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Decreased Locomotor Activity in Mice Carrying Transgenic Fyn Tyrosine Kinase
Biochemical and Biophysical Research Communications 276, 707–709 (2000) doi:10.1006/bbrc.2000.3534, available online at http://www.idealibrary.com on
Decreased Locomotor Activity in Mice Carrying Transgenic Fyn Tyrosine Kinase Hidetoshi Ishibashi, 1 Nobuhiko Kojima, and Kunihiko Obata Department of Physiological Sciences, Graduate University for Advanced Studies and Laboratory of Neurochemistry, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
Received August 21, 2000
We found that mice carrying constitutively active Fyn tyrosine kinase show low levels of spontaneous locomotor activity and that this activity increases when the mice are treated with the NMDA receptor antagonist MK-801. These findings indicate that the tyrosine phosphorylation of the NMDA receptors by Fyn participates in the control of locomotor activity. © 2000 Academic Press Key Words: Fyn; transgenic mice; locomotor activity; MK-801; NMDA receptor; tyrosine phosphorylation.
The functions of the striatum are largely unknown, but it seems that spontaneous locomotor activity might be modulated by the activation and inhibition of striatal N-methyl-D-aspartate (NMDA) type glutamate receptors: NMDA receptor antagonists infused into the anterodorsal striatum produce behavioral activation (1), whereas the infusion of NMDA itself has behavioral depressant effects (2). The function of ion channels associated with NMDA receptors has in turn been shown to be regulated by tyrosine phosphorylation. The NMDA-receptor-mediated ion current in cultured spinal cord neurons is reduced by the tyrosine kinase inhibitor genistein and is increased by the tyrosine kinase c-Src and by the tyrosine phosphatase inhibitor orthovanadate (3). Fyn, another Src-family tyrosine kinase, phosphorylates tyrosine residues of the NR2A and NR2B modulatory subunits of the NMDA receptor (4) and increases glutamate-activated NMDA currents (5). It is thus reasonable to hypothesize that this phosphorylation of tyrosine residues on NMDA receptors plays a role in the modulation of locomotor activity. We have tested this hypothesis by using transgenic mice carrying constitutively active Fyn tyrosine kinase. One 1
To whom correspondence should be addressed at present address: Section of Cognitive Neurobiology, Department of Maxillofacial Biology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8549 Japan. Fax: ⫹81-3-5803-0186. E-mail: [email protected].
advantage of this experimental model is that intrinsic Fyn and transgenic Fyn are expressed in the striatum as well as in the neocortex, hippocampus, and amygdala—three regions containing glutamatergic neurons innervating the striatum (6, 7). Another advantage is that transgenic Fyn is not expressed in the brainstem or spinal cord, other nonstriatal regions involved in the initiation and control of locomotion. And a third advantage of this experimental model is that Fyn-transgenic mice show a 30% increase in the tyrosine-phosphorylation of the NR2A subunit and a 100% increase in the tyrosine-phosphorylation of the NR2B subunit (7). We therefore examined the effect of Fyn-transgene expression on spontaneous locomotor activity and also examined the effect of a NMDA receptor antagonist on the locomotor activity of fyntransgenic mice. MATERIALS AND METHODS The production of Fyn-transgenic mice has been described elsewhere (7). Briefly, to get constitutively active Fyn tyrosine kinase, we introduced a point mutation to fyn cDNA such that the tyrosine residue 531 of the transcript, the presumed key amino acid in negative regulation, was replaced by phenylalanine. The mutated cDNA was then connected downstream to the promoter for the alpha subunit of calcium-calmodulin-dependent kinase II to restrict its expression to postnatal neurons. The transgene was microinjected to the pronuclei of fertilized eggs collected from C57BL6/CBA/F1 females and these eggs were transferred into the oviducts of pseudopregnant females. The mouse line we used was established by crossing founders with the hybrid strain (C57BL/6J and 129SV). The F1 offspring were crossed with nontransgenic littermates, and the F2 offspring and later generations were crossed with C57BL6 mice. In the present work we used a total of 87 male mice, 3 to 5 months old at the beginning of the experiment, of the F5 offspring of the transgenic line denoted M27 in the previous paper (7). Siblings not having the transgene were used as wild-type control animals. The animals were kept in a room with the lights turned on at 06:00 and off at 18:00. The room temperature was kept to 24 ⫾ 1°C. The room used in this experiment was used only for this experiment and disturbances during the experiment were minimized. Each of the experimental mice was housed individually, beginning at least 3 days before measurements, in a polycarbonate cage having attached to the lid an infrared beam sensor used to measure locomotor activity. The
707
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 276, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Spontaneous locomotor activity. Data are means ⫾ s.e.m. of locomotion counts for 16 wild-type control mice and 16 fyn-transgenic mice. (A) Counts for 24 h. Non-paired t test result is shown in the text. (B) Circadian rhythm of spontaneous locomotor activity. The letters D and L above the black-and-white bar indicate dark and light periods.
spontaneous locomotor activity was measured for 48 h and records were stored on a personal computer for off-line analysis using an ABsystem motion analyzer (Neuroscience, Tokyo). In drug injection experiments, either saline or the NMDA receptor antagonist MK-801 (RBI) dissolved in saline had been injected intraperitoneally at 11:30, and locomotor activity was measured from 12:00 to 12:10. Each animal was used only once, and the experimental procedures had been approved by the institutional review committee.
RESULTS AND DISCUSSION The daily count of spontaneous locomotor activity of the Fyn-transgenic mice was significantly smaller than that of the wild-type control mice (Fig. 1A; non-paired two-tailed t test; t ⫽ 2.89; df ⫽ 30; P ⫽ 0.0071). The activity pattern displayed by the wild-type mice, greatest activity at beginning and end of the dark period, was also displayed by the Fyn-transgenic mice (Fig. 1B). These results indicate that the Fyn-transgenic mice are hypokinesic but have the normal circadian rhythm. Intraperitoneal saline injection did not affect the locomotor activity (compare the value in Fig. 2 with that shown in Fig. 1B for 12:00). The MK-801 did not effect the locomotor activity of the wild-type control mice but increased that of the Fyn-transgenic mice in a dose-dependent manner (Fig. 2). Two-way ANOVA revealed no significant effect of transgene expression (F ⫽ 3.45; df ⫽ 1,49; P ⫽ 0.069), a significant effect across MK-801 doses (F ⫽ 13.48; df ⫽ 2,49; P ⬍ 0.0001), and significant interaction between transgene expression and dose (F ⫽ 6.26; df ⫽ 2,49 P ⫽ 0.0038), thus indicating that the effect of the NMDA receptor
antagonist was stronger on transgenic mice than on wild-type mice. These results suggest that the hypokinesia in Fyn-transgenic mice resulted from the activation of NMDA receptors. Other than the locomotor enhancing effect, MK-801 has a convulsant effect at high doses (for mice, 5 mg/kg or more), and Fyn-transgenic mice are susceptible to seizures induced by pentylenetetrazol or an electrical kindling stimulus (7). To evaluate the effect of MK-801 on locomotor activity and to avoid the interference from
FIG. 2. Effect of MK-801 on locomotor activity. Data are means ⫾ s.e.m. of locomotion counts for 8 –12 mice. Two-way ANOVA results are shown in the text.
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Vol. 276, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the convulsion, we set the doses of MK-801 much lower than that elicits convulsion. Indeed, preliminary experiments showed that MK-801 at 100 g/kg did not elicit convulsions in any mice. Thus, the convulsive character of the drug had little effect on the locomotor activity measured 30 min after the drug injection. It is postulated that glutamatergic neurons modulate locomotor activity by affecting GABAergic or dopaminergic activity or both (8). Lu and his colleagues have shown reduced GABAergic synaptic suppression in the hippocampus of Fyn-transgenic mice compare to that of wild-type control mice (9). An in situ hybridization experiment revealed no difference in the level of glutamic acid decarboxylase mRNA between Fyntransgenic mice and wild-type control mice (data not shown), indicating that GABA is synthesized in Fyntransgenic mice. Thus, the reduced locomotor activity of fyn-transgenic mice may be due to a modification of the GABA receptor. In summary, we have found that Fyn-transgenic mice show less spontaneous locomotor activity than do wild-type control mice and that the locomotor activity of Fyn-transgenic mice is influenced more by the NMDA receptor antagonist MK-801 than is the locomotor activity of wild-type controls. Although there maybe many factors, both internal and external, affecting locomotor activity, our results show that one of these factors is the phosphorylation of tyrosine residues in NMDA receptors.
REFERENCES
709
1. Pierce, R. C., and Rebec, G. V. (1993) Intraneostriatal administration of glutamate antagonists increases behavioral activation and decreases neostriatal ascorbate via nondopaminergic mechanisms. J. Neurosci. 13, 4272– 4280. 2. Schmidt, W. J., and Bury, D. (1988) Behavioural effects of N-methyl-D-aspartate in the anterodorsal striatum of the rat. Life Sci. 43, 545–549. 3. Wang, Y. T., and Salter, M. W. (1994) Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369, 233–235. 4. Suzuki, T., and Okumura-Noji, K. (1995) NMDA receptor subunits epsilon 1 (NR2A) and epsilon 2 (NR2B) are substrates for Fyn in the postsynaptic density fraction isolated from the rat brain. Biochem. Biophys. Res. Commun. 216, 582–588. 5. Ko¨hr, G., and Seeburg, G. H. (1996) Subtype-specific regulation of recombinant NMDA receptor-channels by protein tyrosine kinases of the src family. J. Physiol. 492, 445– 452. 6. Albin, R. L., Makowiec, R. L., Hollingworth, Z. R., Dure, L. S., IV, Penny, J. B., and Young, A. B. (1992) Excitatory amino acid binding sites in the basal ganglia of the rat: Quantitative autoradiographic study. Neuroscience 46, 135–148. 7. Kojima, N., Ishibashi, H., Obata, K., and Kandel, E. R. (1998) Higher seizure susceptibility and enhanced tyrosine phosphorylation of N-methyl-D-aspartate receptor subunit 2B in fyntransgenic mice. Learn. Mem. 5, 429 – 445. 8. DeLong, M. R. (1990) Primate models of movement disorders of basal ganglia origin. Trends Neurosci. 13, 281–285. 9. Lu, Y.-F., Kojima, N., Tomizawa, K., Moriwaki, A., Matsushita, M., Obata, K., and Matsui, H. (1999) Enhanced synaptic transmission and reduced threshold for LTP induction in fyntransgenic mice Eur. J. Neurosci. 11, 75– 82.
Decreased Locomotor Activity in Mice Carrying Transgenic Fyn Tyrosine Kinase Hidetoshi Ishibashi, 1 Nobuhiko Kojima, and Kunihiko Obata Department of Physiological Sciences, Graduate University for Advanced Studies and Laboratory of Neurochemistry, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
Received August 21, 2000
We found that mice carrying constitutively active Fyn tyrosine kinase show low levels of spontaneous locomotor activity and that this activity increases when the mice are treated with the NMDA receptor antagonist MK-801. These findings indicate that the tyrosine phosphorylation of the NMDA receptors by Fyn participates in the control of locomotor activity. © 2000 Academic Press Key Words: Fyn; transgenic mice; locomotor activity; MK-801; NMDA receptor; tyrosine phosphorylation.
The functions of the striatum are largely unknown, but it seems that spontaneous locomotor activity might be modulated by the activation and inhibition of striatal N-methyl-D-aspartate (NMDA) type glutamate receptors: NMDA receptor antagonists infused into the anterodorsal striatum produce behavioral activation (1), whereas the infusion of NMDA itself has behavioral depressant effects (2). The function of ion channels associated with NMDA receptors has in turn been shown to be regulated by tyrosine phosphorylation. The NMDA-receptor-mediated ion current in cultured spinal cord neurons is reduced by the tyrosine kinase inhibitor genistein and is increased by the tyrosine kinase c-Src and by the tyrosine phosphatase inhibitor orthovanadate (3). Fyn, another Src-family tyrosine kinase, phosphorylates tyrosine residues of the NR2A and NR2B modulatory subunits of the NMDA receptor (4) and increases glutamate-activated NMDA currents (5). It is thus reasonable to hypothesize that this phosphorylation of tyrosine residues on NMDA receptors plays a role in the modulation of locomotor activity. We have tested this hypothesis by using transgenic mice carrying constitutively active Fyn tyrosine kinase. One 1
To whom correspondence should be addressed at present address: Section of Cognitive Neurobiology, Department of Maxillofacial Biology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, 113-8549 Japan. Fax: ⫹81-3-5803-0186. E-mail: [email protected].
advantage of this experimental model is that intrinsic Fyn and transgenic Fyn are expressed in the striatum as well as in the neocortex, hippocampus, and amygdala—three regions containing glutamatergic neurons innervating the striatum (6, 7). Another advantage is that transgenic Fyn is not expressed in the brainstem or spinal cord, other nonstriatal regions involved in the initiation and control of locomotion. And a third advantage of this experimental model is that Fyn-transgenic mice show a 30% increase in the tyrosine-phosphorylation of the NR2A subunit and a 100% increase in the tyrosine-phosphorylation of the NR2B subunit (7). We therefore examined the effect of Fyn-transgene expression on spontaneous locomotor activity and also examined the effect of a NMDA receptor antagonist on the locomotor activity of fyntransgenic mice. MATERIALS AND METHODS The production of Fyn-transgenic mice has been described elsewhere (7). Briefly, to get constitutively active Fyn tyrosine kinase, we introduced a point mutation to fyn cDNA such that the tyrosine residue 531 of the transcript, the presumed key amino acid in negative regulation, was replaced by phenylalanine. The mutated cDNA was then connected downstream to the promoter for the alpha subunit of calcium-calmodulin-dependent kinase II to restrict its expression to postnatal neurons. The transgene was microinjected to the pronuclei of fertilized eggs collected from C57BL6/CBA/F1 females and these eggs were transferred into the oviducts of pseudopregnant females. The mouse line we used was established by crossing founders with the hybrid strain (C57BL/6J and 129SV). The F1 offspring were crossed with nontransgenic littermates, and the F2 offspring and later generations were crossed with C57BL6 mice. In the present work we used a total of 87 male mice, 3 to 5 months old at the beginning of the experiment, of the F5 offspring of the transgenic line denoted M27 in the previous paper (7). Siblings not having the transgene were used as wild-type control animals. The animals were kept in a room with the lights turned on at 06:00 and off at 18:00. The room temperature was kept to 24 ⫾ 1°C. The room used in this experiment was used only for this experiment and disturbances during the experiment were minimized. Each of the experimental mice was housed individually, beginning at least 3 days before measurements, in a polycarbonate cage having attached to the lid an infrared beam sensor used to measure locomotor activity. The
707
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 276, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Spontaneous locomotor activity. Data are means ⫾ s.e.m. of locomotion counts for 16 wild-type control mice and 16 fyn-transgenic mice. (A) Counts for 24 h. Non-paired t test result is shown in the text. (B) Circadian rhythm of spontaneous locomotor activity. The letters D and L above the black-and-white bar indicate dark and light periods.
spontaneous locomotor activity was measured for 48 h and records were stored on a personal computer for off-line analysis using an ABsystem motion analyzer (Neuroscience, Tokyo). In drug injection experiments, either saline or the NMDA receptor antagonist MK-801 (RBI) dissolved in saline had been injected intraperitoneally at 11:30, and locomotor activity was measured from 12:00 to 12:10. Each animal was used only once, and the experimental procedures had been approved by the institutional review committee.
RESULTS AND DISCUSSION The daily count of spontaneous locomotor activity of the Fyn-transgenic mice was significantly smaller than that of the wild-type control mice (Fig. 1A; non-paired two-tailed t test; t ⫽ 2.89; df ⫽ 30; P ⫽ 0.0071). The activity pattern displayed by the wild-type mice, greatest activity at beginning and end of the dark period, was also displayed by the Fyn-transgenic mice (Fig. 1B). These results indicate that the Fyn-transgenic mice are hypokinesic but have the normal circadian rhythm. Intraperitoneal saline injection did not affect the locomotor activity (compare the value in Fig. 2 with that shown in Fig. 1B for 12:00). The MK-801 did not effect the locomotor activity of the wild-type control mice but increased that of the Fyn-transgenic mice in a dose-dependent manner (Fig. 2). Two-way ANOVA revealed no significant effect of transgene expression (F ⫽ 3.45; df ⫽ 1,49; P ⫽ 0.069), a significant effect across MK-801 doses (F ⫽ 13.48; df ⫽ 2,49; P ⬍ 0.0001), and significant interaction between transgene expression and dose (F ⫽ 6.26; df ⫽ 2,49 P ⫽ 0.0038), thus indicating that the effect of the NMDA receptor
antagonist was stronger on transgenic mice than on wild-type mice. These results suggest that the hypokinesia in Fyn-transgenic mice resulted from the activation of NMDA receptors. Other than the locomotor enhancing effect, MK-801 has a convulsant effect at high doses (for mice, 5 mg/kg or more), and Fyn-transgenic mice are susceptible to seizures induced by pentylenetetrazol or an electrical kindling stimulus (7). To evaluate the effect of MK-801 on locomotor activity and to avoid the interference from
FIG. 2. Effect of MK-801 on locomotor activity. Data are means ⫾ s.e.m. of locomotion counts for 8 –12 mice. Two-way ANOVA results are shown in the text.
708
Vol. 276, No. 2, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the convulsion, we set the doses of MK-801 much lower than that elicits convulsion. Indeed, preliminary experiments showed that MK-801 at 100 g/kg did not elicit convulsions in any mice. Thus, the convulsive character of the drug had little effect on the locomotor activity measured 30 min after the drug injection. It is postulated that glutamatergic neurons modulate locomotor activity by affecting GABAergic or dopaminergic activity or both (8). Lu and his colleagues have shown reduced GABAergic synaptic suppression in the hippocampus of Fyn-transgenic mice compare to that of wild-type control mice (9). An in situ hybridization experiment revealed no difference in the level of glutamic acid decarboxylase mRNA between Fyntransgenic mice and wild-type control mice (data not shown), indicating that GABA is synthesized in Fyntransgenic mice. Thus, the reduced locomotor activity of fyn-transgenic mice may be due to a modification of the GABA receptor. In summary, we have found that Fyn-transgenic mice show less spontaneous locomotor activity than do wild-type control mice and that the locomotor activity of Fyn-transgenic mice is influenced more by the NMDA receptor antagonist MK-801 than is the locomotor activity of wild-type controls. Although there maybe many factors, both internal and external, affecting locomotor activity, our results show that one of these factors is the phosphorylation of tyrosine residues in NMDA receptors.
REFERENCES
709
1. Pierce, R. C., and Rebec, G. V. (1993) Intraneostriatal administration of glutamate antagonists increases behavioral activation and decreases neostriatal ascorbate via nondopaminergic mechanisms. J. Neurosci. 13, 4272– 4280. 2. Schmidt, W. J., and Bury, D. (1988) Behavioural effects of N-methyl-D-aspartate in the anterodorsal striatum of the rat. Life Sci. 43, 545–549. 3. Wang, Y. T., and Salter, M. W. (1994) Regulation of NMDA receptors by tyrosine kinases and phosphatases. Nature 369, 233–235. 4. Suzuki, T., and Okumura-Noji, K. (1995) NMDA receptor subunits epsilon 1 (NR2A) and epsilon 2 (NR2B) are substrates for Fyn in the postsynaptic density fraction isolated from the rat brain. Biochem. Biophys. Res. Commun. 216, 582–588. 5. Ko¨hr, G., and Seeburg, G. H. (1996) Subtype-specific regulation of recombinant NMDA receptor-channels by protein tyrosine kinases of the src family. J. Physiol. 492, 445– 452. 6. Albin, R. L., Makowiec, R. L., Hollingworth, Z. R., Dure, L. S., IV, Penny, J. B., and Young, A. B. (1992) Excitatory amino acid binding sites in the basal ganglia of the rat: Quantitative autoradiographic study. Neuroscience 46, 135–148. 7. Kojima, N., Ishibashi, H., Obata, K., and Kandel, E. R. (1998) Higher seizure susceptibility and enhanced tyrosine phosphorylation of N-methyl-D-aspartate receptor subunit 2B in fyntransgenic mice. Learn. Mem. 5, 429 – 445. 8. DeLong, M. R. (1990) Primate models of movement disorders of basal ganglia origin. Trends Neurosci. 13, 281–285. 9. Lu, Y.-F., Kojima, N., Tomizawa, K., Moriwaki, A., Matsushita, M., Obata, K., and Matsui, H. (1999) Enhanced synaptic transmission and reduced threshold for LTP induction in fyntransgenic mice Eur. J. Neurosci. 11, 75– 82.