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Modulation of MK-801-elicited mouse popping behavior by galantamine is complex and dose-dependent
Life Sciences 73 (2003) 2355 – 2361 www.elsevier.com/locate/lifescie
Modulation of MK-801-elicited mouse popping behavior by galantamine is complex and dose-dependent Stephen I. Deutsch a,b,c,d,*, Richard B. Rosse a,b, Eddie N. Billingslea a, Alan S. Bellack a,c, John Mastropaolo a a
Mental Health Service Line, Veterans Integrated Service Network (VISN) 5, 849 International Drive, Suite 275, Linthicum, MD 21090, USA b Department of Psychiatry, Georgetown University School of Medicine, 3800 Reservoir Road, NW, Washington, DC 20007, USA c Department of Psychiatry, University of Maryland School of Medicine, 701 West Pratt Street, Suite 388, Baltimore, MD 21201, USA d Department of Veterans Affairs Medical Center, Mental Health Service Line, VISN 5, 50 Irving Street, NW, Washington, DC 20422, USA Received 2 April 2003; accepted 9 April 2003
Abstract The ability of phencyclidine (PCP), a noncompetitive antagonist of NMDA receptor-mediated neurotransmission, to precipitate a schizophreniform psychosis in susceptible individuals is consistent with the hypothesized pathologic occurrence of NMDA receptor hypofunction in this disorder. Because the psychosis caused by PCP resembles schizophrenia in all of the relevant domains of psychopathology, investigators have sought to characterize animal models of NMDA receptor hypofunction . MK-801 (dizocilpine) binds to the same hydrophobic channel domain in the NMDA receptor-associated ionophore as PCP, and has been shown to elicit intense irregular episodes of jumping behavior in mice, termed ‘‘popping.’’ MK-801-elicited mouse popping is an animal model of NMDA receptor hypofunction that has been used to screen novel candidate compounds for the treatment of schizophrenia. Recently, a selective abnormality in the transduction of the acetylcholine signal at the level of the a7 nicotinic receptor has been described in schizophrenia. The existence of a nicotinic cholinergic abnormality in schizophrenia has stimulated interest in a potential therapeutic role for positive allosteric modulation of nicotinic receptors. Galantamine is a compound that possesses two interesting properties: inhibition of acetylcholinesterase and positive allosteric modulation of nicotinic neurotransmission. Theoretically, galantamine would be expected to increase the efficiency or likelihood that acetylcholine will promote channel
* Corresponding author. Department of Veterans Affairs Medical Center, Mental Health Service Line, VISN 5, 50 Irving Street, NW, Washington, DC 20422, USA. Tel.: +1-202-745-8156; fax: +1-202-745-8169. E-mail address: [email protected] (S.I. Deutsch). 0024-3205/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/S0024-3205(03)00642-8
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opening and ionic conductance at nicotinic receptors. As expected, in the current investigation statistically significant popping behavior was elicited by MK-801 in mice (T(22) = 2.16, P < 0.05). This MK-801-elicited popping was significantly attenuated by 100 mg/kg of galantamine (T(22) = 2.24, P < 0.05). The data show that nicotinic interventions can influence NMDA receptor-mediated neurotransmission in the intact mouse. D 2003 Published by Elsevier Inc. Keywords: Galantamine; MK-801 (dizocilpine); NMDA receptor; Popping; Schizophrenia
Introduction NMDA receptor hypofunction and its ‘‘downstream’’ consequences may be the pathophysiological basis for much of the psychopathology manifested in schizophrenia (Deutsch et al., 1989, 2001; Coyle, 1996; Farber et al., 1998; Tamminga, 1998). PCP is a noncompetitive antagonist of the NMDA receptor that binds to a hydrophobic channel domain associated with this glutamate-gated receptor (Jentsch and Roth, 1999). The downstream consequences of NMDA receptor hypofunction are also likely to include both diminished GABAergic neurotransmission and excitotoxic stimulation of the a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA)/kainate class of glutamate-gated channel receptors. Ultimately, these consequences of NMDA receptor hypofunction could explain the progressive ventriculomegaly and psychosocial deterioration that is seen in a subgroup of patients with schizophrenia (Deutsch et al., 2001). Recently, in addition to NMDA receptor hypofunction and its consequences, interest has focused on the contribution of defective neurotransmission mediated by the a7 subtype of nicotinic receptor, an ancestral homopentameric protein complex, to the pathophysiology of schizophrenia. For example, autopsy studies of brains of patients with schizophrenia have shown reduced and abnormal a7 polypeptide receptor subunit protein and messenger RNA, and decreased binding of radiolabeled alpha-bungarotoxin, a specific ligand for the ‘‘low-affinity’’ a7 subtype (Freedman et al., 1995; Guan et al., 1999). Acetylcholine, a nonselective agonist, and choline, a relatively selective agonist, are two naturally occurring ligands that gate the a7 nicotinic receptor, producing fast type 1A nicotinic responses that are characterized by rapid decay (Albuquerque et al., 1997; Alkondon et al., 1997). Disturbed signal transduction at the a7 nicotinic receptor stimulated interest in potential salutary therapeutic properties of pharmacological enhancement of signal transduction by this subtype of nicotinic receptor. As noted above, the induction of NMDA receptor hypofunction by PCP is a pharmacological model of schizophrenia that mimics accurately the various domains of psychopathology observed in this illness, including positive (e.g., hallucinations), negative (e.g., affective flattening), and cognitive (e.g., concrete thinking) symptoms (Deutsch et al., 1989). In the current investigation, the ability of galantamine, an acutely administered nicotinic intervention with positive allosteric modulatory properties, to modulate NMDA receptor hypofunction in the intact mouse was studied. The allosteric modulatory effects of galantamine are presumed to occur very rapidly and at low nanomolar concentrations (Samochocki et al., 2000; Maelicke et al., 2001). In prior work, we showed that nicotine, a nonselective direct agonist, and mecamylamine, a relatively selective competitive antagonist for the ‘‘high-affinity’’ nicotinic receptor (e.g., a4h2), modulated MK-801-elicited popping behavior (Tizabi et al., 1998). For a variety of reasons, positive allosteric modulation of nicotinic receptors (i.e., improving the efficiency of signal transduction) may be preferable to their direct stimulation by an agonist, in a condition like schizophrenia. Firstly, the allosteric modulator may improve the faulty signal transduction associated
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with defective or reduced receptors, while helping to maintain the receptor in a sensitized and responsive state. For example, tolerance does not develop to the anti-conflict effects of benzodiazepines; this action of benzodiazepines may result from positive allosteric modulation of the population of GABAA receptors located in the frontal cortex (Braestrup and Nielsen, 1986; Haefely and Polc, 1986; Wamsley et al., 1986). Ideally, galantamine may act in a manner analogous to this presumed action of benzodiazepines in frontal cortex (i.e., anti-conflict effect), improving the transduction of acetylcholine at the a7 nicotinic receptor, without causing desensitization of the receptor and tolerance to its action. In any event, the current study showed that acute administration of galantamine is able to modulate pharmacologically-induced NMDA receptor hypofunction in the intact mouse.
Materials and methods Subjects Experimentally naı¨ve male NIH Swiss mice (an outbred strain obtained from the National Cancer Institute, Frederick, MD) weighing 20–30 g were used. Mice were housed in hanging clear Plexiglass cages in groups of five and maintained on a cycle of 12 h of light followed by 12 h of darkness in an American Association for the Accreditation of Laboratory Animal Care (AAALAC) approved animal facility. The mice had free access to food and water. The animals were weighed individually prior to drug administration and behavioral testing. Because animal subjects were employed in these experiments, all experimental protocols had to be approved by our institutional review board prior to being initiated. All experiments were conducted in accordance to these protocols. No procedures that were not approved were part of these experiments and the number of animals did not exceed those approved. Group sizes of 12 mice per group were tested at each of the treatment conditions. Drugs MK-801 (dizocilpine; Research Biochemical International; Natick, MA) and galantamine (galanthamine hydrobromide, Sigma Chemical, St. Louis, MO) were dissolved in 0.9% saline and prepared on the day of the experiment. Groups of mice were injected intraperitoneally with vehicle or one of several doses (1, 3.2, 10, 32, 100 mg/kg) of galantamine 10 min prior to the injection of MK-801. Subsequently, mice were injected with MK-801 (0.56 mg/kg), or its vehicle, 5 min prior to the 30 min monitoring period for the assessment of popping behavior. All injections were in a volume of 0.01 ml/g of body weight. Computerized assessment of MK-801-elicited popping The recording of MK-801-elicited popping behavior was divided into two phases: a baseline period of five min and an outcome recording period of 30 min, which immediately followed an injection of either MK-801 or its vehicle. The automated system for measuring MK-801-elicited mouse popping is based on the detection and measurement of vertical displacements of a platform related to mouse movements (Rosse et al., 1995). The vertical displacements resulting from mouse ‘‘pops’’ are detected and converted to electrical signals (S72-25 Type A Transducer Coupler and S75-01 Modified Contour Following
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Integrator; Coulbourn Instruments, Allentown, PA, U.S.A.), and are then transformed into a digital signal (L25-12 A/D Converter; Coulbourn Instruments). The chamber, which houses the animals for the experimental session, measured 16.5 cm long, 8.9 cm wide, and 8.9 cm high. A discrete count of popping is defined as a vertical displacement of the platform of more than 150% of body weight. The computer is able to determine the total number of popping counts, force (in g equivalents) of individual pops, and duration of an episode of popping (in seconds). Reverberations or ‘‘aftershock’’ movement of the platform after jumps are removed automatically by the system in the manner used in the measurement of startle responses in laboratory animals (Coulbourn Instruments, Inc., Allentown, PA).
Fig. 1. This figure depicts groups of mice (n = 12/group) defined by a pretreatment injection of vehicle or one of several doses (1, 3.2, 10, 32 or 100 mg/kg) of galantamine, followed 10 min later by an injection of vehicle or 0.56 mg/kg of MK-801. The bars represent the mean of each group, while variability is depicted as the standard error of the mean (S.E.M.). Statistical analysis revealed a significant main effect for both MK-801 dose (F(1,143) = 37.47, P < 0.001) and galantamine dose (F(5,143) = 4.29, P = 0.01). Specifically, MK-801 elicited significant popping (T(22) = 2.16, P < 0.05). This was significantly attenuated by 100 mg/kg of galantamine (T(22) = 2.24, P < 0.05). The bar labeled A (the group injected with vehicle followed by 0.56 mg/kg of MK-801) represents a significant difference from the group injected with vehicle/vehicle. The group labeled B (the group injected with 100 mg/kg of galantamine followed by an injection of 0.56 mg/kg of MK-801) is significantly different from the group injected with vehicle and 0.56 mg/kg of MK-801.
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Results The data were subjected to a two-way analysis of variance with MK-801 dose and galantamine dose as the independent variables. Following this, post-hoc t-tests were performed. The analysis revealed a significant main effect for both MK-801 dose (F(1,143) = 37.47, P < 0.001) and galantamine dose (F(5,143) = 4.29, P = 0.01). As can be seen in Fig. 1, none of the groups injected with the MK-801 vehicle differed from each other, independent of the dose of galantamine, demonstrating that galantamine alone does not produce popping at any of the doses tested. As expected, the group of mice injected with vehicle followed by 0.56 mg/kg of MK-801 was significantly different (T(22) = 2.16, P < 0.05) from the group injected with vehicle followed by vehicle, indicating that the popping behavior produced by this dose of MK-801 was significant. In addition, there was a significant difference (T(22) = 2.24, P < 0.05) between the group injected with vehicle followed by 0.56 mg/kg of MK-801 and the group injected with 100 mg/kg of galantamine followed by 0.56 mg/kg of MK-801. This demonstrates that at this dose of galantamine, popping elicited by MK-801 was antagonized.
Discussion The creation of NMDA receptor hypofunction in the intact mouse pharmacologically with MK-801 is reflected in the elicitation of irregular episodes of intense jumping behavior (Rosse et al., 1995). Prior to this induction of NMDA receptor hypofunction in the normal mouse, there is no reason to presume the existence of any imbalance of neurotransmission mediated by multiple neurotransmitters. This is in marked distinction to the hypothesized condition of the brain in a patient with schizophrenia; in this condition, a pathological imbalance between the actions of multiple neurotransmitters, including dopamine, serotonin, glutamate, and acetylcholine, among others, is thought to exist. In fact, imbalance in neurotransmission mediated by multiple neurotransmitters is one explanation provided for the therapeutic efficacy of clozapine, an atypical antipsychotic medication (Meltzer et al., 1989). In the current study, galantamine, which was administered 10 min before MK-801, modulated the intensity of popping in a dose-dependent manner. Lower doses of galantamine appeared to potentiate the ability of MK-801 to elicit popping, although this lacked statistical significance. Higher doses of galantamine appeared to attenuate this action of MK-801, reaching statistical significance at the highest dose of galantamine tested (100 mg/kg). Given the acute nature of galantamine’s administration in this study, these possible bidirectional effects may relate more to complex allosteric modulatory properties than increasing the neurotransmitter pool size of acetylcholine. Also, a bidirectional effect on popping could be related to the heterogeneous nature of nicotinic receptors, including high (a4h2)- and low-affinity (a7) types, that are not distributed homogeneously throughout the brain. The allosteric modulatory properties of galantamine are not restricted to the a7 nicotinic receptor (Samochocki et al., 2000). In summary, these data lend support to both the existence of a delicate balance between NMDA and nicotinic receptor-mediated neurotransmission and development of allosteric modulatory strategies of nicotinic neurotransmission for influencing NMDA receptor hypofunction. Our data suggest that development of galantamine for a schizophrenia indication will require finding a dosing strategy for addressing the hypothesized NMDA receptor hypofunction in the brains of patients. Moreover,
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there may be important differences between effects observed after both acute and chronic administration. With prolonged administration, there may be increases in the available neurotransmitter pool size of acetylcholine, which may have a variety of nonselective consequences. In addition, prolonged inhibition of acetylcholinesterase by galantamine may lead to the reduced production and availability of choline, a selective ligand for the a7 nicotinic receptor; choline is derived from the hydrolytic cleavage of acetylcholine. In any event, galantamine may be a therapeutic agent to target deficient or defective a7 nicotinic neurotransmission in the brains of patients with schizophrenia. Dosing and schedules of administration for this indication will be important areas for human investigation. Acknowledgements The VISN 5 Mental Illness Research, Education, and Clinical Center (MIRECC; Alan S. Bellack, Ph.D., Director) supported this work. References Albuquerque, E.X., Alkondon, M., Pereira, E.F.R., Castro, N.G., Schrattenholz, A., Barbosa, C.T.F., Bonfante-Cabarcas, R., Aracava, Y., Eisenberg, H.M., Maelicke, A., 1997. Properties of neuronal nicotine acetylcholine receptors: pharmacological characterization and modulation of synaptic function. J Pharmacol Exp Ther 280, 1117 – 1136. Alkondon, M., Pereira, E.F.R., Cortes, W.S., Maelicke, A., Albuquerque, E.X., 1997. Choline is a selective agonist of a7 nicotinic acetylcholine receptors in the rat brain neurons. European Journal of Neuroscience 9, 2734 – 2742. Braestrup, C., Nielsen, M., 1986. Benzodiazepine receptor binding in vivo and efficacy. In: Olsen, R.W., Venter, J.C. (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, pp. 167 – 184. Coyle, J.T., 1996. The glutamatergic dysfunction hypothesis for schizophrenia. Harvard Rev Psychiatry 3, 241 – 253. Deutsch, S.I., Mastropaolo, J., Schwartz, B.L., Rosse, R.B., Morihisa, J.M., 1989. A ‘‘glutamatergic hypothesis’’ of schizophrenia: rationale for pharmacotherapy with glycine. Clin Neuropharmacol 12 (1), 1 – 13. Deutsch, S.I., Rosse, R.B., Schwartz, B.L., Mastropaolo, J., 2001. A revised excitotoxic hypothesis of schizophrenia: therapeutic implications. Clin Neuropharmacol 24, 43 – 49. Farber, N.B., Newcomer, J.W., Olney, J.W., 1998. The glutamate synapse in neuropsychiatric disorders. Prog Brain Res 116, 421 – 437. Freedman, R., Hall, M., Adler, L.E., Leonard, S., 1995. Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia. Biol Psychiatry 38 (1), 22 – 33. Guan, Z.Z., Zhang, X., Blennow, K., Nordberg, A., 1999. Decreased protein level of nicotinic receptor a7 subunit in the frontal cortex from schizophrenic brain. Neuroreport 10, 1779 – 1782. Haefely, W., Polc, P., 1986. Physiology of GABA enhancement by benzodiazepines and barbiturates. In: Olsen, R.W., Venter, J.C. (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, pp. 97 – 133. Jentsch, J.D., Roth, R.H., 1999. The neuropsychopharmacology of phencyclidine: from NMDA receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 20, 201 – 225. Maelicke, A., Samochocki, M., Jostock, R., Fehrenbacher, A., Ludwig, J., Albuquerque, E.X., Zerlin, M., 2001. Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer’s disease. Biol Psychiatry 49 (3), 279 – 288 (Feb 1) . Meltzer, H.Y., Matsubara, S., Lee, J.C., 1989. Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-1, D-2 and serotonin pKi values. J Pharmacol Exp Ther 251, 238 – 246. Rosse, R.B., Mastropaolo, J., Koetzner, L., Morn, C.B., Sussman, D.M., Deutsch, S.I., 1995. Computer measurement of MK801-elicited hyperactivity and popping in mice. Clin Neuropharmacol 18, 448 – 457. Samochocki, M., Zerlin, M., Jostock, R., Groot Kormelink, P.J., Luyten, W.H., Albuquerque, E.X., Maelicke, A., 2000.
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Galantamine is an allosterically potentiating ligand of the human alpha4/beta2 nAChR. Acta Neurol Scand. (Suppl.) 176, 68 – 73. Tamminga, C.A., 1998. Schizophrenia and glutamatergic transmission. Crit Rev Neurobiol 12, 21 – 36. Tizabi, Y., Mastropaolo, J., Park, C.H., Riggs, R.L., Powell, D., Rosse, R.B., Deutsch, S.I., 1998. Both nicotine and mecamylamine block dizocilpine-induced explosive jumping behavior in mice: psychiatric implications. Psychopharmacology 140 (2), 202 – 205. Wamsley, J.K., Gehlert, D.R., Olsen, R.W., 1986. The benzodiazepine/barbiturate-sensitive convulsant/GABA receptor/ chloride ionophore complex: autoradiographic localization of individual components. In: Olsen, R.W., Venter, J.C. (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, pp. 299 – 313.
Modulation of MK-801-elicited mouse popping behavior by galantamine is complex and dose-dependent Stephen I. Deutsch a,b,c,d,*, Richard B. Rosse a,b, Eddie N. Billingslea a, Alan S. Bellack a,c, John Mastropaolo a a
Mental Health Service Line, Veterans Integrated Service Network (VISN) 5, 849 International Drive, Suite 275, Linthicum, MD 21090, USA b Department of Psychiatry, Georgetown University School of Medicine, 3800 Reservoir Road, NW, Washington, DC 20007, USA c Department of Psychiatry, University of Maryland School of Medicine, 701 West Pratt Street, Suite 388, Baltimore, MD 21201, USA d Department of Veterans Affairs Medical Center, Mental Health Service Line, VISN 5, 50 Irving Street, NW, Washington, DC 20422, USA Received 2 April 2003; accepted 9 April 2003
Abstract The ability of phencyclidine (PCP), a noncompetitive antagonist of NMDA receptor-mediated neurotransmission, to precipitate a schizophreniform psychosis in susceptible individuals is consistent with the hypothesized pathologic occurrence of NMDA receptor hypofunction in this disorder. Because the psychosis caused by PCP resembles schizophrenia in all of the relevant domains of psychopathology, investigators have sought to characterize animal models of NMDA receptor hypofunction . MK-801 (dizocilpine) binds to the same hydrophobic channel domain in the NMDA receptor-associated ionophore as PCP, and has been shown to elicit intense irregular episodes of jumping behavior in mice, termed ‘‘popping.’’ MK-801-elicited mouse popping is an animal model of NMDA receptor hypofunction that has been used to screen novel candidate compounds for the treatment of schizophrenia. Recently, a selective abnormality in the transduction of the acetylcholine signal at the level of the a7 nicotinic receptor has been described in schizophrenia. The existence of a nicotinic cholinergic abnormality in schizophrenia has stimulated interest in a potential therapeutic role for positive allosteric modulation of nicotinic receptors. Galantamine is a compound that possesses two interesting properties: inhibition of acetylcholinesterase and positive allosteric modulation of nicotinic neurotransmission. Theoretically, galantamine would be expected to increase the efficiency or likelihood that acetylcholine will promote channel
* Corresponding author. Department of Veterans Affairs Medical Center, Mental Health Service Line, VISN 5, 50 Irving Street, NW, Washington, DC 20422, USA. Tel.: +1-202-745-8156; fax: +1-202-745-8169. E-mail address: [email protected] (S.I. Deutsch). 0024-3205/$ - see front matter D 2003 Published by Elsevier Inc. doi:10.1016/S0024-3205(03)00642-8
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opening and ionic conductance at nicotinic receptors. As expected, in the current investigation statistically significant popping behavior was elicited by MK-801 in mice (T(22) = 2.16, P < 0.05). This MK-801-elicited popping was significantly attenuated by 100 mg/kg of galantamine (T(22) = 2.24, P < 0.05). The data show that nicotinic interventions can influence NMDA receptor-mediated neurotransmission in the intact mouse. D 2003 Published by Elsevier Inc. Keywords: Galantamine; MK-801 (dizocilpine); NMDA receptor; Popping; Schizophrenia
Introduction NMDA receptor hypofunction and its ‘‘downstream’’ consequences may be the pathophysiological basis for much of the psychopathology manifested in schizophrenia (Deutsch et al., 1989, 2001; Coyle, 1996; Farber et al., 1998; Tamminga, 1998). PCP is a noncompetitive antagonist of the NMDA receptor that binds to a hydrophobic channel domain associated with this glutamate-gated receptor (Jentsch and Roth, 1999). The downstream consequences of NMDA receptor hypofunction are also likely to include both diminished GABAergic neurotransmission and excitotoxic stimulation of the a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA)/kainate class of glutamate-gated channel receptors. Ultimately, these consequences of NMDA receptor hypofunction could explain the progressive ventriculomegaly and psychosocial deterioration that is seen in a subgroup of patients with schizophrenia (Deutsch et al., 2001). Recently, in addition to NMDA receptor hypofunction and its consequences, interest has focused on the contribution of defective neurotransmission mediated by the a7 subtype of nicotinic receptor, an ancestral homopentameric protein complex, to the pathophysiology of schizophrenia. For example, autopsy studies of brains of patients with schizophrenia have shown reduced and abnormal a7 polypeptide receptor subunit protein and messenger RNA, and decreased binding of radiolabeled alpha-bungarotoxin, a specific ligand for the ‘‘low-affinity’’ a7 subtype (Freedman et al., 1995; Guan et al., 1999). Acetylcholine, a nonselective agonist, and choline, a relatively selective agonist, are two naturally occurring ligands that gate the a7 nicotinic receptor, producing fast type 1A nicotinic responses that are characterized by rapid decay (Albuquerque et al., 1997; Alkondon et al., 1997). Disturbed signal transduction at the a7 nicotinic receptor stimulated interest in potential salutary therapeutic properties of pharmacological enhancement of signal transduction by this subtype of nicotinic receptor. As noted above, the induction of NMDA receptor hypofunction by PCP is a pharmacological model of schizophrenia that mimics accurately the various domains of psychopathology observed in this illness, including positive (e.g., hallucinations), negative (e.g., affective flattening), and cognitive (e.g., concrete thinking) symptoms (Deutsch et al., 1989). In the current investigation, the ability of galantamine, an acutely administered nicotinic intervention with positive allosteric modulatory properties, to modulate NMDA receptor hypofunction in the intact mouse was studied. The allosteric modulatory effects of galantamine are presumed to occur very rapidly and at low nanomolar concentrations (Samochocki et al., 2000; Maelicke et al., 2001). In prior work, we showed that nicotine, a nonselective direct agonist, and mecamylamine, a relatively selective competitive antagonist for the ‘‘high-affinity’’ nicotinic receptor (e.g., a4h2), modulated MK-801-elicited popping behavior (Tizabi et al., 1998). For a variety of reasons, positive allosteric modulation of nicotinic receptors (i.e., improving the efficiency of signal transduction) may be preferable to their direct stimulation by an agonist, in a condition like schizophrenia. Firstly, the allosteric modulator may improve the faulty signal transduction associated
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with defective or reduced receptors, while helping to maintain the receptor in a sensitized and responsive state. For example, tolerance does not develop to the anti-conflict effects of benzodiazepines; this action of benzodiazepines may result from positive allosteric modulation of the population of GABAA receptors located in the frontal cortex (Braestrup and Nielsen, 1986; Haefely and Polc, 1986; Wamsley et al., 1986). Ideally, galantamine may act in a manner analogous to this presumed action of benzodiazepines in frontal cortex (i.e., anti-conflict effect), improving the transduction of acetylcholine at the a7 nicotinic receptor, without causing desensitization of the receptor and tolerance to its action. In any event, the current study showed that acute administration of galantamine is able to modulate pharmacologically-induced NMDA receptor hypofunction in the intact mouse.
Materials and methods Subjects Experimentally naı¨ve male NIH Swiss mice (an outbred strain obtained from the National Cancer Institute, Frederick, MD) weighing 20–30 g were used. Mice were housed in hanging clear Plexiglass cages in groups of five and maintained on a cycle of 12 h of light followed by 12 h of darkness in an American Association for the Accreditation of Laboratory Animal Care (AAALAC) approved animal facility. The mice had free access to food and water. The animals were weighed individually prior to drug administration and behavioral testing. Because animal subjects were employed in these experiments, all experimental protocols had to be approved by our institutional review board prior to being initiated. All experiments were conducted in accordance to these protocols. No procedures that were not approved were part of these experiments and the number of animals did not exceed those approved. Group sizes of 12 mice per group were tested at each of the treatment conditions. Drugs MK-801 (dizocilpine; Research Biochemical International; Natick, MA) and galantamine (galanthamine hydrobromide, Sigma Chemical, St. Louis, MO) were dissolved in 0.9% saline and prepared on the day of the experiment. Groups of mice were injected intraperitoneally with vehicle or one of several doses (1, 3.2, 10, 32, 100 mg/kg) of galantamine 10 min prior to the injection of MK-801. Subsequently, mice were injected with MK-801 (0.56 mg/kg), or its vehicle, 5 min prior to the 30 min monitoring period for the assessment of popping behavior. All injections were in a volume of 0.01 ml/g of body weight. Computerized assessment of MK-801-elicited popping The recording of MK-801-elicited popping behavior was divided into two phases: a baseline period of five min and an outcome recording period of 30 min, which immediately followed an injection of either MK-801 or its vehicle. The automated system for measuring MK-801-elicited mouse popping is based on the detection and measurement of vertical displacements of a platform related to mouse movements (Rosse et al., 1995). The vertical displacements resulting from mouse ‘‘pops’’ are detected and converted to electrical signals (S72-25 Type A Transducer Coupler and S75-01 Modified Contour Following
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Integrator; Coulbourn Instruments, Allentown, PA, U.S.A.), and are then transformed into a digital signal (L25-12 A/D Converter; Coulbourn Instruments). The chamber, which houses the animals for the experimental session, measured 16.5 cm long, 8.9 cm wide, and 8.9 cm high. A discrete count of popping is defined as a vertical displacement of the platform of more than 150% of body weight. The computer is able to determine the total number of popping counts, force (in g equivalents) of individual pops, and duration of an episode of popping (in seconds). Reverberations or ‘‘aftershock’’ movement of the platform after jumps are removed automatically by the system in the manner used in the measurement of startle responses in laboratory animals (Coulbourn Instruments, Inc., Allentown, PA).
Fig. 1. This figure depicts groups of mice (n = 12/group) defined by a pretreatment injection of vehicle or one of several doses (1, 3.2, 10, 32 or 100 mg/kg) of galantamine, followed 10 min later by an injection of vehicle or 0.56 mg/kg of MK-801. The bars represent the mean of each group, while variability is depicted as the standard error of the mean (S.E.M.). Statistical analysis revealed a significant main effect for both MK-801 dose (F(1,143) = 37.47, P < 0.001) and galantamine dose (F(5,143) = 4.29, P = 0.01). Specifically, MK-801 elicited significant popping (T(22) = 2.16, P < 0.05). This was significantly attenuated by 100 mg/kg of galantamine (T(22) = 2.24, P < 0.05). The bar labeled A (the group injected with vehicle followed by 0.56 mg/kg of MK-801) represents a significant difference from the group injected with vehicle/vehicle. The group labeled B (the group injected with 100 mg/kg of galantamine followed by an injection of 0.56 mg/kg of MK-801) is significantly different from the group injected with vehicle and 0.56 mg/kg of MK-801.
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Results The data were subjected to a two-way analysis of variance with MK-801 dose and galantamine dose as the independent variables. Following this, post-hoc t-tests were performed. The analysis revealed a significant main effect for both MK-801 dose (F(1,143) = 37.47, P < 0.001) and galantamine dose (F(5,143) = 4.29, P = 0.01). As can be seen in Fig. 1, none of the groups injected with the MK-801 vehicle differed from each other, independent of the dose of galantamine, demonstrating that galantamine alone does not produce popping at any of the doses tested. As expected, the group of mice injected with vehicle followed by 0.56 mg/kg of MK-801 was significantly different (T(22) = 2.16, P < 0.05) from the group injected with vehicle followed by vehicle, indicating that the popping behavior produced by this dose of MK-801 was significant. In addition, there was a significant difference (T(22) = 2.24, P < 0.05) between the group injected with vehicle followed by 0.56 mg/kg of MK-801 and the group injected with 100 mg/kg of galantamine followed by 0.56 mg/kg of MK-801. This demonstrates that at this dose of galantamine, popping elicited by MK-801 was antagonized.
Discussion The creation of NMDA receptor hypofunction in the intact mouse pharmacologically with MK-801 is reflected in the elicitation of irregular episodes of intense jumping behavior (Rosse et al., 1995). Prior to this induction of NMDA receptor hypofunction in the normal mouse, there is no reason to presume the existence of any imbalance of neurotransmission mediated by multiple neurotransmitters. This is in marked distinction to the hypothesized condition of the brain in a patient with schizophrenia; in this condition, a pathological imbalance between the actions of multiple neurotransmitters, including dopamine, serotonin, glutamate, and acetylcholine, among others, is thought to exist. In fact, imbalance in neurotransmission mediated by multiple neurotransmitters is one explanation provided for the therapeutic efficacy of clozapine, an atypical antipsychotic medication (Meltzer et al., 1989). In the current study, galantamine, which was administered 10 min before MK-801, modulated the intensity of popping in a dose-dependent manner. Lower doses of galantamine appeared to potentiate the ability of MK-801 to elicit popping, although this lacked statistical significance. Higher doses of galantamine appeared to attenuate this action of MK-801, reaching statistical significance at the highest dose of galantamine tested (100 mg/kg). Given the acute nature of galantamine’s administration in this study, these possible bidirectional effects may relate more to complex allosteric modulatory properties than increasing the neurotransmitter pool size of acetylcholine. Also, a bidirectional effect on popping could be related to the heterogeneous nature of nicotinic receptors, including high (a4h2)- and low-affinity (a7) types, that are not distributed homogeneously throughout the brain. The allosteric modulatory properties of galantamine are not restricted to the a7 nicotinic receptor (Samochocki et al., 2000). In summary, these data lend support to both the existence of a delicate balance between NMDA and nicotinic receptor-mediated neurotransmission and development of allosteric modulatory strategies of nicotinic neurotransmission for influencing NMDA receptor hypofunction. Our data suggest that development of galantamine for a schizophrenia indication will require finding a dosing strategy for addressing the hypothesized NMDA receptor hypofunction in the brains of patients. Moreover,
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there may be important differences between effects observed after both acute and chronic administration. With prolonged administration, there may be increases in the available neurotransmitter pool size of acetylcholine, which may have a variety of nonselective consequences. In addition, prolonged inhibition of acetylcholinesterase by galantamine may lead to the reduced production and availability of choline, a selective ligand for the a7 nicotinic receptor; choline is derived from the hydrolytic cleavage of acetylcholine. In any event, galantamine may be a therapeutic agent to target deficient or defective a7 nicotinic neurotransmission in the brains of patients with schizophrenia. Dosing and schedules of administration for this indication will be important areas for human investigation. Acknowledgements The VISN 5 Mental Illness Research, Education, and Clinical Center (MIRECC; Alan S. Bellack, Ph.D., Director) supported this work. References Albuquerque, E.X., Alkondon, M., Pereira, E.F.R., Castro, N.G., Schrattenholz, A., Barbosa, C.T.F., Bonfante-Cabarcas, R., Aracava, Y., Eisenberg, H.M., Maelicke, A., 1997. Properties of neuronal nicotine acetylcholine receptors: pharmacological characterization and modulation of synaptic function. J Pharmacol Exp Ther 280, 1117 – 1136. Alkondon, M., Pereira, E.F.R., Cortes, W.S., Maelicke, A., Albuquerque, E.X., 1997. Choline is a selective agonist of a7 nicotinic acetylcholine receptors in the rat brain neurons. European Journal of Neuroscience 9, 2734 – 2742. Braestrup, C., Nielsen, M., 1986. Benzodiazepine receptor binding in vivo and efficacy. In: Olsen, R.W., Venter, J.C. (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, pp. 167 – 184. Coyle, J.T., 1996. The glutamatergic dysfunction hypothesis for schizophrenia. Harvard Rev Psychiatry 3, 241 – 253. Deutsch, S.I., Mastropaolo, J., Schwartz, B.L., Rosse, R.B., Morihisa, J.M., 1989. A ‘‘glutamatergic hypothesis’’ of schizophrenia: rationale for pharmacotherapy with glycine. Clin Neuropharmacol 12 (1), 1 – 13. Deutsch, S.I., Rosse, R.B., Schwartz, B.L., Mastropaolo, J., 2001. A revised excitotoxic hypothesis of schizophrenia: therapeutic implications. Clin Neuropharmacol 24, 43 – 49. Farber, N.B., Newcomer, J.W., Olney, J.W., 1998. The glutamate synapse in neuropsychiatric disorders. Prog Brain Res 116, 421 – 437. Freedman, R., Hall, M., Adler, L.E., Leonard, S., 1995. Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia. Biol Psychiatry 38 (1), 22 – 33. Guan, Z.Z., Zhang, X., Blennow, K., Nordberg, A., 1999. Decreased protein level of nicotinic receptor a7 subunit in the frontal cortex from schizophrenic brain. Neuroreport 10, 1779 – 1782. Haefely, W., Polc, P., 1986. Physiology of GABA enhancement by benzodiazepines and barbiturates. In: Olsen, R.W., Venter, J.C. (Eds.), Benzodiazepine/GABA Receptors and Chloride Channels: Structural and Functional Properties, pp. 97 – 133. Jentsch, J.D., Roth, R.H., 1999. The neuropsychopharmacology of phencyclidine: from NMDA receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 20, 201 – 225. Maelicke, A., Samochocki, M., Jostock, R., Fehrenbacher, A., Ludwig, J., Albuquerque, E.X., Zerlin, M., 2001. Allosteric sensitization of nicotinic receptors by galantamine, a new treatment strategy for Alzheimer’s disease. Biol Psychiatry 49 (3), 279 – 288 (Feb 1) . Meltzer, H.Y., Matsubara, S., Lee, J.C., 1989. Classification of typical and atypical antipsychotic drugs on the basis of dopamine D-1, D-2 and serotonin pKi values. J Pharmacol Exp Ther 251, 238 – 246. Rosse, R.B., Mastropaolo, J., Koetzner, L., Morn, C.B., Sussman, D.M., Deutsch, S.I., 1995. Computer measurement of MK801-elicited hyperactivity and popping in mice. Clin Neuropharmacol 18, 448 – 457. Samochocki, M., Zerlin, M., Jostock, R., Groot Kormelink, P.J., Luyten, W.H., Albuquerque, E.X., Maelicke, A., 2000.
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