- Email: [email protected]
Effect of dibutyryl cyclic AMP on axoplasmic transport in the hippocampus
Brain Research 755 Ž1997. 343–346
Short communication
Effect of dibutyryl cyclic AMP on axoplasmic transport in thehippocampus Yoko Hashimoto a
a, )
, Hideaki Hori a , Tadashi Kawakami b , Tatsumi Kusakabe c , Toshifumi Takenaka a
Department of Physiology, Yokohama City UniÕersity, 3-9 Fukuura Kanazawa-ku, Yokohama, 236, Japan b Department of Physiology, Kitasato UniÕersity School of Medicine, Sagamihara, Kanagawa, Japan c Department of Anatomy, Yokohama City UniÕersity, Fukuura Kanazawa-ku, Yokohama, Japan Accepted 11 February 1997
Abstract The effect of dibutyryl cyclic AMP ŽdbcAMP. on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95 " 1.01 mmrs Ž n s 55. to 2.66 " 1.26 mmrs Ž n s 58. and from 1.94 " 0.85 Ž n s 57. to 2.39 " 0.93 Ž n s 57.. Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport. Keywords: Axonal transport; Dibutyryl cyclic AMP; Hippocampus; Cell culture; Organelle motion; Neurotransmitter
Cyclic AMP is an important second messenger, which mediates many functions caused by neurotransmitters and hormones. Some experiments have been reported on the action of cAMP on axoplasmic transport. For example, Azderian et al. reported that cAMP stimulates axoplasmic transport in the neurons of Aplysia bag cell neurons w2x. Forscher et al. also reported that cAMP induced regulation of organelle transport in bag cell growth cones of aplysia w6x. We reported that dibutyryl cyclic AMP ŽdbcAMP. increased axoplasmic transport in superior cervical ganglion ŽSCG. cells w10,19–21x. In the hippocampus, cAMP induces later and longer long-term-potentiation w7x. However, experiments on the effects of cAMP on hippocampal axoplasmic transport have not been reported. Therefore, we examined the effect of cAMP on neurons in the hippocampus, which is thought to be important for memory and learning w17x. We followed the methods of Hatanaka et al. w9x for cell culture. Primary cultures of dissociated hippocampal CA1 region neurons were prepared from the brains of 1-day-old c57BLr6 mice. The location of the area was determined
)
Corresponding author. Fax: q81 Ž45. 787-2509.
0006-8993r96r$17.00 Published by Elsevier Science B.V. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 3 0 - 8
according to a developmental rat atlas w15x. Dissection was carried out on ice under a microscope. Tissue fragments were added to 0.5 ml of a freshly prepared Ca2q, Mg 2q-free phosphate buffered saline ŽCMF-PBS. containing papain Ž10 U., DNase I ŽSigma, 50 U., L-cystein-HCl ŽSigma, 0.1 mg., bovine serum albumin ŽSigma, 0.1 mg. and glucose Ž2.5 mg., and then incubated twice, with a change of buffer, for 20 min at 378C and 150 r.p.m. Next the papain digestion was arrested by the addition of fetal calf serum, and the tissue fragments were washed with a culture medium, consisting of 10% Žvrv. fetal calf serum, 90% of Dulbecco’s modified Eagle’s medium, 50 Urml penicillin G, 0.05 mgrml streptomycin sulfate and 100 ngrml 7 s NGF. Cells were then dissociated by gentle drawing through small pipettes Ž0.3–0.5 mm diameter. whose tips had been rounded in a flame. The dissociated cells were seeded onto 30 = 40 mm cover glasses whose center Ž1 cm2 of culture surface area. was coated with poly-L-lysine. All cell culture was done at 378C in a 5% Žvrv. CO 2 atmosphere at saturated humidity, in an incubator. After 1 week culture the cover glasses were attached with waterproof tape to the underside of a 0.5 mm thick stainless-steel plate Ž50 = 80 mm. with a lozenge-shaped hole. The upper side of the steel plate was covered with another cover
344
Y. Hashimoto et al.r Brain Research 755 (1997) 343–346
glass, leaving small openings on both sides to perfuse solutions through. The plate was placed on an inverted Zeiss Axiovart Microscope equipped with a computer-assisted video-enhanced microscope w1,18x, with an oil-immersed plan-apochromat 100 = 1.40 objective lens. The individual particle flow in neurites was observed in real time video-enhanced differential interference contrast optic recording. The video camera was a Hamamatsu Photonics Harpicon. The test solution was 0.5 mM dbcAMP ŽSigma USA. dissolved in high concentration glucose physiological salt solution ŽHG-PSS: NaCl 135.0 mM, KCl 5.0 mM, CaCl 2 1.0 mM, MgCl 2 1.0 mM, glucose 25 mM, HEPES 10 mM, pH 7.3.. Fig. 1 is the average time course of 0.5 mM dbcAMP effect during five 23-min applications. The ordinate shows the percent of transported particles passing through one region of a neurite per min relative to the control. The asterisks show P values. The dbcAMP was applied by changing from normal HG-PSS to HG-PSS containing 0.5 mM dbcAMP at 7 min. After the application of dbcAMP, the number of flowing particles increased and reached a peak about 5 min after application in both anterograde and retrograde transport. The number peaked at about 160%. In this connection, the actual numbers were about 35–50 at control and 60–80 at peak in both directions. This effect gradually decreased with time, although the number had not returned to control level at 30 min. This gradual return was more sluggish than in superior cervical ganglion cells.
To produce a physiological action, the concentration of intracellular cAMP must be greater than 0.01 mM w13x. We used 0.01 mM to 1.0 mM solutions of dbcAMP, and a similar increasing effect was clearly observed at concentrations of 0.1 to 1.0 mM. At a concentration of 0.01 mM, this effect could not be observed. The instantaneous velocity of axoplasmic transport was also increased by dbcAMP in both directions. The average velocity in anterograde and retrograde directions changed respectively from 1.95 " 1.01 mmrs Ž n s 55. to 2.66 " 1.26 mmrs Ž n s 58. and from 1.94 " 0.85 Ž n s 57. to 2.39 " 0.93 Ž n s 57.. Rates were 136.1 and 123.1%, respectively. The difference of average velocity between control and 5 min after application was statistically significant at P - 0.01 in both anterograde and retrograde directions. Table 1 shows these data. Fig. 2 shows the relation between instantaneous velocity and histogram of the number of particles moving at that velocity 5 min after application of dbcAMP and in the control. Ordinates show the number of particles. Abscissae show instantaneous velocity. In both anterograde and retrograde directions, the curve was shifted to the right, that is, to the faster side. This result also suggested that the velocity of individual particles was increased by dbcAMP. In comparing the histograms of anterograde and retrograde particles, the anterograde histogram has a broader distribution, although both have almost the same average. This result also can be seen in Table 1, where anterograde velocity has a larger S.D. than retrograde velocity.
Fig. 1. The average time course of 0.5 mM dbcAMP increase in anterograde and retrograde transport. Dibutyryl cyclic AMP was applied at 7 min. About 5 min after the perfusion, relative number of particles reached a peak. The ordinate shows the percentage of particle flow compared with the initial value of 100%. Ž ) P - 0.10, ) ) P - 0.05, ) ) ) P - 0.02, ) ) ) ) P - 0.01..
Y. Hashimoto et al.r Brain Research 755 (1996) 343–346
Fig. 2. Instantaneous velocity histogram of anterograde ŽAntero.. and retrograde ŽRetro.. axoplasmic transport particles. Before Ž – Ø – . and during Ž – ( – . application Žfrom 4 to 6 min after perfusion..
The fact that dibutyryl cyclic AMP increased the number of particles in both anterograde and retrograde direction not only in the SCG but also in hippocampal neurons suggests that endogenous cAMP takes part in the control system of axoplasmic transport in the hippocampus as a second messenger. We have investigated the control system of axoplasmic transport in the SCG, which is part of the sympathetic and peripheral nervous system, and have shown that acetylcholine depresses and adrenaline enhances the axoplasmic transport in SCG cells w10,19–21x. These effects were related to the quantity of endogeneous cAMP. The binding of acetylcholine to m2-acetylcholine-receptors depresses adenyl cyclase and the binding of adrenaline to b 2-adrenaline-receptor activates adenyl cyclase through inhibitory and stimulatory GTP-binding proteins, respectively. These resultant changes in the amount of endogenous cAMP initiate a sequence of metabolic processes which presumably includes the phosphorylation of specific proteins by cAMP-dependent protein kinase, as suggested for the decrease and increase of the axoplasmic transport.
345
The hippocampus, to which cholinergic nuclei of the medial septal area and the nucleus of the diagonal band project w11x, is considered of crucial importance for learning and memory processes. Electrical stimulation of the medial septal area increases both ACh release and the neuronal activity of the hippocampus w5x. Other studies have reported that acquisition of an operant behavior enhances the ACh release in the hippocampus w4,14x. Muscarinic acetylcholine receptors ŽmACh-Rs. have subtypes known as m1, m2, m3, m4, and m5 w16x. Subtypes m1, m3, and m5 which mediate the stimulation of phosphoinositide hydrolysis, are related to activation of PKC and Ca2qrcalmodulin kinases. On the other hand, subtypes m2 and m4 inhibit the effect of adenyl cyclase activity through inhibitory GTP-binding protein. Subtypes m1 and m3 are abundant in the hippocampus. Subtype m4 is less abundant and m2 subtype is said not to exist in the hippocampus w3x. Therefore, it appears that ACh binding to m2ACh-R does not suppress the axoplasmic transport of hippocampal neurons, but does suppress that of SCG neurons. Mons et al. w12x reported that two functional types of adenyl cyclase are detected in the brain that differ by being sensitive or insensitive to Ca2qrcalmodulin stimulation, and are known as Type I and Type II, respectively. In the CA1 region, Type II expression was higher than that of Type I. These studies provide support for the following hypothesis. In the hippocampal CA1 region, binding of ACh to subtypes m1, m3, and m5 raises the endogeneous cAMP by phosphoinositide hydrolysis producing diacylglycerol and IP3, which release Ca2q from an internal Ca2q then binds to calmodulin ŽCa2qrcalmodulin., and activation of Type II adenyl cyclase by this Ca2qrcalmodulin, raises the endogeneous cAMP. Regarding hippocampal long-term-potentiation ŽLTP. in the CA1 region, the later and longer lasting stage ŽL-LTP. is mediated by synthesis of cAMP w7x, and according to another report, coactivation of metabolic glutamate receptors and b-adrenergic receptors is mediated by cAMP and activation of cAMP-dependent protein kinases w8x. If hippocampal LTP is indeed related to mammalian leaning and memory, then activation of hippocampal axoplasmic transport may also take part in learning and memory, since raising of the endogeneous cAMP level both initiates L-LTP and at the some time intensifies the axoplasmic transport.
Acknowledgements Table 1 Velocity change 5 min after application of 0.5 mM dbcAMP Velocity Control Žmmrs. After 5 min Žmmrs. Rate a
P - 0.01.
Anterograde 1.95"1.01 2.66"1.26 136.1%
a a
Retrograde 1.94"0.85 a 2.39"0.93 a 123.1%
We wish to thank Dr. R.C. Goris of the Department of Anatomy, Yokohama City University School of Medicine, Dr. Masato Kano of their help in preparing the manuscript and Mr. Satoshi Yuki of Mitsubishi Chemical Corporation for his advice of cell culture. This study was supported by Grants-in Aid for Scientific Research Projects 07557005 from The Japanese Ministry of Education.
346
Y. Hashimoto et al.r Brain Research 755 (1997) 343–346
References w1x R.D. Allen, N.S. Allen, J.L. Travis, Video-enhanced contrast, differential interference contrast ŽAVEC-DIC. micro-scopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris, Cell Motil. 1 Ž1989. 291–302. w2x E. Azderian, C.H. Lin, D. Hefner, P. Forscher and L.K. Kaczmarek, Cyclic AMP increases the quantity of ELH-containing proteins in the neurites of Aplysia bag cell neurons, 21st Ann. Meet. Soc. for Neurosci. Abstr., 59 Ž1991.. w3x N.J. Buckley, T.I. Bonner, M.R. Brann, Localization of a family of muscarinic receptor mRNAs in rat brain, J. Neurosci. 8 Ž1988. 4646–4652. w4x J. Day, G. Damsma, H.C. Fibiger, Cholinergic activity in the rat hippocampus, cortex and striatum correlates with locomotor activity: An in vivo microdialysis study, Pharmacol. Biochem. BehaÕ. 38 Ž1991. 723–729. w5x J.D. Dudar, The effect of septal nuclei release ofacetylcholine from the rabbit hippocampus, Brain Res. 83 Ž1975. 123–133. w6x P. Forscher, L.K. Kaczmarek, J.-A. Buchanan, S.J. Smith, Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cells neurons, J. Neurosci. 7 Ž1987. 3600–3611. w7x U. Frey, Y.Y. Huang, E.R. Kandel, Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons, Science 260 Ž1993. 1661–1664. w8x R.W. Gereau IV, P.J. Conn, A cyclic AMP-dependent form of associative synaptic plasticity induced by coactivation of b-adrenergic receptors and metabotropic glutamate receptors in rat hippocampus, J. Neurosci. 14 Ž1994. 3310–3318. w9x H. Hatanaka, C. Nishio, Y. Kushima, H. Tsukui, Nerve-growth-factor-dependent and cell-density independent survival of septal cholinergic neurons in culture from postnatal rats, Neurosci. Res. 8 Ž1990. 69–82. w10x T. Kawakami, T. Takenaka, H. Hori, Y. Bandou, T. Kusakabe, Effects of acetylcholine and adrenaline on axoplasmic transport at
w11x
w12x
w13x w14x
w15x w16x
w17x
w18x
w19x
w20x
w21x
different regions of mouse superior cervical ganglion cells in culture, Brain Res. 683 Ž1995. 88–92. P.R. Lewis, C.C.D. Shute, The cholinergic limbic system: projections to hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system, and the subfonical organ and supra-optic crest, Brain 90 Ž1967. 521–542. N. Mons, M. Yoshimura, D.M.F. Cooper, Discrete expression of Ca2qrcalmodulin-sensitive and Ca2q-insensitive adenylyl cyclases in the rat brain, Synapse 14 Ž1993. 51–59. J.A. Nathanson, Cyclic nucleotides and nervous systems function, Physiol. ReÕ. 57 Ž1977. 157–256. M. Orsetti, F. Casamenti, G. Pepeu, Enhanced acetylcholine release in the hippocampus and cortex during acquisition of an operant behavior, Brain Res. 724 Ž1996. 89–96. G. Paxions and C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1982. E.G. Peralta, A. Ashkenazi, J.W. Winslow, J. Ramachandran, D.J. Capon, Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes, Nature 334 Ž1988. 434–437. W.B. Scoville, B. Milner, Loss of recent memory after bilateral hippocampal lesion, J. Neurol. Neurosurg. Psychiatry 20 Ž1957. 11–21. T. Takenaka, K. Kawakami, N. Hikawa, H. Gotoh, Axoplasmic transport of mitochondria in cultured dorsal root ganglion cells, Brain Res. 528 Ž1990. 285–290. T. Takenaka, T. Kawakami, N. Hikawa, H. Gotoh, Y. Bandou, Neurotransmitter regulation of axoplasmic transport and neuronal growth, Biomedic. Res. (Suppl.) 2 Ž1991. 171–172. T. Takenaka, K. Kawakami, N. Hikawa, Y. Bandou, H. Gotoh, Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells, Brain Res. 588 Ž1992. 212–216. T. Takenaka, T. Kawakami, H. Hori, Y. Bandou, Effect of neurotransmitters on axoplasmic transport: how adrenaline affects superior cervical ganglion cells, Brain Res. 643 Ž1994. 81–85.
Short communication
Effect of dibutyryl cyclic AMP on axoplasmic transport in thehippocampus Yoko Hashimoto a
a, )
, Hideaki Hori a , Tadashi Kawakami b , Tatsumi Kusakabe c , Toshifumi Takenaka a
Department of Physiology, Yokohama City UniÕersity, 3-9 Fukuura Kanazawa-ku, Yokohama, 236, Japan b Department of Physiology, Kitasato UniÕersity School of Medicine, Sagamihara, Kanagawa, Japan c Department of Anatomy, Yokohama City UniÕersity, Fukuura Kanazawa-ku, Yokohama, Japan Accepted 11 February 1997
Abstract The effect of dibutyryl cyclic AMP ŽdbcAMP. on axoplasmic transport of cultured hippocampal neuron cells from postnatal 1-day mice was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. Dibutyryl cyclic AMP increased the axoplasmic transport in both anterograde and retrograde directions. The number of particles flowing in the neurites was increased by 0.5 mM dbcAMP. The peak reached about 160% of the initial value. The instantaneous velocity of axoplasmic transport was also increased by 0.5 mM dbcAMP. The average velocity of anterograde and retrograde direction changed respectively from 1.95 " 1.01 mmrs Ž n s 55. to 2.66 " 1.26 mmrs Ž n s 58. and from 1.94 " 0.85 Ž n s 57. to 2.39 " 0.93 Ž n s 57.. Rates were 136.1 and 123.1%, respectively. Previously, we have found that acetylcholine suppressed and adrenaline increased the axoplasmic transport in superior cervical ganglion cells. These effects are related to the amount of endogeneous cAMP. The results of the present report suggest that endogeneous cAMP is also related to hippocampal axoplasmic transport. Keywords: Axonal transport; Dibutyryl cyclic AMP; Hippocampus; Cell culture; Organelle motion; Neurotransmitter
Cyclic AMP is an important second messenger, which mediates many functions caused by neurotransmitters and hormones. Some experiments have been reported on the action of cAMP on axoplasmic transport. For example, Azderian et al. reported that cAMP stimulates axoplasmic transport in the neurons of Aplysia bag cell neurons w2x. Forscher et al. also reported that cAMP induced regulation of organelle transport in bag cell growth cones of aplysia w6x. We reported that dibutyryl cyclic AMP ŽdbcAMP. increased axoplasmic transport in superior cervical ganglion ŽSCG. cells w10,19–21x. In the hippocampus, cAMP induces later and longer long-term-potentiation w7x. However, experiments on the effects of cAMP on hippocampal axoplasmic transport have not been reported. Therefore, we examined the effect of cAMP on neurons in the hippocampus, which is thought to be important for memory and learning w17x. We followed the methods of Hatanaka et al. w9x for cell culture. Primary cultures of dissociated hippocampal CA1 region neurons were prepared from the brains of 1-day-old c57BLr6 mice. The location of the area was determined
)
Corresponding author. Fax: q81 Ž45. 787-2509.
0006-8993r96r$17.00 Published by Elsevier Science B.V. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 2 3 0 - 8
according to a developmental rat atlas w15x. Dissection was carried out on ice under a microscope. Tissue fragments were added to 0.5 ml of a freshly prepared Ca2q, Mg 2q-free phosphate buffered saline ŽCMF-PBS. containing papain Ž10 U., DNase I ŽSigma, 50 U., L-cystein-HCl ŽSigma, 0.1 mg., bovine serum albumin ŽSigma, 0.1 mg. and glucose Ž2.5 mg., and then incubated twice, with a change of buffer, for 20 min at 378C and 150 r.p.m. Next the papain digestion was arrested by the addition of fetal calf serum, and the tissue fragments were washed with a culture medium, consisting of 10% Žvrv. fetal calf serum, 90% of Dulbecco’s modified Eagle’s medium, 50 Urml penicillin G, 0.05 mgrml streptomycin sulfate and 100 ngrml 7 s NGF. Cells were then dissociated by gentle drawing through small pipettes Ž0.3–0.5 mm diameter. whose tips had been rounded in a flame. The dissociated cells were seeded onto 30 = 40 mm cover glasses whose center Ž1 cm2 of culture surface area. was coated with poly-L-lysine. All cell culture was done at 378C in a 5% Žvrv. CO 2 atmosphere at saturated humidity, in an incubator. After 1 week culture the cover glasses were attached with waterproof tape to the underside of a 0.5 mm thick stainless-steel plate Ž50 = 80 mm. with a lozenge-shaped hole. The upper side of the steel plate was covered with another cover
344
Y. Hashimoto et al.r Brain Research 755 (1997) 343–346
glass, leaving small openings on both sides to perfuse solutions through. The plate was placed on an inverted Zeiss Axiovart Microscope equipped with a computer-assisted video-enhanced microscope w1,18x, with an oil-immersed plan-apochromat 100 = 1.40 objective lens. The individual particle flow in neurites was observed in real time video-enhanced differential interference contrast optic recording. The video camera was a Hamamatsu Photonics Harpicon. The test solution was 0.5 mM dbcAMP ŽSigma USA. dissolved in high concentration glucose physiological salt solution ŽHG-PSS: NaCl 135.0 mM, KCl 5.0 mM, CaCl 2 1.0 mM, MgCl 2 1.0 mM, glucose 25 mM, HEPES 10 mM, pH 7.3.. Fig. 1 is the average time course of 0.5 mM dbcAMP effect during five 23-min applications. The ordinate shows the percent of transported particles passing through one region of a neurite per min relative to the control. The asterisks show P values. The dbcAMP was applied by changing from normal HG-PSS to HG-PSS containing 0.5 mM dbcAMP at 7 min. After the application of dbcAMP, the number of flowing particles increased and reached a peak about 5 min after application in both anterograde and retrograde transport. The number peaked at about 160%. In this connection, the actual numbers were about 35–50 at control and 60–80 at peak in both directions. This effect gradually decreased with time, although the number had not returned to control level at 30 min. This gradual return was more sluggish than in superior cervical ganglion cells.
To produce a physiological action, the concentration of intracellular cAMP must be greater than 0.01 mM w13x. We used 0.01 mM to 1.0 mM solutions of dbcAMP, and a similar increasing effect was clearly observed at concentrations of 0.1 to 1.0 mM. At a concentration of 0.01 mM, this effect could not be observed. The instantaneous velocity of axoplasmic transport was also increased by dbcAMP in both directions. The average velocity in anterograde and retrograde directions changed respectively from 1.95 " 1.01 mmrs Ž n s 55. to 2.66 " 1.26 mmrs Ž n s 58. and from 1.94 " 0.85 Ž n s 57. to 2.39 " 0.93 Ž n s 57.. Rates were 136.1 and 123.1%, respectively. The difference of average velocity between control and 5 min after application was statistically significant at P - 0.01 in both anterograde and retrograde directions. Table 1 shows these data. Fig. 2 shows the relation between instantaneous velocity and histogram of the number of particles moving at that velocity 5 min after application of dbcAMP and in the control. Ordinates show the number of particles. Abscissae show instantaneous velocity. In both anterograde and retrograde directions, the curve was shifted to the right, that is, to the faster side. This result also suggested that the velocity of individual particles was increased by dbcAMP. In comparing the histograms of anterograde and retrograde particles, the anterograde histogram has a broader distribution, although both have almost the same average. This result also can be seen in Table 1, where anterograde velocity has a larger S.D. than retrograde velocity.
Fig. 1. The average time course of 0.5 mM dbcAMP increase in anterograde and retrograde transport. Dibutyryl cyclic AMP was applied at 7 min. About 5 min after the perfusion, relative number of particles reached a peak. The ordinate shows the percentage of particle flow compared with the initial value of 100%. Ž ) P - 0.10, ) ) P - 0.05, ) ) ) P - 0.02, ) ) ) ) P - 0.01..
Y. Hashimoto et al.r Brain Research 755 (1996) 343–346
Fig. 2. Instantaneous velocity histogram of anterograde ŽAntero.. and retrograde ŽRetro.. axoplasmic transport particles. Before Ž – Ø – . and during Ž – ( – . application Žfrom 4 to 6 min after perfusion..
The fact that dibutyryl cyclic AMP increased the number of particles in both anterograde and retrograde direction not only in the SCG but also in hippocampal neurons suggests that endogenous cAMP takes part in the control system of axoplasmic transport in the hippocampus as a second messenger. We have investigated the control system of axoplasmic transport in the SCG, which is part of the sympathetic and peripheral nervous system, and have shown that acetylcholine depresses and adrenaline enhances the axoplasmic transport in SCG cells w10,19–21x. These effects were related to the quantity of endogeneous cAMP. The binding of acetylcholine to m2-acetylcholine-receptors depresses adenyl cyclase and the binding of adrenaline to b 2-adrenaline-receptor activates adenyl cyclase through inhibitory and stimulatory GTP-binding proteins, respectively. These resultant changes in the amount of endogenous cAMP initiate a sequence of metabolic processes which presumably includes the phosphorylation of specific proteins by cAMP-dependent protein kinase, as suggested for the decrease and increase of the axoplasmic transport.
345
The hippocampus, to which cholinergic nuclei of the medial septal area and the nucleus of the diagonal band project w11x, is considered of crucial importance for learning and memory processes. Electrical stimulation of the medial septal area increases both ACh release and the neuronal activity of the hippocampus w5x. Other studies have reported that acquisition of an operant behavior enhances the ACh release in the hippocampus w4,14x. Muscarinic acetylcholine receptors ŽmACh-Rs. have subtypes known as m1, m2, m3, m4, and m5 w16x. Subtypes m1, m3, and m5 which mediate the stimulation of phosphoinositide hydrolysis, are related to activation of PKC and Ca2qrcalmodulin kinases. On the other hand, subtypes m2 and m4 inhibit the effect of adenyl cyclase activity through inhibitory GTP-binding protein. Subtypes m1 and m3 are abundant in the hippocampus. Subtype m4 is less abundant and m2 subtype is said not to exist in the hippocampus w3x. Therefore, it appears that ACh binding to m2ACh-R does not suppress the axoplasmic transport of hippocampal neurons, but does suppress that of SCG neurons. Mons et al. w12x reported that two functional types of adenyl cyclase are detected in the brain that differ by being sensitive or insensitive to Ca2qrcalmodulin stimulation, and are known as Type I and Type II, respectively. In the CA1 region, Type II expression was higher than that of Type I. These studies provide support for the following hypothesis. In the hippocampal CA1 region, binding of ACh to subtypes m1, m3, and m5 raises the endogeneous cAMP by phosphoinositide hydrolysis producing diacylglycerol and IP3, which release Ca2q from an internal Ca2q then binds to calmodulin ŽCa2qrcalmodulin., and activation of Type II adenyl cyclase by this Ca2qrcalmodulin, raises the endogeneous cAMP. Regarding hippocampal long-term-potentiation ŽLTP. in the CA1 region, the later and longer lasting stage ŽL-LTP. is mediated by synthesis of cAMP w7x, and according to another report, coactivation of metabolic glutamate receptors and b-adrenergic receptors is mediated by cAMP and activation of cAMP-dependent protein kinases w8x. If hippocampal LTP is indeed related to mammalian leaning and memory, then activation of hippocampal axoplasmic transport may also take part in learning and memory, since raising of the endogeneous cAMP level both initiates L-LTP and at the some time intensifies the axoplasmic transport.
Acknowledgements Table 1 Velocity change 5 min after application of 0.5 mM dbcAMP Velocity Control Žmmrs. After 5 min Žmmrs. Rate a
P - 0.01.
Anterograde 1.95"1.01 2.66"1.26 136.1%
a a
Retrograde 1.94"0.85 a 2.39"0.93 a 123.1%
We wish to thank Dr. R.C. Goris of the Department of Anatomy, Yokohama City University School of Medicine, Dr. Masato Kano of their help in preparing the manuscript and Mr. Satoshi Yuki of Mitsubishi Chemical Corporation for his advice of cell culture. This study was supported by Grants-in Aid for Scientific Research Projects 07557005 from The Japanese Ministry of Education.
346
Y. Hashimoto et al.r Brain Research 755 (1997) 343–346
References w1x R.D. Allen, N.S. Allen, J.L. Travis, Video-enhanced contrast, differential interference contrast ŽAVEC-DIC. micro-scopy: a new method capable of analyzing microtubule-related motility in the reticulopodial network of Allogromia laticollaris, Cell Motil. 1 Ž1989. 291–302. w2x E. Azderian, C.H. Lin, D. Hefner, P. Forscher and L.K. Kaczmarek, Cyclic AMP increases the quantity of ELH-containing proteins in the neurites of Aplysia bag cell neurons, 21st Ann. Meet. Soc. for Neurosci. Abstr., 59 Ž1991.. w3x N.J. Buckley, T.I. Bonner, M.R. Brann, Localization of a family of muscarinic receptor mRNAs in rat brain, J. Neurosci. 8 Ž1988. 4646–4652. w4x J. Day, G. Damsma, H.C. Fibiger, Cholinergic activity in the rat hippocampus, cortex and striatum correlates with locomotor activity: An in vivo microdialysis study, Pharmacol. Biochem. BehaÕ. 38 Ž1991. 723–729. w5x J.D. Dudar, The effect of septal nuclei release ofacetylcholine from the rabbit hippocampus, Brain Res. 83 Ž1975. 123–133. w6x P. Forscher, L.K. Kaczmarek, J.-A. Buchanan, S.J. Smith, Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cells neurons, J. Neurosci. 7 Ž1987. 3600–3611. w7x U. Frey, Y.Y. Huang, E.R. Kandel, Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons, Science 260 Ž1993. 1661–1664. w8x R.W. Gereau IV, P.J. Conn, A cyclic AMP-dependent form of associative synaptic plasticity induced by coactivation of b-adrenergic receptors and metabotropic glutamate receptors in rat hippocampus, J. Neurosci. 14 Ž1994. 3310–3318. w9x H. Hatanaka, C. Nishio, Y. Kushima, H. Tsukui, Nerve-growth-factor-dependent and cell-density independent survival of septal cholinergic neurons in culture from postnatal rats, Neurosci. Res. 8 Ž1990. 69–82. w10x T. Kawakami, T. Takenaka, H. Hori, Y. Bandou, T. Kusakabe, Effects of acetylcholine and adrenaline on axoplasmic transport at
w11x
w12x
w13x w14x
w15x w16x
w17x
w18x
w19x
w20x
w21x
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