Tacrine and Bis(7)-Tacrine Attenuate Cycloheximide-Induced Amnesia in Mice, with Attention to Acute Toxicity

Authors


Author for correspondence: Si-Yuan Pan, Department of Pharmacology, Beijing University of Chinese Medicine, Beijing 100102, China (fax +86 010 847 38627, e-mail siyuan-pan@163.com).

Abstract

Abstract:  Effects of tacrine and bis(7)-tacrine (0.25–20 μmol/kg, s.c.) on cognitive behaviour in cycloheximide (CYH)-treated mice were investigated. Cognitive behaviour was assessed by open-field test and step-through task with a 24-hr retention interval. Drugs or vehicle was given 30 min. prior to the first session. Although CYH treatment (110 mg/kg, i.p.) alone did not affect the locomotor activity of mice, CYH treatment in combination with tacrine (20 μmol/kg) decreased the locomotor activity by 37% in the acquisition session, when compared with mice treated with CYH alone. Bis-(7) tacrine cotreatment did not produce any detectable effect on locomotor activity. During the retention trial, tacrine (5 μmol/kg) or bis(7)-tacrine (1 μmol/kg) enhanced the retention latency (by 3.8- or 1.4-fold, respectively) in CYH-treated mice. In both training and retention trials, CYH treatment increased the number of footshocks (by 50% and 11.3-fold, respectively). However, during the retention (but not training) trial, tacrine (5 μmol/kg) or bis(7)-tacrine (1 μmol/kg) decreased the footshocks (by 8.6-fold or 39%, respectively) in CYH-treated mice. Combined treatment with CYH and bis(7)-tacrine (but not tacrine) resulted in an increased mortality rate in mice. The results indicated that tacrine and bis(7)-tacrine improved the amnesia caused by CYH treatment. However, the combined treatment with bis(7)-tacrine and CYH administration caused acute toxicity.

Acetylcholine (ACh) is widely distributed in the central and peripheral nervous system. ACh released from cholinergic nerve terminals activates receptors at both pre-synaptic and post-synaptic sites. It is then hydrolysed by acetylcholinesterase (AChE) into choline and acetyl coenzyme A. Cholinergic nerve in the brain may play an important role in many cognitive functions, such as cortical modulation of sensory information processing, attention, memory and learning [1–3]. Therapeutic strategies adopting cholinergic precursor loading and AChE inhibitors, which can increase ACh concentration in vivo, were used for treating cognitive impairment occurring in Alzheimer’s disease (AD) and geriatric memory dysfunction [4–6].

The cognitive enhancement and AChE inhibition afforded by tacrine, a reversible AChE inhibitor, has been widely studied. The administration of tacrine is often accompanied by abnormal behaviour and a decline in physical strength in patients [7,8]. Bis(7)-tacrine is a novel anti-AD agent that can reversibly inhibit AChE 150-fold more potently than tacrine in rat brains [9,10]. The cognitive enhancement of bis(7)-tacrine was observed in rats treated with scopolamine, a muscarinic cholinergic receptor antagonist that causes memory loss, and AF64A, a cholinergic neuron-specific neurotoxin [11,12]. Our previous works have shown that both tacrine and bis(7)-tacrine inhibited the locomotor activity in normal mice and improved the impairment of open-field and passive avoidance response memory in mice treated with scopolamine [13–15]. Cycloheximide (CYH) is a protein synthesis inhibitor widely used for investigating the biochemical mechanism(s) underlying the consolidation of long-term memory [16–19]. In this study, we examined the effect of CYH per se or CYH in combination with tacrine or bis(7) tacrine on cognitive behaviour using open-field test and step-through task in the mice.

Materials and Methods

Animal care.  Male ICR mice (Grade II, certificate No. SCXK(jing)2007-0001) weighing 15–18 g were supplied by Vital River Lab Animal Co. Ltd. (Beijing, China). Eight animals were housed in one cage (28 × 18 × 12 cm) and allowed free access to food and water in a room kept at 23–25°C, with a relative humidity of 50–55% and a 12-hr light : dark cycle (lights on at 07:00). Mouse chow and tap water were available ad libitum. Experiments were performed when the animals had grown up to a body weight of 23–25 g. All experiments were conducted according to the guidelines of the Committee on Care and Use of Laboratory Animals of the Beijing University of Chinese Medicine. Every effort was made to minimize the number of animals used and the suffering of animals.

Drug treatment.  Tacrine hydrochloride was purchased from Sigma Chemical Company (St. Louis, MO, USA). Bis(7)-tacrine hydrochloride was synthesized and structurally characterized according to method Pang et al. [20]. CYH was obtained from Fluka AG (Buchs, Switzerland). Drugs were dissolved in distilled water and given in a single subcutaneous injection at increasing doses for tacrine (1–20 μmol/kg) and bis(7)-tacrine (0.25–20 μmol/kg). The dose range of tacrine or bis(7) tacrine adopted in this study was based on findings in previous studies regarding the memory enhancing effect of the drugs [11–15]. CYH was administered intraperitoneally at a dose of 110 mg/kg. Mice in the drug-untreated group were given the same volume of vehicle (s.c. or i.p.).

Cognitive behavioural assessment.  The cognitive behavioural performance was assessed by an open-field test and a step-through task, as previously described [13]. For the open-field test, the locomotor activity within a fixed period of time was measured in an open-field chamber. Each mouse was placed in this chamber for 3 min. in two successive days. When the mouse was placed in the open-field chamber, the locomotor activity counts were automatically recorded using an Activity Meter (MK-ANIMEX, Muromachi Kikai Co., Tokyo, Japan). The open-field memory was indicated by the reduced locomotor activity in the recall session, when compared with that measured in the acquisition session. After the open-field test, the mouse was proceeded immediately to perform the step-through task. Preliminary studies confirmed that the prior open-field test did not affect the outcome of the step-through task in control mice. When entering the dark compartment, the mouse was electrified (1 Hz, 0.5 sec. and 40 V DC) and thus returned the light (safe) one. During the training and retention trial, the latency and number of received footshocks were recorded within 5 min. The retention trial was conducted 24 hr after the training trial. If a mouse did not receive a shock within 5 min., it was assigned a zero score for the number of footshocks and 300 sec. for the retention latency. The learning and memory on the step-through task was reflected by the number of footshocks in the training trials and the retention latency as well as the number of footshocks recorded in the retention trials, respectively. Both drug and vehicle were administered 30 min. before the acquisition session in the open-field test. No drug and vehicle was given before the recall sessions and retention trials. Experiments were carried out between 08:00 and 13:00.

Statistical analysis.  Data were expressed as mean ± S.E.M, with the indicated number of animals. They were analysed by one-way anova followed by Dunnett’s test to detect the significant difference between two groups, using SPSS13.0 statistical software, IBM Corporation (Somers, New York, USA). The difference with < 0.05 was considered as significant.

Results

Locomotor activity in open-field test.

During the acquisition session, while CYH at a dose of 110 mg/kg did not affect the locomotor activity (F1,19 = 0.048, > 0.05), the cotreatment with tacrine at 20 μmol/kg markedly decreased the locomotor activity in CYH-treated mice by 37% (F4,36 = 14.075, < 0.001), when compared with the mice treated with CYH alone (fig. 1A). Bis(7)-tacrine cotreatment (0.25–20 μmol/kg) did not affect the locomotor activity in the CYH-treated mice (F1,19 = 0.215, > 0.05), when compared with the mice treated with CYH alone (fig. 1B). During the recall session, the locomotor activity in both vehicle- and drug-treated mice was significantly reduced (< 0.01 or 0.001), except for tacrine at 20 μmol/kg, when compared with that observed in the corresponding acquisition session. However, pre-acquisition administration of CYH alone (F1,19 = 1.414, > 0.05) and CYH in combination with tacrine (F3,36 = 2.399, > 0.05) or bis(7)-tacrine (F4,40 = 1.279, > 0.05) did not change the motor function of mice in the recall session (fig. 1).

Figure 1.

 Effects of tacrine and bis(7)-tacrine treatment on locomotor activity in cycloheximide-treated mice in the open-field test. Mice were administered with tacrine (s.c.), bis(7)-tacrine (s.c.) or cycloheximide (i.p.) 30 min. prior to the acquisition session. Control animals were administered with vehicle (both s.c. and i.p.). The locomotor activity in each mouse was monitored for 3 min. in the acquisition and recall session, with the latter being conducted 24 hr after the former. Each bar represents the mean ± S.E.M., with n = 10. The number inside the bar in panel (B) indicates the number of animals. ***< 0.001 versus the vehicle-treated mice, using Student’s t test; < 0.05, ††< 0.01, †††< 0.001 versus the corresponding group in the acquisition session, using one-way anova followed by Dunnett’s t test.

Latency in step-through task.

Drug-treated mice did not show any detectable changes in latency in the training trial, when compared with the vehicle-treated control. During the retention trial, the latency was markedly prolonged in vehicle-treated mice (up to 10-fold) (F1,19 = 128.496, < 0.001), when compared with those observed in the training trial. The retention latency was shortened by 82% (F1,19 = 65.427, < 0.001) in the mice treated with CYH, when compared with the mice receiving vehicle. Mice cotreated with CYH and tacrine (5 μmol/kg only) (F1,19 = 46.422, < 0.001) or bis(7)-tacrine (0.25 μmol/kg) (F1,19 = 11.416, < 0.01) showed a prolonged retention latency (3.8- or 1.4-fold, respectively), when compared with that of the mice treated with CYH alone (fig. 2A,B).

Figure 2.

 Effects of tacrine and bis(7)-tacrine on latency in cycloheximide-treated mice in the step-through task. Following the open-field test described as fig. 1, the mice were proceeded immediately to perform the step-through task. The latencies (s) in each mouse were measured for 5 min. in both training and retention trials, with the latter being conducted 24 hr after the former. Each bar represents the mean ± S.E.M., with n = 10. The number inside the bar in panel (b) indicates the number of animals. ***< 0.001 versus the vehicle-treated mice; #< 0.05, ###< 0.001 versus cycloheximide-treated mice; †††< 0.001 versus the corresponding group in the training session, using one-way anova followed by Dunnett’s t test.

Footshocks in step-through task.

Fig. 3 shows that CYH treatment increased the number of footshocks (by 50% and 11.3-fold, respectively) during both training (F1,19 = 6.194, < 0.05) and retention trials (F1,19 = 8.656, < 0.01), when compared with the vehicle-treated control. During the retention trial, the number of footshocks was markedly decreased in vehicle-treated mice (up to 95%) (F1,19 = 48.210, < 0.001). Although tacrine or bis(7)-tacrine cotreatment did not significantly suppress the CYH-induced increase in footshocks in the training session, tacrine (5 μmol/kg) (F1,19 = 44.100, < 0.001) or bis(7)-tacrine (1 μmol/kg) (F1,18 = 18.361, < 0.001) cotreatment ameliorated the increase in the number of footshocks (by 89% or 39%, respectively) in the retention trial, when compared with the CYH treatment alone (fig. 3A,B).

Figure 3.

 Effects of tacrine and bis(7)-tacrine on the number of footshocks in the cycloheximide-treated mice in the step-through task. Experimental procedures are described in fig. 2. The number of footshocks in each mouse was measured for 5 min. in both training and retention sessions. Each bar represents the mean ± S.E.M., with n = 10. The number inside the bar in panel (B) indicates the number of animals. *< 0.05, **< 0.01 versus the vehicle-treated mice; ##< 0.01 versus cycloheximide -treated mice; †††< 0.001 versus the corresponding group in the training session, using one-way anova followed by Dunnett’s t test.

Acute toxicity.

Mice were treated with CYH or tacrine/bis(7)-tacrine alone or CYH in combination with tacrine or bis(7)tacrine. The mortality was not affected by the treatment with CYH, tacrine or bis(7)tacrine alone in mice. However, bis(7)-tacrine cotreatment (1–20 μmol/kg) dose dependently increased the mortality of CYH-treated mice, when compared with that of mice treated with CYH or bis(7)-tacrine alone. No acute toxicity was observed when CYH and tacrine were coadministered in mice (table 1).

Table 1. 
Acute toxicity of tacrine or bis(7)-tacrine and cycloheximide cotreatment in mice.
GroupsDose (μmol/kg)Number of animalsMortality rate (%)
  1. Mice were treated with tacrine, bis(7)-tacrine and cycloheximide, as described in fig. 1. The mortality rate was recorded 24 hr following the drug administration.

Non-cycloheximide
Tacrine1100
5100
20100
Bis(7)-tacrine0.25100
1100
5100
20100
Cycloheximide (110 mg/kg)
Non-tacrine or bis(7)-tacrine 100
Tacrine1100
5100
20100
Bis(7)-tacrine0.25100
11010
51020
201060

Discussion

Open-field test is commonly used for evaluating the effect of drugs on motor function of animals. While CYH treatment alone did not inhibit the spontaneous locomotor activity of mice, cotreatment with tacrine suppressed the locomotor activity in the acquisition (but not recall) session. However, bis(7)-tacrine treatment did not significantly affect the locomotor activity in the mice treated with CYH in both acquisition and recall sessions. Despite the fact that the inhibition of AChE by bis(7)-tacrine was much more potent than tacrine [9], our finding indicated that the inhibition of motor function by tacrine was more potent than bis(7)-tacrine in the CYH-treated mice. Consistent with this, our recent studies also showed that tacrine and bis(7)-tacrine treatment alone inhibited the locomotor activity, with the former being more potent [13,14]. Apparently, the AChE inhibitor-induced decrease in locomotor activity may not be causally related to the inhibition of AChE.

The decrease in locomotor activity of mice during the recall session reflects a habituation or remembrance to the conditioning apparatus, which is a manifestation of open-field memory [21–23]. Memory loss, as assessed by the open-field test and manifested in hyperlocomotion in the recall session, has been observed in mice treated with high doses of scolopamine and tacrine [14,24,25]. The modification of neuronal connections in response to stimuli is generally believed to be the basis of long-term memory formation, in that local protein synthesis contributes critically to the site-restricted modulation of individual synapses [26]. While the administration of CYH at the pre-acquisition time did not cause the loss of open-field memory loss in mice, it impaired the passive avoidance memory. The remnant CYH present in blood, if there was any, during the retention session of the step-through task unlikely influenced the experimental outcome because the retention latency and the number of footshocks should have been prolonged and decreased, respectively, by the possible sedative effect of CYH. These findings therefore suggest that the mechanism underlying open-field memory may be distinct from that of passive avoidance memory. Possibly, the open-field memory does not require protein synthesis that may be needed in acquiring passive avoidance memory. Nevertheless, we cannot exclude the possibility that a higher dose of CYH is required for impairing an open-field memory than that for passive avoidance memory. As the acute treatment with CYH did not affect the sensitivity of mice to footshock (data not shown), the effect of CYH pre-treatment on passive avoidance memory, as assessed by the number of footshocks, was unlikely because of the direct action of CYH on the sensitivity of mice to electric footshock. Consistently, the cognition-enhancing drugs such as tacrine and bis(7)tacrine can reverse the action of CYH.

The inhibition of learning and memory by CYH treatment was completely blocked by tacrine (at 5 μmol/kg). However, the CYH-induced cognitive dysfunction was only partially antagonized by bis(7)-tacrine treatment (at 0.25 or 1 μmol/kg). Consistently, Nabeshima et al. [27] reported that tacrine treatment for 7 days improved the amnesia caused by CYH administration (1.5 mg/kg, s.c.), as assessed by passive avoidance task in rats. In addition, a novel muscarinic agonist was also found to ameliorate the impairment of passive avoidance response in senescence-accelerated mice [28]. As a cholinesterase inhibitor, tacrine and bis(7)-tacine are expected to attenuate the cognitive impairment induced by antagonists of ACh function. The two AChE inhibitors used in the present study were also found to produce a similar cognition-enhancing effect in CYH-treated mice. However, higher doses of bis(7)-tacrine were found to cause acute toxicity in CYH-treated mice. As the combined treatment with tacrine and CYH did not cause an increase in mortality in mice, it is unlikely that the acute toxicity caused by the combined treatment with bis(7)-tacrine and CYH is because of the increase in the extent of AChE inhibition. In this regard, bis(7)-tacrine may act differently from tacrine, as was the case in producing protection against transient focal cerebral ischaemia in rats [29]. While the biochemical mechanism involved in the cognition enhancement afforded by tacrine in CYH-treated mice remains to be determined, it is likely that the cognitive disorder induced by CYH may be causally related to dysfunction of cholinergic nerves in the brain. Alternatively, tacrine may improve protein synthesis under the inhibitory action of CYH. In this regard, it was found that CYH seemed to act pre-synaptically by inhibiting ACh release from nerve terminals [30].

Acetylcholinesterase is broadly distributed in various brain regions, and the inhibition of AChE can increase the synaptic concentration of ACh, resulting in the prolongation of ACh action. Interestingly, the inhibition of CYH on learning and memory was completely blocked by tacrine at 5 μmol/kg but not 1 and 20 μmol/kg. As a result, the attenuation of memory impairment by tacrine showed an inverted U-shape dose response in CYH-treated mice, which is not unusual for AChE inhibitor in improving cognitive function. Under the condition of AChE inhibition, the high concentration of ACh causes over-stimulation of synapses, which may in turn affect the formation of a longer-term memory [31]. Nevertheless, the biphasic dose-dependent effect of tacrine on CYH-induced impairment on cognitive function remains to be investigated.

In summary, tacrine and bis(7)-tacrine treatment at doses that did not cause motor dysfunction reduced the number of footshocks and enhanced the retention latency in step-through task in CYH-treated mice. A high dose of tacrine but not bis(7)-tacrine caused motor dysfunction in CYH-treated mice. The results indicated that AChE inhibitor could attenuate the learning and memory deficit induced by CYH, a protein synthesis inhibitor. When administered in combination, CYH and bis(7)-tacrine but not tacrine caused acute toxicity, with the resultant increase in mortality in mice. Bis(7)-tacrine and tacrine may act differently in vivo aside from the inhibition of AChE.

Acknowledgement

This work was supported by a grant from the National Natural Science Foundation of China (Grant No 31071989) and HKUST 9441/06M.

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