INTRODUCTION

The importance of forebrain cholinergic systems for cognitive functions has been known for decades. Systemic administration of the nonselective muscarinic receptor antagonists atropine and scopolamine causes cognitive deficits in humans (Christensen et al, 1992; Ebert and Kirch, 1998; Wesnes et al, 1991), nonhuman primates (Rupniak et al, 1989) and rodents (Patel and Tariot, 1991; Sunderland et al, 1986). In human volunteers, the pattern of cognitive impairment caused by scopolamine mimics in some aspects the cognitive symptomology seen in Alzheimer's disease (AD) (Ebert and Kirch, 1998) which is associated with the degeneration of cholinergic neurons in the basal forebrain resulting in a reduction of cholinergic neurotransmission in the forebrain (Araujo et al, 1988; DeKosky et al, 1996; Kuhl et al, 1999; Shinotoh et al, 2000; Shiozaki et al, 1999; Whitehouse et al, 1982). On the other hand, acetylcholinesterase (AChE) inhibitors, which increase synaptic acetylcholine (ACh) levels, are effective in the treatment of some cognitive symptoms of AD (Francis et al, 1999).

In addition to a deficit in cholinergic transmission, dementia is also related to degeneration of glutamatergic hippocampal and cortical pyramidal neurons (Aronica et al, 1998; Francis et al, 1999), which are targets for cholinergic and serotonergic innervation. Furthermore, the activity of glutamatergic neurons in the hippocampus and neocortex is modulated by cholinergic systems, which have facilitatory effects mediated via muscarinic receptors (Cole and Nicoll, 1984; Segal, 1982), and serotonergic systems, which have inhibitory effects partly mediated via 5-HT1A receptors (Beck et al, 1992; Davies et al, 1987; Newberry et al, 1999; Pugliese et al, 1998). Thus, 5-HT1A receptor agonists such as 8-hydroxy-2-(di-n-propyloamino)tetralin hydrobromide (8-OH-DPAT) have been found to cause a hyperpolarization of limbic (hippocampus and entorhinal cortex) (Grunschlag et al, 1997; Sprouse and Aghajanian, 1988; Tada et al, 1999) and neocortical pyramidal cells (Hajos et al, 1999). These effects are blocked by the 5-HT1A receptor antagonists NAN-190 and WAY 100635 (Hajos et al, 1999; Schmitz et al, 1998).

Passive avoidance (PA) is one of the most frequently used animal models for studying the role of neurotransmitters in learning and memory processes (Bammer, 1982; Sarter et al, 1992a,1992b). This aversive learning task, which is based on classical (Pavlovian) fear conditioning, can be modified by alterations in both serotonergic and cholinergic transmission (Bammer, 1982; Misane and Ögren, 2000; Ögren, 1985). For instance, nonselective muscarinic receptor antagonists such as scopolamine injected prior to PA training cause a dose-dependent impairment of PA retention when tested 24 h after training (Cole and Jones, 1995; Meyers, 1965; Rush and Streit, 1992; Wilson and Cook, 1994). Conversely, pretraining administration of cholinomimetics such as AChE inhibitors (physostigmine, tacrine, donepezil) have been shown to antagonize the impairment of PA retention caused by scopolamine (Kojima et al, 1997; Rush and Streit, 1992; Yoshida and Suzuki, 1993). Unlike changes in cholinergic transmission, treatments that increase serotonergic (5-HT) activity in the brain, for example, the 5-HT-releasing compound p-chloroamphetamine, were shown to disrupt PA retention (Misane and Ögren, 2000; Ögren, 1985). The impairment of PA by p-chloroamphetamine was related to 5-HT release and subsequent activation of the postsynaptic 5-HT1A receptors, but not the 5-HT2A or 5-HT2C receptors (Misane and Ögren, 2000).

Several studies using the selective 5-HT receptor agonists and antagonists have revealed the particular importance of 5-HT1A receptor subtype in PA. The selective 5-HT1A agonist 8-OH-DPAT given subcutaneously (s.c.) prior to PA training was found to produce a dose-dependent impairment of retention in the rat when examined 24 h later (Carli et al, 1992; Misane et al, 1998a; Misane and Ögren, 2000). The PA impairment caused by 8-OH-DPAT was related to stimulation of post- but not presynaptic 5-HT1A receptors in the brain (Misane et al, 1998a; Misane and Ögren, 2000).

The role of muscarinic receptors and 5-HT1A receptors themselves in PA has been extensively studied, but the potential functional interactions between these two receptor systems have received little attention. There is some evidence that 5-HT1A and muscarinic receptors can interact in the regulation of PA performance. Coadministration of a subthreshold dose of 8-OH-DPAT (0.03 mg/kg s.c.) and scopolamine (0.1 mg/kg i.p.) before PA training resulted in an impairment of PA retention when tested 24 h later (Riekkinen, 1994). However, since this study was based on the use of the 5-HT1A agonist 8-OH-DPAT and scopolamine and did not include a 5-HT1A antagonist, the results do not allow conclusions as to the possible interaction between 5-HT1A-receptor-mediated transmission and muscarinic-receptor-mediated transmission.

The present study examined whether a blockade of central 5-HT1A receptors could modify the impairment of PA caused by muscarinic receptor blockade. For that purpose, the studies were performed under high-training conditions producing almost maximal (cutoff) retention latencies in control animals. This design allows a reliable detection of impairment of PA retention caused by muscarinic receptor blockade, but is not suitable for studying the facilitation of PA retention.

The effects of the highly selective 5-HT1A receptor antagonists WAY 100635 and NAD-299 (robalzotan) on PA were examined alone or in combination with the nonspecific muscarinic receptor antagonist scopolamine in the rat. Robalzotan is a more selective 5-HT1A receptor antagonist than WAY 100635, with a weak affinity for both NA and DA receptors (Johansson et al, 1997). Robalzotan is also a more short-acting drug in the rat compared with WAY 100635. For this reason, the temporal kinetics of WAY 100635 and robalzotan was compared in relation to scopolamine. In view of these data suggestive of the therapeutic potential of the 5-HT1A antagonists, the two AChE inhibitors tacrine and donepezil (E2020) used in the treatment of AD were included as ‘reference compounds’.

MATERIALS AND METHODS

Animals

Adult male Sprague–Dawley rats (2 months of age), weighing 300–350 g at the time of testing, were obtained from B&K UNIVERSAL AB (Sollentuna, Sweden). The animals were allowed at least a 5-day adaptation period at the animal maintenance facilities of the department before the start of the experiments. The animals were housed four per cage in standard plastic type IV Macrolon cages (57×35×19 cm, with 2 l wood-cuttings as bedding) and maintained at an ambient room temperature of 20±0.5°C with 40–50% relative humidity. A 12-h light/dark schedule (lights on at 06:00 h) was used throughout the experiment. The animals had free access to standard lab chow (Ewos R36, Ewos AB, Sweden) and tap water up to the time of the experiments. The cages were changed twice a week during the adaptation period. In order to decrease the influence of stress factors on performance, the cages were not cleaned during the days of PA training and retention. On the experimental days, the animals were brought to the experimental room and allowed to habituate to the environmental conditions for a period of approximately 60 min. Animal housing and all experimental procedures followed the provisions and general recommendations of the Swedish animal protection legislation. The experimental procedures were approved by the local Animal Ethics Committee (ethical N 80/96 and 116/00).

PA Procedure

PA was conducted as described earlier (Misane and Ögren, 2000; Misane et al, 1998b). A standard shuttle box (Ugo Basile, Comerio-Varese, Italy), with two communicating (7×7 cm sliding door built in the separating wall) compartments of equal size and a stainless-steel bar floor, was used. The right-hand compartment (shock compartment) was painted black to obtain a dark chamber. The left-hand compartment was illuminated by a bulb (24 V; 5 W) installed on the top Plexiglas cover.

PA training was conducted in a single session (day 1) during the light phase of a 12-h day/night cycle (09:00–16:00). In all the experiments, the animals (n=7–16) were treated with the test compounds prior to training according to the schedule described below. After the selected time interval following injection (day 1), the rat was placed in the light compartment with no access to the dark compartment and allowed to explore for 2 min. During the exploration phase in the PA apparatus, the behavior of the animals, including number of full rearings (move of the body through the vertical plane) and locomotor activity, was noted by the experimenter.

When 2 min expired, the sliding door was automatically opened by pressing a pedal and the rat was allowed to cross over into the dark compartment. Once the rat had entered the dark compartment, the sliding door was automatically closed and an inescapable, constant current, scrambled shock (5 s, 0.6 mA) was delivered through the grid floor. Latency to cross into the dark compartment (training latency) was recorded. If a rat failed to move into the dark compartment within 300 s (cutoff latency), the door was reopened and the rat was gently moved into the dark compartment by the experimenter, where it received footshock. Following training, the rat was immediately removed from the PA apparatus.

Retention performance was examined 24 h after training (day 2). The animal was placed in the light (safe) compartment, with access to the dark compartment (within 15 s) for a period of 300 s. The latency to enter the dark compartment with all four feet (retention latency) was automatically measured. If the rat failed to enter the dark compartment within 300 s, it was removed and assigned a maximum test latency score of 300 s.

Drugs

The following compounds were used in the present study: (−)-scopolamine hydrobromide (Sigma, St Louis, MO, USA); N-2-4-(2-methoxyphenyl)-1-piperazinylethyl-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride (WAY 100635) (Wyeth Research, Taplow, UK); (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-3H-1-benzopy-ran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate (NAD-299, robalzotan) (AstraZeneca, SÖdertälje, Sweden); tacrine hydrochloride (Sigma Chemical Co., St Louis, MO, USA); and 1-benzyl-4-(5,6-dimethoxy-1-indanon)-2-yl-methylpiperidine hydrochloride (donepezil, E2020) (kindly supplied by Anne-Lie Svensson, Karolinska Institutet, Huddinge University Hospital).

All drugs were dissolved in saline (NaCl 0.9%) and administered s.c. or i.p. (as indicated in the Results section and the figure legends) in volumes of 2 or 5 ml/kg, respectively.

The compounds were administered at the following doses and times before PA training: scopolamine (0.03–0.3 mg/kg) 40 min; WAY 100635 (0.03–1.0 mg/kg) 30 or 50 min; robalzotan (0.01–3.0 mg/kg) 15, 30 or 50 min; tacrine (0.3–3.0 mg/kg) 60 min; and donepezil (0.1–2.0 mg/kg) 30 or 90 min.

Statistical Analysis

The overall treatment effects in the PA studies were examined using one-way analysis of variance (ANOVA). For each significant F-ratio, Fisher's protected least significant difference test (Fisher's PLSD test) was used to analyze the statistical significance of appropriate multiple comparisons (Kirk, 1968). Subsequently, to analyze the possible dose-dependent effect of scopolamine itself and the dose dependency of 5-HT1A antagonists and AChE inhibitors effects on the scopolamine effects, the analysis of polynomial regression was used and the coefficients of determination (R2) were calculated to show the possible linearity between treatment and measured variables (training and retention latencies and number of rearings). In the drug combination studies with scopolamine, the respective scopolamine groups were used as control groups. Both when one-way ANOVA and regression ANOVA were applied, a probability level of P<0.05 was accepted as statistically significant. The post hoc tests were two tailed.

RESULTS

Effects of Scopolamine on PA

When examined 24 h after training, the retention latency in the saline-treated control group was close to 300 s with a small distribution in the responses (Figure 1), indicating that animals had acquired the task. When injected 40 min before PA training, scopolamine (0.03–0.3 mg/kg s.c.) caused an impairment of PA retention (F3,28=13.65, P<0.01) with a significant effect at the 0.3 mg/kg dose (P<0.01 vs saline control group) (Figure 1). Polynomial regression ANOVA showed that the scopolamine effect was dose-dependent (F2,29=21.12, P<0.01), although the treatment-latency relation was not strictly linear (R2=0.59). Training latencies were not affected by scopolamine: F3,28=1.36, P>0.27 (Table 1). Behavioral observations in the PA apparatus during the 2-min exploration period indicated that locomotor activity was increased while the number of full rearings was markedly decreased (P<0.01 vs saline control group) by the 0.3 mg/kg dose of scopolamine. Based on the dose–response experiments, the 0.3 mg/kg dose of scopolamine was used in most of the subsequent interaction studies with 5-HT1A antagonists and AChE inhibitors.

Figure 1
figure 1

Dose-dependent effects of scopolamine on PA retention in the rat. Rats were injected with scopolamine (0.03–0.3 mg/kg s.c.) 40 min before the training session (exposure to inescapable foot shock). The saline (s.c. 2 ml/kg) control group was run concurrently with scopolamine-treated groups. The retention test was performed 24 h later. Vertical bars represent means (±SEM) of retention latencies. The maximal time of latency was set at 300 s (cutoff time). The statistical analysis was performed by one-way ANOVA followed by Fisher's PLSD test. **P<0.01 vs saline control group, n=8. SCOP, scopolamine in all figures.

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Table 1 Effects of Scopolamine, the 5-HT1A Antagonists and AChE Inhibitors on PA Training in the Rat
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Effects of the 5-HT1A Antagonists and the AChE Inhibitors on PA

Both WAY 100635 (0.03–1.0 mg/kg s.c.) and robalzotan (0.01–1.0 mg/kg s.c.), and tacrine (0.3–3.0 mg/kg i.p.) and donepezil (0.1–2.0 mg/kg s.c.) failed to affect PA retention (Table 2). However, robalzotan caused an impairment of PA retention at the highest dose tested (3.0 mg/kg) (P<0.01 vs saline control group). Only donepezil caused a significant increase in PA training latency at the 1.0 and 2.0 mg/kg doses (Table 1).

Table 2 Effects of the 5-HT1A Antagonists and AChE Inhibitors on PA Retention in the Rat
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WAY 100635 or robalzotan did not cause any apparent behavioral changes during the 2-min exploration phase in the PA apparatus, except for a decrease in rearing noted at the 3.0 mg/kg of robalzotan (P<0.05 vs saline control group).

At the highest doses tested, both tacrine (3.0 mg/kg IP) and donepezil (2.0 mg/kg s.c.) decreased rearing (P<0.01 and 0.05 vs saline control group, respectively). In addition, slight salivation, jaw and head movements resembling orofacial dyskinesias, and an increase in defecation (diarrhea) was observed in rats treated with the 2.0 mg/kg dose of donepezil and the 3.0 mg/kg dose of tacrine. In addition, at the 3.0 mg/kg dose, tacrine had an increased reactivity to sudden noise (such as opening the door in the PA apparatus). Importantly, no tremor was observed in the tacrine- or donepezil-treated rats.

Effects of WAY 100635 on Impairment of PA Caused by Scopolamine

The effects of WAY 100635 on scopolamine-induced impairment of PA retention were examined using two different injection schedules: WAY 100635 was given either 10 min after scopolamine or 10 min before scopolamine, that is 30 and 50 min before PA training, respectively.

When injected 10 min after scopolamine (0.3 mg/kg s.c.), WAY 100635 (0.03–1.0 mg/kg s.c.) failed to reverse the impairment of PA retention (Figure 2a). In contrast, when injected 10 min before scopolamine (0.3 mg/kg s.c.), WAY 100635 (0.3 and 1.0 mg/kg s.c.) almost completely blocked the impairment of PA retention (P<0.01 vs saline+scopolamine group and P>0.15 and 0.13 vs saline+saline control group for the 0.3 and 1.0 mg/kg doses of WAY 100635, respectively) (Figure 2b). Regression ANOVA revealed a highly significant treatment effect (F2,21=15.72, P<0.01), although there was no strictly linear relation between the dose of WAY 100635 effect and retention latency (R2=0.60). It is notable that regardless of the injection schedule, WAY 100635 failed to normalize rearing, which was nearly abolished because of the scopolamine treatment (data not shown).

Figure 2
figure 2

Combined effects of WAY 100635 and scopolamine on PA retention in the rat. (a) Rats were injected with scopolamine (0.3 mg/kg s.c.) and WAY 100635 (0.03–1.0 mg/kg s.c.) 40 and 30 min before the training session, respectively. (b) Rats were injected with WAY 100635 (0.3–1.0 mg/kg s.c.) and scopolamine (0.3 mg/kg s.c.) 50 and 40 min before the training session, respectively. The saline+saline (s.c. 2 ml/kg) control groups were run concurrently with scopolamine- and WAY 100635-treated groups. The retention test was performed 24 h later. Vertical bars represent means (±SEM) of retention latencies. **P<0.01 vs corresponding saline control group; ♯P<0.01 vs saline+scopolamine-treated group, n=8. For details of statistical analysis and general information, see the legend to Figure 1 and the Methods section.

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Effects of Robalzotan on Impairment of PA Caused by Scopolamine

Owing to the relatively short half-life of robalzotan in the rat (Stenfors et al, 1998), the effects of robalzotan on scopolamine-induced impairment of PA retention were examined using four different injection schedules; the compound was given either 25 or 10 min after scopolamine and/or 10 min before scopolamine, that is 15, 30, and 50 min before PA training, respectively.

When injected 15 min before training, robalzotan (0.03–0.3 mg/kg s.c.) failed to attenuate the inhibitory effect of scopolamine (0.3 mg/kg s.c.) on PA retention (data not shown), and it did not alter training latencies (F4,35=0.69, P>0.60).

A significant overall treatment effect in the retention test (F6,65=21.18, P<0.01) was found when robalzotan (0.01–3.0 mg/kg s.c.) was injected 30 min before PA training (Figure 3a). Under these conditions, robalzotan displayed an inverse ‘U-shape’ type of activity. Regression ANOVA revealed a significant attenuation of scopolamine (0.3 mg/kg s.c.)-induced PA retention deficit by robalzotan at the 0.01–0.1 mg/kg dose range (F2,37= 3.60, P<0.05), although the dose–latency relation was not linear (R2=0.16). It is notable that the drug effect was not significant at the highest 0.3–3.0 mg/kg dose ranges of robalzotan tested. A similar ‘U-shape’ pattern was also found for training latencies. While scopolamine itself did not affect the training latency (P>0.95 vs saline+saline control group), a profound (P<0.01 vs saline+saline control and scopolamine+saline groups) increase in training latencies was seen when scopolamine was combined with robalzotan at the 0.03–0.3 mg/kg dose range. The maximum effect was found at the 0.1 mg/kg dose.

Figure 3
figure 3

Combined effects of robalzotan and scopolamine on PA retention in the rat. (a) Rats were injected with scopolamine (0.3 mg/kg s.c.) and robalzotan (0.01–3.0 mg/kg s.c.) 40 and 30 min before the training session, respectively. (b) Rats were injected with robalzotan (0.1–3.0 mg/kg s.c.) and scopolamine (0.3 mg/kg s.c.) 50 and 40 min before the training session, respectively. (c) Rats were injected with robalzotan (0.03–0.3 mg/kg s.c.) 50 and 30 min before the training session. Scopolamine (0.3 mg/kg s.c.) was given 40 min before training. The saline (double or triple injections, s.c. 2 ml/kg) control groups were run concurrently with scopolamine- and robalzotan-treated groups. The retention test was performed 24 h later. Vertical bars represent means (±SEM) of retention latencies. *P<0.05 and **P<0.01 vs corresponding saline control group; ♯P<0.05 and ♯♯P<0.01 vs corresponding scopolamine control group, n=8–16. For details of statistical analysis and general information, see the legend to Figure 1 and the Methods section.

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Robalzotan (0.1–3.0 mg/kg s.c.) injected 50 min before training failed to attenuate the inhibitory effect of scopolamine (0.3 mg/kg s.c.) on PA retention (Figure 3b). In addition, no significant overall treatment effect was found for training latencies (F4,35=0.78, P>0.51). However, when robalzotan (0.03–0.3 mg/kg s.c.) was injected twice both 50 and 30 min before PA training (Figure 3c), ANOVA revealed a significant overall treatment effect on the retention test (F4,35=5.56, P<0.01). In this experiment, robalzotan attenuated the PA retention deficit caused by scopolamine (0.3 mg/kg s.c.) at the 0.3 mg/kg dose (P<0.05 vs control group and saline+scopolamine+saline-treated group). However, regression ANOVA showed that the robalzotan effect was not dose dependent (F2,29=2.48, P>0.10, R2=0.15). It is notable that regardless of the injection schedule (before and/or after scopolamine), robalzotan failed to normalize rearing, which was nearly abolished because of the scopolamine treatment (data not shown).

Effects of Tacrine on Impairment of PA Caused by Scopolamine

The activity of tacrine was tested vs two doses of scopolamine (0.2 and 0.3 mg/kg s.c.).

Figure 4a shows that tacrine (3.0 mg/kg i.p.) attenuated the deficit of PA caused by the 0.3 mg/kg dose of scopolamine (P<0.05 vs saline+saline control and P<0.05 vs saline+scopolamine-treated group), while the 0.3 mg/kg dose of tacrine did not have any significant effect. Regression ANOVA revealed that the tacrine effect was dose dependent (F2,18=4.28, P<0.05) without the linear treatment–latency relation (R2=0.32). No overall treatment effect on training was found (F3,24=0.71, P>0.55).

Figure 4
figure 4

Combined effects of tacrine and scopolamine on PA retention in the rat. (a) Rats were injected with tacrine (0.3–3.0 mg/kg i.p,) and scopolamine (0.3 mg/kg s.c.) 60 and 40 min before the training session, respectively. (b) Rats were injected with tacrine (0.3–3.0 mg/kg i.p.) and scopolamine (0.2 mg/kg s.c.) 60 and 40 min before the training session, respectively. The saline (i.p. 5 ml/kg)+saline (s.c. 2 ml/kg) control groups were run concurrently with tacrine- and scopolamine-treated groups. The retention test was performed 24 h later. Vertical bars represent means (±SEM) of retention latencies. *P<0.05 and **P<0.01 vs corresponding saline+saline control group; ♯P<0.05 vs corresponding saline+scopolamine-treated group, n=7–8. For details of statistical analysis and general information, see the legend to Figure 1 and the Methods section.

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Tacrine (3.0 mg/kg i.p.) also attenuated the impairment of PA retention caused by the lower 0.2 mg/kg dose of scopolamine (P>0.06 vs saline+saline control and P<0.05 vs saline+scopolamine-treated group) (Figure 4b); however, the tacrine effect was not dose dependent (F2,29=1.65, P=0.21, R2=0.10). In this experiment, ANOVA revealed a significant effect on training latencies (F4,35=2.70, P<0.05).

Tacrine failed to normalize rearing, which was nearly abolished following treatment with either the 0.2 or 0.3 mg/kg dose of scopolamine (data not shown).

Effects of Donepezil on Impairment of PA Caused by Scopolamine

The effects of donepezil on the scopolamine-induced impairment of PA retention were examined using two different injection schedules. Donepezil was given either before or after scopolamine, that is 90 and 30 min before PA.

When injected 30 or 90 min before training, donepezil (0.1–2.0 mg/kg s.c.) failed to block the inhibitory effect of scopolamine (0.3 mg/kg s.c.) on PA retention (Figures 5a and b) and it did not alter training latencies (F5,42=0.96, P>0.44 and F5,58=0.94, P>0.45, respectively).

Figure 5
figure 5

Combined effects of donepezil and scopolamine on PA retention in the rat. (a) Rats were injected with scopolamine (0.3 mg/kg s.c.) and donepezil (0.1–2.0 mg/kg s.c.) 40 min and 30 min before the training session, respectively. (b) Rats were injected with donepezil (0.1–2.0 mg/kg s.c.) and scopolamine (0.3 mg/kg s.c.) 90 and 40 min before the training session, respectively. (c) Rats were injected with donepezil (0.5–1.0 mg/kg s.c.) and scopolamine (0.2 mg/kg s.c.) 90 and 40 min before the training session, respectively. The saline+saline (s.c. 2 ml/kg) control groups were run concurrently with donepezil- and scopolamine-treated groups. The retention test was performed 24 h later. Vertical bars represent means (±SEM) of retention latencies. *P<0.05 and **P<0.01 vs corresponding saline+saline control group; ♯P<0.05 vs saline+scopolamine-treated group, n=8–16. For details of statistical analysis and general information, see the legend to Figure 1 and the Methods section.

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In view of the inability of donepezil to counteract the impairment of PA retention caused by the 0.3 mg/kg dose of scopolamine, donepezil was also examined using the 0.2 mg/kg dose of scopolamine. Under this condition, when given 90 min before PA training, donepezil (0.5–1.0 mg/kg s.c.) almost completely blocked the inhibitory effects of scopolamine (Figure 5c), although this effect was not dose dependent (F2,21=2.57, P>0.10, R2=0.20). No overall treatment-effect was found for training latencies (F3,28=0.75, P>0.53).

Regardless of the injection time (90 or 30 min before training), donepezil (0.1–2.0 mg/kg s.c.) failed to normalize rearing in the PA apparatus, which was nearly abolished following the 0.2 and 0.3 mg/kg doses of scopolamine (data not shown).

DISCUSSION

In agreement with previous studies (Bammer, 1982), systemic (s.c.) scopolamine administration before training produced a dose-related impairment of step-through PA retention when examined 24 h later. The interval between the s.c. administration of scopolamine and training in this study was 40 min, which is longer than in most previous PA studies (varying usually between 5 and 30 min) (Bammer, 1982). The choice of this time interval was based on in vivo microdialysis studies, in which s.c. scopolamine produced an increase in the ACh levels in the ventral hippocampus with a peak effect after 40–60 min (Antoniou et al, 1997; Ögren et al, 1996; Toide and Arima, 1989).

Scopolamine markedly elevates extrasynaptic ACh at the time of PA training mainly because of the blockade of presynaptic M2 muscarinic receptors (Ögren et al, 1996; Stillman et al, 1996). At the same time scopolamine blocks several postsynaptic muscarinic (M1–M5) receptors (Bymaster et al, 1993), which explains its inhibitory effects on cognitive processing. Thus, the action of scopolamine on pre-/postsynaptic muscarinic receptors is of critical importance for its effects on learning and memory and the ability of AChE inhibitors to attenuate these effects.

The most important finding of this study is the observation that muscarinic and 5-HT1A receptors can interact in the regulation of aversive learning. The ‘prototypic’ selective 5-HT1A receptor antagonist WAY 100635 and the selective 5-HT1A receptor antagonist robalzotan both attenuated the inhibitory effects of scopolamine. However, WAY 100635 was active vs scopolamine when injected 50 but not 30 min before PA training. In contrast, robalzotan was found to attenuate the PA impairment by scopolamine when injected 30 but not 50 min before PA training. This attenuating effect was even more pronounced when robalzotan was injected twice, that is, both 30 and 50 min before PA training, which is consistent with the kinetic concentrations of robalzotan in the mouse brain (Stenfors et al, 1998). In contrast to robalzotan, WAY 100635 failed to reverse the deficit of PA retention caused by scopolamine. This suggests potential differences in the action of the two 5-HT1A receptor antagonists, and it is possible that the temporal kinetics of the 5-HT1A receptor antagonists in relationship to muscarinic receptor blockade by scopolamine is of importance. In addition, unlike WAY 100635, robalzotan itself at the highest dose tested (3.0 mg/kg) impaired retention performance ( Table 2) and this dose failed to attenuate scopolamine effects (Figure 3a). This finding suggests that at higher doses, robalzotan can increase 5-HT1A-mediated transmission probably via increased 5-HT release or partial agonistic 5-HT1A receptor activity.

The role of 5-HT in learning and memory seems to be mainly related to the acquisition or the encoding phase (Ögren, 1985). Thus, the activation of 5-HT1A receptors before but not immediately after training has been shown to impair retention in associative learning tasks such as step-through passive avoidance and fear conditioning (Misane et al, 1998a; Misane and Ögren, 2000; Stiedl et al, 2000). It is notable that inhibitory effects of pretraining administration of 8-OH-DPAT on PA retention could not be explained by state-dependent factors (Misane et al, 1998a). Our recent studies show that scopolamine injected s.c. immediately after training at the doses used in this study failed to impair PA retention tested 24 h later.

Thus, both alterations in 5-HT1A- and muscarinic-receptor-mediated transmission appear to interfere with the formation of contextual short-term representation and, thereby, possibly produce a deficit in the long-term memory representation of the context. This suggests that the modulatory action of 5-HT and 5-HT1A receptors on muscarinic-receptor-mediated transmission is mainly related to encoding mechanisms important for acquisition. The exact neurobiological mechanisms underlying the anterograde amnestic effect of scopolamine are still not well understood, but probably involve multiple forebrain areas important for learning and memory.

The mechanisms and the sites of action mediating the functional interaction between muscarinic and 5-HT1A receptors are presently unknown. The use of the double immunocytochemical technique has shown that the 5-HT1A receptors are colocalized with choline acetyltransferase (ChAT) in the medial septum and diagonal band of Broca (MS-dBB) in the rat (Kia et al, 1996). About 25% of the cholinergic neurons in the septal complex were found to express 5-HT1A receptor immunoreactivity. In the dorsal and ventral cholinergic cell groups of the MS–dBB, the number of neurons coexpressing both ChAT and 5-HT1A receptors was in the range of 40–44% (Kia et al, 1996). Triple labeling immunofluorescent techniques have also identified 5-HT-immunoreceptive neurons in close proximity to the soma and dendrites of cholinergic neurons in the nucleus basalis (NB) of the guinea pig (Fort et al, 1998; Khateb et al, 1993). Interestingly, 5-HT and the 5-HT1A agonist 8-OH-DPAT were also found to hyperpolarize the ChAT-immunoreactive cells in vitro, suggesting an ability to inhibit tonic firing and also to modulate the low threshold bursting of the cholinergic NB neurons (Khateb et al, 1993). Because the NB neurons provide the main cholinergic innervation of the cerebral cortex (Lehmann et al, 1980; Lewis and Shute, 1967; Rye et al, 1984), changes in the activity of these neurons will influence cortical functioning, for example, the sleep–waking cycle (Stewart et al, 1984). It seems likely that a blockade of endogenous 5-HT1A-receptor-mediated transmission in the basal forebrain could result in a disinhibition of cholinergic MS–dBB and/or NB neurons. These findings support the view that the postsynaptic 5-HT1A receptors in the basal forebrain can modulate cholinergic transmission and, thus, influence learning and memory. Direct support for this view has recently been provided by in vivo microdialysis studies in awake rats performed in our laboratory. Both robalzotan and WAY 100635 have been found to increase basal ACh release in the hippocampus and cortex of the rat (J Kehr, personal communications). Robalzotan produced a two-fold increase in cortical and hippocampal ACh in the dose range that attenuated the PA deficit caused by scopolamine in this study. Also WAY 100635 increased ACh release in the same dose range as that blocking the action of scopolamine in the PA test. However, quantitatively robalzotan was more effective than WAY 100635 in its elevations of extracellular ACh. In view of the anatomical localization of brain 5-HT1A receptors, this finding suggests that the 5-HT1A antagonists can enhance cholinergic transmission in the forebrain by blocking 5-HT1A receptors located on the ascending cholinergic neuronal systems.

Muscarinic and 5-HT1A receptors are localized on the pyramidal hippocampal cells (Azmitia et al, 1996; van der Zee and Luiten, 1999; van der Zee et al, 1989), and electrophysiological studies (Grunschlag et al, 1997; Kobayashi et al, 1997; Sprouse and Aghajanian, 1988; Stewart et al, 1992; Tada et al, 1999; Wang and Tang, 1998) have shown that muscarinic and 5-HT1A receptors are involved in the regulation of pyramidal cell activity. These effects might explain the marked inhibitory effects of the 5-HT1A agonists in various types of learning in rodents ranging from aversive conditioning (Carli et al, 1993; Misane et al, 1998a; Misane and Ögren, 2000) to spatial learning (Bertrand et al, 2000; Carli et al, 1992) (see Meneses, 1999). Therefore, it is likely that another site for the 5-HT1A–muscarinic receptor interactions might involve populations of cortico-limbic pyramidal cells. In support of this hypothesis, subcutaneous WAY 100635 was found to prevent the impairment of spatial learning caused by intrahippocampal scopolamine or 7-chloro-kynurenic acid (an NMDA receptor antagonist), suggesting that blockade of 5-HT1A receptors can compensate for the loss of cholinergic or NMDA-receptor-mediated excitatory input to pyramidal cells in the hippocampus (Carli et al, 1997). This assumption receives support from other learning tasks in rodents. Thus, the selective 5-HT1A antagonist WAY 100635 when injected both s.c. and intrahippocampally reversed the impairment of the two-platform discrimination task caused by intrahippocampal scopolamine (Carli et al, 1997). However, detailed hypotheses on the potential interactive sites of muscarinic and 5-HT1A receptors and their therapeutical relevance must await a more detailed experimental analysis.

It is generally believed that AChE inhibitors such as physostigmine act by increasing synaptic ACh (Kawashima et al, 1994; Scali et al, 1997), resulting in competition with scopolamine at the postsynaptic muscarinic receptors. In agreement with this view, tacrine and donepezil attenuated or completely blocked the inhibitory effects of scopolamine depending on the dose of the muscarinic receptor antagonist. It is clear, however, that noncompetitive AChE inhibitors including physostigmine and tacrine in general can only attenuate but not fully block the cognitive impairments caused by scopolamine in different learning and memory tasks (Chopin and Briley, 1992; Kojima et al, 1997; Rush and Streit, 1992; Yoshida and Suzuki, 1993). The more potent effect of tacrine compared to donepezil seen in this study might be because of differences in the mechanism of action of these two AChE inhibitors. Donepezil is a piperidine-based mixed (competitive/noncompetitive)-type AChE inhibitor that has higher selectivity for AChE vs butyrylcholinesterase in the CNS than tacrine (Rogers, 1998). In addition, tacrine acts directly on both muscarinic and nicotinic receptors and potassium channels (Flynn and Mash, 1989; Halliwell and Grove, 1989; Hirai et al, 1997; Nilsson et al, 1987).

The relatively low potency of tacrine and particularly donepezil may seem paradoxical because the enzyme inhibitors will essentially increase cholinergic transmission at all muscarinic synapses. However, such a mechanism might be counterproductive since AChE inhibitors can also affect ‘noncholinergic’ neurons via the mode of extrasynaptic transmission (Mrzljak et al, 1998, Umbriaco et al, 1994,1995). In addition, at higher doses AChE inhibitors might ‘overstimulate’ ACh receptor systems in the brain. Thus, adequate performance of PA retention seems to depend on an optimal level of cholinergic receptor stimulation (Deutsch, 1971). This means that a delicate balance between pre- and postsynaptic ACh receptor mechanisms is probably required to compete with the functional muscarinic receptor blockade caused by the high-affinity receptor antagonist scopolamine.

In view of its nonselectivity for muscarinic receptors, the effects of systemic scopolamine administration on PA are probably resulting from impairments of a range of information processes such as attention, learning and memory. In addition, results obtained with the local administration of scopolamine suggest that its effects on aversive learning are mediated by limbic forebrain circuits involving entorhinal and parietal cortices, hippocampus, amygdala and septum (Izquierdo et al, 1992,1998; Nomura et al, 1994; Riekkinen et al, 1995). In this context, the ability of the 5-HT1A receptor antagonists to attenuate the impairment of PA caused by systemic scopolamine administration in a manner similar to tacrine is both unexpected and intriguing.

Taken together, these data support the hypothesis that cholinergic transmission in cortico-limbic brain regions can be enhanced by 5-HT1A receptor blockade. The 5-HT1A receptor antagonists might, therefore, be of value for the treatment of human psychopathologies associated with a reduction in ACh transmission such as AD. The lack of peripheral cholinergic side effects (salivation, tremor, etc) by this type of compounds may be of particular importance in human therapy.