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Comparative studies of huperzine A, donepezil, and rivastigmine on brain acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine levels in freely-moving rats



To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats.


Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters.


Our results showed that huperzine A (0.25, 0.5, and 0.75 μmol/kg, po) dose-dependently elevated extracellular acetylcholine (ACh) levels in the medial prefrontal cortex (mPFC) and hippocampus. Oral administration of donepezil (5.4 μmol/kg) or rivastigmine (1 μmol/kg) also elicited significant increases in ACh in the mPFC and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of ACh at the same dose. The marked elevation of ACh after oral administration of huperzine A (0.5 μmol/kg) and donepezil (5.4 μmol/kg) was associated with a significantly increased release of dopamine (DA) in the mPFC or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the mPFC and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase ACh levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 μmol/kg, once per day for 30 d.


The present findings showed that, in molar terms, huperzine A had similar potency on increasing mPFC ACh and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing.


  1. 1

    Alhainen K, Helkala EL, Reinikainen K, Riekkinen, P Sr . The relationship of cerebrospinal fluid monoamine metabolites with clinical response to tetrahydroaminoacridine in patients with Alzheimer's disease. J Neural Transm 1993; 5: 185–92.

    CAS  Article  Google Scholar 

  2. 2

    Barili P, De Carolis G, Zaccheo D, Amenta F . Sensitivity to ageing of the limbic dopaminergic system: a review. Mech Ageing Dev 1998; 106: 57–92.

    CAS  Article  Google Scholar 

  3. 3

    Bar-On P, Millard CB, Harel M, Dvir H, Enz A, Sussman JL, et al. Kinetic and structural studies on the interaction of cholinesterase with the anti-Alzheimer drug rivastigmine. Biochemistry 2002; 41: 3555–64.

    CAS  Article  Google Scholar 

  4. 4

    Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–54.

    CAS  Article  Google Scholar 

  5. 5

    Brozoski T, Brown RM, Rosvold HE, Goldman PS . Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey. Science 1979; 205: 929–32.

    CAS  Article  Google Scholar 

  6. 6

    Costa LG, Schwab BW, Murphy SD . Tolerance to anticholinest-erase compounds in mammals. Toxicology 1982; 25: 79–97.

    CAS  Article  Google Scholar 

  7. 7

    Coyle JT, Price DL, DeLong MR . Alzheimer's disease: a disorder of cortical cholinergic innervation. Science 1983; 219: 1184–90.

    CAS  Article  Google Scholar 

  8. 8

    Cuadra G, Summers K, Giacobini E . Cholinesterase inhibitor effects on neurotransmitters in rat cortex in vivo. J Pharmacal Exp Ther 1994; 270: 277–84.

    CAS  Google Scholar 

  9. 9

    Davidson M, Bierer LM, Kaminsky R, Ryan TM, Davis KL . Combined administration of physostigmine and clonidine to patients with dementia of the Alzheimer type: a pilot safety study. Alzheimer Disease Assoc Disorders 1989; 3: 224–7.

    CAS  Article  Google Scholar 

  10. 10

    Day J, Fibiger HC . Dopaminergic regulation of cortical acetylcholine release. Synapse 1992; 12: 281–6.

    CAS  Article  Google Scholar 

  11. 11

    Decker MW, McGaugh JL . The role of interactions between the cholinergic system and other neuromodulatory systems in learning and memory. Synapse 1991; 7: 151–68.

    CAS  Article  Google Scholar 

  12. 12

    Ellman GL, Courtney KD, Andre V Jr, Featherstone RM . A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961; 7: 88–95.

    CAS  Article  Google Scholar 

  13. 13

    Floresco SB, Seamans JK, Phillips AG . Selective roles for hippocampal, refrontal cortical, and ventral striatal circuits in radial-arm maze tasks with or without a delay. J Neurosci 1997; 17: 1880–90.

    CAS  Article  Google Scholar 

  14. 14

    Giacobini E, Becker R . Development of drugs for Alzheimer therapy: a decade of progress. In: Giacobini E, Becker, R Alzheimer Disease: Therapeutic Strategies. Boston, MA: Birkhäuser Press; 1994. p 1–7.

    Chapter  Google Scholar 

  15. 15

    Giacobini E, Cuadra G . Second and third generation cholinesterase inhibitors: from preclinical studies to clinical efficacy. In: Giacobini E, Becker, R Alzheimer Disease: Therapeutic Strategies. Boston, MA: Birkhäuser Press; 1994. p 155–71.

    Chapter  Google Scholar 

  16. 16

    Giacobini E, Zhu XD, Williams E, Sherman KA . The effect of the selective reversible acetylcholinesterase inhibitor E2020 on extracellular acetylcholine and biogenic amine levels in rat cortex. Neuropharmacology 1996; 35: 205–11.

    CAS  Article  Google Scholar 

  17. 17

    Hallak M, Giacobini E . Physostigmine, tacrine and metrifonate: the effect of multiple doses on acetylcholine metabolism in rat brain. Neuropharmacology 1989; 28: 199–206.

    CAS  Article  Google Scholar 

  18. 18

    Haroutunian V, Santucci AC, Davis KL . Implications of multiple transmitter system lesions for cholinominetic therapy in Alzheimer's disease. Prog Brain Res 1990; 84: 333–46.

    CAS  Article  Google Scholar 

  19. 19

    Hersi AI, Rowe W, Gaudreau P, Quirion R . Dopamine D1 receptor ligands modulate cognitive performance and hippocampal acetylcholine release in memory-impaired aged rats. Neuroscience 1995; 69: 1067–74.

    CAS  Article  Google Scholar 

  20. 20

    Jann MW . Rivastigmine, a new-generation cholinesterase inhibitor for the treatment of Alzheimer's disease. Pharmacotherapy 2000; 20: 1–12.

    CAS  Article  Google Scholar 

  21. 21

    Kosasa T, Kuriya Y, Matsui K, Yamanishi Y . Effect of donepezil hydrochloride (E2020) on basal concentration of extracellular acetylcholine in the hippocampus of rats. Eur J Pharmacol 1999; 380: 101–7.

    CAS  Article  Google Scholar 

  22. 22

    Laganiere S, Corey J, Tang XC, Wulfert E, Hanin I . Acute and chronic studies with the anticholinesterase huperzine A: effect on central nervous system cholinergic parameters. Neuropharmacology 1991; 30: 763–9.

    CAS  Article  Google Scholar 

  23. 23

    Levy A, Kong RM, Stillman MJ, Shukitt-Hale B, Kadar T, Rauch TM, et al. Nimodipine improves spatial working memory and elevates hippocampal acetylcholine in young rats. Pharmacol Biochem Behav 1991; 39: 781–6.

    CAS  Article  Google Scholar 

  24. 24

    Marien MR, Colpaert FC, Rosenquist AC . Noradrenergic mechanisms in neurodegenerative diseases: a theory. Brain Res Rev 2004; 45: 38–78.

    CAS  Article  Google Scholar 

  25. 25

    McCarthy G, Blamire AM, Puce A . Functional magnetic resonance imaging of human prefrontal cortex activation during a spatial working memory task. Proc Natl Acad Sci USA 1994; 91: 8690–4.

    CAS  Article  Google Scholar 

  26. 26

    Pang YP, Kozikowski AP . Prediction of the binding site of huperzine A in acetylcholinesterase by docking studies. J Comput Aided Mol Des 1994; 8: 669–81.

    CAS  Article  Google Scholar 

  27. 27

    Paxinos G, Watson, C The rat brain in stereotaxic coordinates. Sydney: Academic Press; 1997.

    Google Scholar 

  28. 28

    Raves ML, Harel M, Pang YP, Silman I, Kozikowski AP, Sussman JL . Structure of acetylcholinesterase complexed with the nootropic alkaloid (-)-huperzine A. Natural Struct Biol 1997; 4: 57–63.

    CAS  Article  Google Scholar 

  29. 29

    Scali C, Casamenti F, Pazzagli M, Bartolini L, Pepeu G . Nerve growth factor increases extracellular acetylcholine levels in the parietal cortex and hippocampus of aged rats and restores object recognition. Neurosci Lett 1994; 170: 117–20.

    CAS  Article  Google Scholar 

  30. 30

    Steckler T, Sahgal A . The role of serotonergic-cholinergic interactions in the mediation of cognitive behaviour. Behav Brain Res 1995; 67: 165–99.

    CAS  Article  Google Scholar 

  31. 31

    Sugimoto H, Ogura H, Arai Y, Limura Y, Yamanishi Y . Research and development of donepezil hydrochloride, a new type of acetylcholinesterase inhibitor. Jpn J Pharmacol 2002; 89: 7–20.

    CAS  Article  Google Scholar 

  32. 32

    Taghzouti K, Le Moal, M, Simon H . Dopaminergic innervation of the lateral septum and learning processes. Neurosci Lett Suppl 1986; 26: s332.

    Google Scholar 

  33. 33

    Tang XC . Huperzine A (Shuangyiping): A promising drug for Alzheimer's disease. Acta Pharmacol Sin 1996; 17: 481–4.

    CAS  Google Scholar 

  34. 34

    Tang XC, De Sarno P, Sugaya K, Giacobini E . Effect of huperzine A, a new cholinesterase inhibitor, on the central cholinergic system of the rat. J Neurosci Res 1989; 24: 276–85.

    CAS  Article  Google Scholar 

  35. 35

    Taylor P . Anticholinesterase agents. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman, AG The pharmacological basis of therapeutics. New York: McGraw-Hill; 1996. p 161–76.

    Google Scholar 

  36. 36

    Trabace L, Coluccia A, Gaetani S, Tattoli M, Cagiano R, Pietra C, et al. In vivo neurochemical effects of the acetylcholinesterase inhibitor ENA713 in rat hippocampus. Brain Res 2000; 865: 268–71.

    CAS  Article  Google Scholar 

  37. 37

    Wang R, Yan H, Tang XC . Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine. Acta Pharmacol Sin 2006; 27: 1–26.

    Article  Google Scholar 

  38. 38

    Weinstock M, Razin M, Chorev M, Enz A . Pharmacological evaluation of phenylcarbamates as CNS-selective acetylcholinesterase inhibitors. J Neural Transm 1994; 43: 219–25.

    CAS  Google Scholar 

  39. 39

    Zhu XD, Giacobini E . Second generation cholinesterase inhibitors: effect of (L)-huperzine A on cortical biogenic amines. J Neurosci Res 1995; 41: 828–35.

    CAS  Article  Google Scholar 

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Correspondence to Xi-can Tang.

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Project supported by the National Natural Science Foundation of China (No 30123005).

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Liang, Yq., Tang, Xc. Comparative studies of huperzine A, donepezil, and rivastigmine on brain acetylcholine, dopamine, norepinephrine, and 5-hydroxytryptamine levels in freely-moving rats. Acta Pharmacol Sin 27, 1127–1136 (2006).

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  • huperzine A
  • donepezil
  • rivastigmine
  • acetylcholine
  • dopamine
  • norepinephrine
  • 5-hydroxy tryptamine
  • Alzheimer's disease
  • rats

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