma ligands (DTG, pentazocine) in the substantia nigra of the rat (Goldstein et al., 1989) the DTG-induced circling being blocked by haloperidol, rimcazole and x-(Cfluorophenyl)4-(fluoro-2-pyrimidinyl)-I-piperazine-butanol (BMY 14802) (Perrault, Bastianetto and Sanger, 1991). In line with these data, circling behaviour, elicited

Neuropharmacology in mice by injection

of DTG and (+)-3PPP in the striatum, a structure for which sigma binding is reportedly associated with intrinsic neurones rather than DA-containing afferents, was investigated (Gundlach, Largent and Snyder, 1986). In agreement with the respective affinities for sigma sites, it was found that DTG and ( +)-3PPP were nearly equipotent in inducing contralateral turning. SR 31742A and haloperidol dose-dependently antagonized both DTG- and (+)-3PPP-induced turning, whereas spiperidol did not affect such behaviour, even at doses larger than those sufficient to abolish DA receptor-dependent rotation (apomorphine-induced turning). It was also found (unpublished results) that a sigma ligand (BMY 14802) devoid of significant affinity for DA receptors (Largcnt, Gundlach and Snyder, 1988) inhibited DTG-induced turning. These data suggest that the function of the nigrostriatum can be controlled at the striatal level by sigma sites, inde~ndently of any direct interaction with DA receptors. The hippocampus is another structure in the brain enriched in sigma sites, binding within this structure being distinct from PCP binding and primarily associated with pyramidal cell layers (Gundlath et al., 1986; Largent et al., 1986; but see MacLean and Weber, 1988). Electrophysiological studies have led to the proposal that sigma sites may modulate the neuronal excitability of the hippocampus through an interaction with excitatory amino acid transmission (Malouf, Swearengen and Chavkin, 1988; Monnet, Debonnel, Junien and De Montigny, 1990). It was found that SR 3 1742A and halo~~dol but not spiroperidol markedly reduced the spontaneous firing rate of hippocampal cells, recorded in the CA, field. In spite of marked discrepancies, the study of Monnet et al. (1990) and the present one converge, to suggest that neuronal activity in the hippocampus could be altered by sigma ligands, independently of any ‘binding to PCP receptors. The second objective was to assess whether SR 31742A exerted some of the main effects shared by conventional antipsychotic drugs. Reversal by neuroleptic drugs of behavioural hyperactivity caused by activating DA function is a commonly observed phenomenon that is thought to be predictive of antipsychotic potential (Moore and Gershon, 1989). In agreement with reports showing that sigma ligands such as BMY 14802 and rimcazole reduced the stimulant effect of .amfonelic acid and cocaine (Matthews, MacMillen, Sallis and Blair, 1986; Menkel, Terry, Pontecorvo, Katz and Witkin, 1991) it was found that SR 31142A dose-dependently antagonized amphetamine-, cocaine- and morphineinduced hyperactivity in mice and methylphenidateinduced hyperactivity in rats. This antagonism was observed at doses smaller than those required to depress spontaneous locomotor activity. Moreover, SR 31742A markedly reduced the stimulation of locomotor activity produced by the intra-accumbens injection of amphetamine in rats. In the light of these

of SR 3 1742A

613

results, it is tempting to speculate, as the localization of the sigma sites might suggest (Gundlach et al., 1986), that SR 3174214, like other sigma ligands (see Costa11 et al., 1987), may modulate the activity of mesolimbic DA systems. Disruption of the conditioned (active) avoidance response is another ~ha~oural effect commonly observed with antipsychotic drugs (Amt, 1982). As reported for another sigma ligand, 6-[6+hydroxypiperidine- 1-yl)hexyloxy]-3-methyl-2-phenyl-4Hlbenzopyran-4-one PPC 16377, Clissold, Hartman, Valentine, Abren, Erickson, Karbon, Pontecorvo and Ferkany (1991)] SR 3 1742A also impaired avoidance behaviour at doses sparing escape responses. Electrophysiological models have been developed which attempt to predict the therapeutic and adverse effects of antipsychotic drugs based upon their effects on midbrain DA neurones. When administered acutely, classical neuroleptic drugs: (i) increase the spontaneous firing rates of DA cells while changing their firing pattern (from single spike to bursting mode); (ii) reverse the depressant effect of DA agonists on rate; and (iii) increase the number of spontaneously active DA neurones (see Chiodo, 1988). SR 3 1742A produced electrophysiological changes very similar to those caused by neuroleptics, except that, like most sigma ligands studied so far, it did not prevent or reverse apomorphine-induced inhibition of firing. Indeed, BMY 14802, rimcazole and trans9-methoxy-4-~nzyl~l,2,3,4,4a,5,6~lOb-~tahydro~~o[f]quinoline (HW 173) failed to prevent and/or reverse (but see Wachtel and White, 1988) the depressant effect of apomorphine on rate (Piontek and Wang, 1986; Wachtel and White, 1988; Engberg and Wikstrom, 1991; Shepard and Romeyn, 1991). The stimulatory effects of SR 31742A on firing rate, though significant, were weak (+ 20%) and only involved “slow” DA neurones. It has already been observed with neuroleptics that the increasing effect of these drugs on rate was inversely correlated with the degree of basal neuronal activity (see Chiodo, 1988) and varied markedly with the degree of anaesthesia: the deeper the anaesthesia, the smaller the neuroleptic-indu~d increase (Mereu, Fanni and Gessa, 1984). These factors may also have some relevance as regards the reported inconsistent effects (but see Steinfels and Tam, 1989) of sigma ligands on the discharge rates of DA neurones (Piontek and Wang, 1986; Wachtel and White; 1988; Engberg and Wikstrom, 1991). Beside this moderate increase in firing rate, a change in the firing pattern of DA cells was observed with SR 31742A. This change from a single spike mode to bursting activity has already been suggested to occur after administration of neuroleptics. Although a similar change was not found with HW 173, this sigma ligand was shown to affect discharge patterns of DA cells by increasing the degree of regularity (Engberg and Wikstriim, 1991).

614

M. PONCELET et al,

Furthe~ore, SR 3 1742A increased the number of spontaneously active.A, and A,, neurones and thus resembled classical neuroleptic drugs, while differing from BMY 14802 and rimcazole. After acute administration, these sigma ligands were found either to reduce (rimcazole) or not to affect (BMY 14802) the number of active DA cells (Piontek and Wang, 1986; Wachtel and White, 1988). After repeated administration, however, the effects of BMY 14802 resembled atypical antipsychotics as this drug reduced the number of A,, but not A9 active DA neurones (Wachtel and White, 1988). After chronic treatment, SR 31742A resembled typical neuroleptic drugs but seemed to lead to an atypical profile. Indeed, the reduction of the number of active DA cells occurring in A, was notably delayed with respect to that seen in A,,,. The inability of SR 3 1742A to block DA receptors may probably account for the fact that the compound only exhibited some of the effects shared by neuroleptic drugs. As an illustration, SR 31742A failed to induce catalepsy or to potentiate neuroleptic-induced catalepsy nor did it, in agreement with the electrophysiological and behavioural ~turning) studies, antagonize a~mo~hine-induced stereotypy (rats) and climbing behaviour (mice). An additional major difference between SR 3174212 and neuroleptic drugs was that SR 31742A did not increase the metabolism of DA in the nigrostriatal and mesolimbic systems, an increase in utilization of DA by neuroleptic drugs being thought to occur as a result of blockade of DA receptors. SR 31742A, although devoid of affinity for DA receptors, produced two types of effects: (i) those shared by conventional antipsychotic DA blockers; and (ii) those exerted only by neuroleptics, with mixed DA/sigma receptor affinity and not by neuroleptics without affinity for sigma sites. In addition, similarity but also heterogeneity can be noticed in the respective profiles of action of SR 3 1742A, rimcazole, BMY 14802, 1-(cy~lopropylmethyl)-4-)~~‘-fluoroethyl)-2’-oxoethyl)piperidine (Dup 734) and NPC 16377. This may be due to the low selectivity of most sigma ligands studied so far for sigma sites (VanderMaelen and Braselton, 1990; Tam, Steinfels, Gilligan, McElroy, De Noble, Johnson and Cook, 1991; Quirion et al., 1992). In conclusion, it would be tempting to speculate that a reduced DA function is a unitary mechanism accounting for the pattern of alterations produced by SR 3 1742A. Although such a hypothesis cannot accommodate the effects of SR 3 1742A on (+ )-3PPPor DTG-induced turning and on ~p~campal firing activity, it may apply to blockade of drug-induced hyperactivity, impairment of avoidance behaviour and changes in firing of DA cells produced by SR 31742A. This compound however failed to antagonize apomorphine-induced effects and to increase the turnover of DA, thus suggesting that an interaction upstream, rather than downstream of the DA receptor may underly such a postulated

reduction of DA function. SR 31742A, however, was found not to markedly affect basal levels of DA or DOPAC nor to potentiate catalepsy-induced by blockade of DA receptors. This would suggest that SR 31742A does reduce enhanced rather than basal DA transmission. Data obtained on amphetamine-induced accumulation of 3-MT are compatible with this assumption. Whatever the exact mechanisms involved, this study showed that SR 31742A, a selective and potent sigma ligand devoid of affinity for DA receptors, displays a pattern of actions which suggest antipsychotic potential. This suggestion is further supported by the recent finding that, like antipsychotic drugs, acute and chronic treatment with BMY 14802 increased the concentrations of neurotensin-like immunoreactivity in the nucleus accumbens and caudate of the rat, neurotensin being a peptide reported to exert a number of effects similar to those of antipsychotic drugs (Levant and Nemeroff, 1990). Beside the antipsychotic hypothesis, the alternative hypothesis, that binding to sigma sites may underly the adverse (mainly motor) effects of antipsychotic drugs has also heen proposed (see Goldstein et al.,

1989). Further studies are thus required to assess the therapeutic and side effect potential linked to the neuropha~acologi~l profile of SR 3 174219 and of selective sigma ligands more generally.

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