Psychopharmacology (2009) 202:165–172 DOI 10.1007/s00213-008-1272-y
MK801- and scopolamine-induced amnesias are reversed by an Amazonian herbal locally used as a “brain tonic” Adriana Lourenço da Silva & Bárbara da Silva Martins & Viviane de Moura Linck & Ana Paula Herrmann & Nathalia Mai & Domingos S. Nunes & Elaine Elisabetsky
Received: 29 April 2008 / Accepted: 21 July 2008 / Published online: 10 August 2008 # Springer-Verlag 2008
Abstract Rationale Traditional remedies prepared from Ptychopetalum olacoides (PO) are used throughout the Amazon to alleviate age-related conditions. These formulas are mainly used by elders, and alleged effects may be related to the anticholinesterase properties identified in a standardized ethanol extract of this species [P. olacoides standardized ethanol extract (POEE)]. Objectives To further characterize the potential of this extract for developing drugs useful to treat cognitive deficits, the effects of POEE on scopolamine (scop)- and MK801-induced amnesias (acquisition, consolidation, and retrieval) in mice were investigated. Results Scop (3.0 mg/kg, ip) significantly impaired memory (all three phases) in the step-down inhibitory avoidance protocol. As expected, MK801 (0.1 mg/kg, ip) was amnesic regarding acquisition and consolidation, but not retrieval. A. L. da Silva : B. d. Silva Martins : V. d. M. Linck : A. P. Herrmann : N. Mai : E. Elisabetsky (*) Laboratório de Etnofarmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Av. Sarmento Leite 500/202, Porto Alegre, RS 90046-900, Brazil e-mail: [email protected]
A. L. da Silva : E. Elisabetsky PPG em Ciências Biológicas-Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Av. Ramiro Barcelos 2600, Porto Alegre, RS 90035-003, Brazil D. S. Nunes Departamento de Química, Universidade Estadual de Ponta Grossa, Campus Uvaranas, Bloco M, Ponta Grossa, PR 84030-310, Brazil
POEE (100 mg/kg, ip) reversed the scop-induced impairment in all three phases of long-term and short memories, whereas only the memory consolidation deficit was reversed with MK801-induced amnesia. Conclusions This study complements previously reported promnesic properties of this plant extract and suggests that POEE may be further developed for treating conditions associated with cognitive deficits, especially those linked with cholinergic malfunction. Keywords Ptychopetalum olacoides . Marapuama . Muirapuama . Acetylcholine . Glutamate . Amnesia . Acquisition . Consolidation . Retrieval . Memory
Introduction Reduced brain cholinergic function, particularly at the level of muscarinic receptors, is a characteristic of patients suffering from dementia. In Alzheimer’s disease (AD) and senile dementia of the Alzheimer’s type, the degree of cholinergic degeneration parallels the functional losses seen in these disorders (Coyle et al. 1983). Such clinical evidence combined with experimental observations strongly supports the crucial role of cholinergic activity in various forms of cognitive function (Matsuoka et al. 1991). Accordingly, there is evidence that learning functionally modifies cholinergic neurons, which becomes progressively more active. For instance, acetylcholine (ACh) release in the hippocampus increases during performance of a learned spatial memory task (Stancampiano et al. 1999), and the increase in ACh is positively correlated with performance improvement during learning (Fadda et al. 2006). Hence, the nonselective muscarine antagonist scopolamine (scop) has been used to induce amnesia in rodents, as an approach
to evaluate the effects of nootropic drugs on experimental memory deficits. Other than acetylcholine, it has long been recognized that glutamate has a pivotal role in neuroplasticity, learning, memory, and neurodegenerative diseases. Specifically in the hippocampus CA1 area, N-methyl-D-aspartic acid (NMDA) receptors are known to regulate synaptic plasticity, long term-potentiation (LTP), and learning and memory processes, including short- and long-term memories (Riedel et al. 2003). Moreover, competitive and noncompetitive NMDA antagonists, as well as lesions in glutamate pathways, have been shown to impair learning and memory processes in various tasks (Myhrer 2000; Jafari-Sabet 2006). Alcoholic infusions of Ptychopetalum olacoides Bentham (PO, Olacaceae), known as “Marapuama,” are consumed in the Amazon region for the treatment of central nervous system (CNS) conditions, and/or during particularly stressful periods (Elisabetsky and Siqueira 1998). We reported that P. olacoides standardized ethanol extract (POEE), a standardized ethanol extract obtained from P. olacoides, possesses various CNS-relevant properties, including antioxidant (Siqueira et al. 2007) and neuroprotective (Siqueira et al. 2004) activities. Regarding cognition, we showed that POEE facilitates short-term and long-term memories (inhibitory avoidance and object recognition) in adult and aged mice (da Silva et al. 2004, 2007). As a likely neurochemical correlate of POEE promnesic effects, we also reported in vitro and in vivo acetylcholinesterase inhibition for the same extract (Siqueira et al. 2003, 2007). The purpose of this study was to investigate if the POEE promnesic effects can offset scop- and MK-801-induced amnesias in mice.
Material and methods Animals Experiments were performed using male (CF1) adult albino mice, received from Fundação Estadual de Produção e Pesquisa em Saúde (FEPPS) at 2.0 months of age (35–40 g). Animals were maintained in our own animal facility under controlled environmental conditions [22±1°C, 12-h light/dark cycle, free access to food (Nuvilab CR1) and water], for at least 2 weeks before the experiments. The “Principles of laboratory animal care” (www.nap.edu/reading groom/books/labrats) were followed; the project was approved by the University Ethical Committee (approval no. 2007834), which follow the NIH guidelines (NIH Guide for Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985). Preparation of Extract Roots of P. olacoides Bentham (Olacaceae) were collected in Pará (Brazil) and identified by Nelson Rosa (MPEG 108.036 voucher at the Goeldi
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Museum Herbarium). P. olacoides standardized ethanol extract (POEE) was prepared and characterized as detailed elsewhere (Elisabetsky and Siqueira 1998); analytical highperformance liquid chromatography fingerprinting can be seen in Siqueira et al. (2007). Drugs Dimethyl sulfoxide (DMSO) was acquired from Delaware (Brazil). Scop and physostigmine (phys) were obtained from Sigma (USA). MK-801 was obtained from RBI (USA). Scop, phys, and MK-801 were dissolved in distilled water. POEE was dissolved in DMSO 20%. Saline (NaCl 0.9%, sal) was used as blank control. All chemicals were of analytical grade. Drug administration Drugs and vehicles were administered intraperitoneally, as 0.1 ml/10 g of body weight. For statistical accuracy, controls were run concurrently with drug-treated groups; graphics therefore display groups run at a given time. Scop (3.0 mg/kg) was given 15 min before training (acquisition) and test (retrieval) or immediately post-training (consolidation). Although it is known that cholinergic compounds might posses state-dependence effects (Boccia et al. 2003), scop was not administrated both pretraining and pretesting, since the aim of this study was to asses POEE cognitive-enhanced effects. Saline, DMSO 20%, phys (0.075 mg/kg, used as positive control), and POEE (50 or 100 mg/kg) were administered 30 min before scop, except for consolidation when treatments were given immediately after scop. MK-801 (0.1 mg/kg) was given 30 min before training (acquisition) and test (retrieval) or immediately post-training (consolidation). Saline, DMSO 20%, phys (0.075 mg/kg), and POEE (100 mg/kg) were administered 30 min before MK-801, except for consolidation where treatments were given immediately after MK-801. For chronic treatment, the protocol was adapted from Parra et al. (2000). Sixty-six mice were randomly distributed into three groups and treated once a day for 21 days with saline (n=21), DMSO 20% (n=23), or POEE 800 mg/kg (n=22) session training 24 h after the last drug administration and tested 24 h later.
Behavioral procedures Step-down inhibitory avoidance The protocol was adapted from Bernaerts et al. (2004). Mice were habituated to the dimly lit room for at least 30 min before the experiments. The inhibitory avoidance training apparatus was a plastic box of 30×30×40 cm, with a platform (5×5×4 cm) fixed in the center of the grid floor. Each mouse was placed on the platform, and the latency to step-down (four paws on the grid) was automatically recorded in training and test
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sessions. In the training session, upon stepping down, the mouse received a 0.4-mA scrambled foot shock for 15 s. Animals with training latencies shorter than 3 s and higher than 30 s were excluded from experiments; less than 5% of the animals met this exclusion criterion. Test sessions were performed 3 h [short-term memory, (STM)] or 24 h [longterm memory, (LTM)] later, with the same procedure except that no shock was administered after stepping down; an upper cutoff time of 300 s was set. N=18–21 per treatment. In order to determine whether the effects of the drugs on step-down inhibitory avoidance could be explained in terms of drug-induced learned aversion or other unspecific drug effects, the following experiment was included: mice were divided in non-shocked groups. POEE 100 mg/kg was given 30 min before training (acquisition), immediately post-training (consolidation), or 30 min before test (retrieval). The non-shocked animals were treated just as described above, except that no foot shock was administered following stepping down. Test sessions were performed 3 (STM) or 24 h (LTM) later. Statistical analysis The Kolmogorov–Smirnov test indicates (p