- Email: [email protected]
Increase in hippocampal acetylcholine after choline administration
Brain Research, 125 (1977) 383-385
383
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Increase in hippocampal acetylcholine after choline administration
MADELYN J. HIRSCH, JOHN H. GROWDON and RICHARD J. WURTMAN Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.)
(Accepted January 7th, 1977)
Choline chloride, administered either by injection 1 or in the diet s, elevates choline and acetylcholine (ACh) levels in homogenates of whole rat b r a i n - - specifically in the caudate nucleus and cerebral cortex. No observations have been described concerning the ability of exogenous choline to increase ACh in brain regions in which the transmitter is known to be localized within nerve terminals (and, thus, to be available for release into synapses). The hippocampus constitutes such a region: its ACh is largely confined within the axons and terminals of the septo-hippocampal tract 4,1°, as shown by the release of ACh from the hippocampal surface after septal stimulation 8,1~ and by the major decline in hippocampal ACh levels after transection of this tract s. (The high-affinity uptake of choline by hippocampal synaptosomes 6 and the activities of choline acetyltransferase 6 and acetylcholinesterase 7 within this brain region also are reduced by septo-hippocampal lesions.) Studies described in this report show that a single intraperitoneal (i.p.) injection of choline markedly elevates ACh levels within the dorsal hippocampus, just as it does in the caudate nuclei I and in whole rat brain 1. In each of two experiments, 30 male Sprague-Dawley rats were given ad libitum access to a choline-deficient diet (Bio-Serv, Inc.) and water for 8 days before choline administration; they were exposed to light (Vita-Lite, 300/~W/sq. cm) from 8 a.m. to 8 p.m. daily. At 9 a.m. on the ninth day, 20 rats were injected with an aqueous solution of choline chloride (60 mg/kg, i.p.) and killed after 20 or 40 min (10 animals each) by microwave irradiation of the head 1,~,12. Ten control rats received the diluent and were killed after 20 or 40 min (5 rats each); since the 20-rain choline and ACh levels did not vary from the 40-min values, the control data were pooled. Portions of the dorsal hippocampi and the caudate nuclei were dissected from each brain 5 and assayed for choline and ACh by a radioenzymatic method 1,9. Student's t-test was used to analyze the data. In both experiments, the choline levels in the hippocampus and caudate were maximally elevated 20 min after choline injection (Table I). Acetylcholine levels also were significantly elevated after 20 min (Table I), but, in one experiment, they were higher 40 min after choline injection than after 20 min. These observations indicate that choline administration markedly elevates ACh levels within a brain region - - the dorsal hippocampus - - in which the transmitter is
384 TABLE I Effect of choline chloride administration on choline and acetylcholine concentrations in rat hippocampus and caudate nuclei
Groups of 10 rats received choline chloride (ChCI) (60 mg/kg, i.p.) or its diluent (water) and were killed 20 or 40 rain after injection. Data are given as means ± S.E.M. Group
Experiment 1 Control Hippocampus Caudate 20 rain after ChCI Hippocampus Caudate 40 rain after ChCI Hippocampus Caudate Experiment 2 Control Hippocampus Caudate 20 min after ChCI Hippocampus Caudate 40 min after ChCI Hippocampus Caudate * P
**P<0.02,
Choline (riM~g)
Acetylcholine (nM/g)
27.29 ± 3.00 34.14 ~ 2.31
14.23 ~ 2.95 48.70 ± 2.00
42.60 ± 1.70" 57.47 ± 5.50*
26.50 ± 2.70** 59.15 ± 2.45**
37.48 ~ 2.99 45.04 -- 3.53***
29.60 ± 3.04** 63.16 ± 3.00**
25.33 :t- 1.27 27.75 ± 4.31
19.92 :t_ 0.83 29.38 ± 1.66
65.21 ± 8.15§ 50.76 ± 4.73*
23.90 ± 1.54"** 46.39 ± 2.45§
36.00 ± 3.05* 38.34 d- 2.50
23.63 :L 3.00 31.60 ± 1.84
***P<0.05,
§P < 0.001, differs from control.
k n o w n to be located presynaptically. W h e t h e r this increase is associated with e n h a n c e d release of A C h into cholinergic synapses remains to be determined. However, indirect evidence obtained from experiments on A C h synapses elsewhere in the body suggests that this m a y be the case : choline a d m i n i s t r a t i o n causes a rapid (2 h) transsynaptic activation of the enzyme tyrosine hydroxylase ( T O H ) in the rat caudate nucleus i4 a n d a slower (24 h) i n d u c t i o n of T O H in the adrenal medulla 13. These effects can be blocked by atropine a d m i n i s t r a t i o n la or by presynaptic denervation 13, respectively. These studies were supported in part by grants from the F o r d F o u n d a t i o n a n d the N a t i o n a l Aeronautics a n d Space A d m i n i s t r a t i o n . Dr. G r o w d o n is the J o h n R. Whittier Fellow of the Committee to C o m b a t H u n t i n g t o n ' s Disease. We t h a n k Mr. Eli W y l e n for excellent technical assistance.
1 Cohen, E. L. and Wurtman, R. J., Brain acetylcholine: increase after systemic choline administration, Life Sci., 16 (1975) 1095-1102. 2 Cohen, E. L. and Wurtman, R. J., Brain acetylcholine: control by dietary choline, Science, 191 (1976) 561-562.
385 3 Dudar, J. D., The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus, Brain Research, 83 (1975) 123-133. 4 Fonnum, F., Topographical and subcellular localization of choline acetyltransferase in rat hippocampal formation, J. Neurochem., 17 (1970) 1029-1037. 5 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain - - I, J. Neurochem., 13 (1966) 655-669. 6 Kuhar, M. J., Sethy, V. H., Roth, R. H. and Aghajanian, G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 7 Mellgren, S. I. and Srebro, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesion in the rat, Brain Research, 52 (1973) 19-36. 8 Sethy, V. H., Roth, R. H., Kuhar, M. J. and Van Woert, M. H., Choline and acetylcholine: regional distribution and effect of degeneration of cholinergic nerve terminals in the rat hippocampus, Neuropharmacology, 12 (1973) 819-823. 9 Shea, P. A. and Aprison, M. H., An enzymatic method for measuring picomole quantities of acetylcholine and choline in CNS tissue, ,4nalyt. Biochem., 56 (1973) 165-177. 10 Shute, C. C. D. and Lewis, P. R., Electron microscopy of cholinergic terminals and acetylcholinesterase-containing neurons in the hippocampal formation of the rat, Z. Zellforsch., 69 (1966) 334-343. 11 Smith, C. M., Acetylcholine release from the cholinergic septohippocampal pathway, Life ScL, 14 (1974) 2159-2166. 12 Stavinoha, W. B., Weintraub, S. T. and Modak, A. T., The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine, J. Neurochem., 20 (1973) 361-371. 13 Ulus, I. H., Hirsch, M. J. and Wurtman, R. J., Trans-synaptic induction of adrenomedullary tyrosine hydroxylase activity by choline: evidence that choline administration increases cholinergic transmission, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 798-800. 14 Ulus, I. H. and Wurtman, R. J., Choline administration: activation of tyrosine hydroxylase in dopaminergic neurons of rat brain, Science, 194 (1976) 1060-1061.
383
© Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands
Increase in hippocampal acetylcholine after choline administration
MADELYN J. HIRSCH, JOHN H. GROWDON and RICHARD J. WURTMAN Laboratory of Neuroendocrine Regulation, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass. 02139 (U.S.A.)
(Accepted January 7th, 1977)
Choline chloride, administered either by injection 1 or in the diet s, elevates choline and acetylcholine (ACh) levels in homogenates of whole rat b r a i n - - specifically in the caudate nucleus and cerebral cortex. No observations have been described concerning the ability of exogenous choline to increase ACh in brain regions in which the transmitter is known to be localized within nerve terminals (and, thus, to be available for release into synapses). The hippocampus constitutes such a region: its ACh is largely confined within the axons and terminals of the septo-hippocampal tract 4,1°, as shown by the release of ACh from the hippocampal surface after septal stimulation 8,1~ and by the major decline in hippocampal ACh levels after transection of this tract s. (The high-affinity uptake of choline by hippocampal synaptosomes 6 and the activities of choline acetyltransferase 6 and acetylcholinesterase 7 within this brain region also are reduced by septo-hippocampal lesions.) Studies described in this report show that a single intraperitoneal (i.p.) injection of choline markedly elevates ACh levels within the dorsal hippocampus, just as it does in the caudate nuclei I and in whole rat brain 1. In each of two experiments, 30 male Sprague-Dawley rats were given ad libitum access to a choline-deficient diet (Bio-Serv, Inc.) and water for 8 days before choline administration; they were exposed to light (Vita-Lite, 300/~W/sq. cm) from 8 a.m. to 8 p.m. daily. At 9 a.m. on the ninth day, 20 rats were injected with an aqueous solution of choline chloride (60 mg/kg, i.p.) and killed after 20 or 40 min (10 animals each) by microwave irradiation of the head 1,~,12. Ten control rats received the diluent and were killed after 20 or 40 min (5 rats each); since the 20-rain choline and ACh levels did not vary from the 40-min values, the control data were pooled. Portions of the dorsal hippocampi and the caudate nuclei were dissected from each brain 5 and assayed for choline and ACh by a radioenzymatic method 1,9. Student's t-test was used to analyze the data. In both experiments, the choline levels in the hippocampus and caudate were maximally elevated 20 min after choline injection (Table I). Acetylcholine levels also were significantly elevated after 20 min (Table I), but, in one experiment, they were higher 40 min after choline injection than after 20 min. These observations indicate that choline administration markedly elevates ACh levels within a brain region - - the dorsal hippocampus - - in which the transmitter is
384 TABLE I Effect of choline chloride administration on choline and acetylcholine concentrations in rat hippocampus and caudate nuclei
Groups of 10 rats received choline chloride (ChCI) (60 mg/kg, i.p.) or its diluent (water) and were killed 20 or 40 rain after injection. Data are given as means ± S.E.M. Group
Experiment 1 Control Hippocampus Caudate 20 rain after ChCI Hippocampus Caudate 40 rain after ChCI Hippocampus Caudate Experiment 2 Control Hippocampus Caudate 20 min after ChCI Hippocampus Caudate 40 min after ChCI Hippocampus Caudate * P
**P<0.02,
Choline (riM~g)
Acetylcholine (nM/g)
27.29 ± 3.00 34.14 ~ 2.31
14.23 ~ 2.95 48.70 ± 2.00
42.60 ± 1.70" 57.47 ± 5.50*
26.50 ± 2.70** 59.15 ± 2.45**
37.48 ~ 2.99 45.04 -- 3.53***
29.60 ± 3.04** 63.16 ± 3.00**
25.33 :t- 1.27 27.75 ± 4.31
19.92 :t_ 0.83 29.38 ± 1.66
65.21 ± 8.15§ 50.76 ± 4.73*
23.90 ± 1.54"** 46.39 ± 2.45§
36.00 ± 3.05* 38.34 d- 2.50
23.63 :L 3.00 31.60 ± 1.84
***P<0.05,
§P < 0.001, differs from control.
k n o w n to be located presynaptically. W h e t h e r this increase is associated with e n h a n c e d release of A C h into cholinergic synapses remains to be determined. However, indirect evidence obtained from experiments on A C h synapses elsewhere in the body suggests that this m a y be the case : choline a d m i n i s t r a t i o n causes a rapid (2 h) transsynaptic activation of the enzyme tyrosine hydroxylase ( T O H ) in the rat caudate nucleus i4 a n d a slower (24 h) i n d u c t i o n of T O H in the adrenal medulla 13. These effects can be blocked by atropine a d m i n i s t r a t i o n la or by presynaptic denervation 13, respectively. These studies were supported in part by grants from the F o r d F o u n d a t i o n a n d the N a t i o n a l Aeronautics a n d Space A d m i n i s t r a t i o n . Dr. G r o w d o n is the J o h n R. Whittier Fellow of the Committee to C o m b a t H u n t i n g t o n ' s Disease. We t h a n k Mr. Eli W y l e n for excellent technical assistance.
1 Cohen, E. L. and Wurtman, R. J., Brain acetylcholine: increase after systemic choline administration, Life Sci., 16 (1975) 1095-1102. 2 Cohen, E. L. and Wurtman, R. J., Brain acetylcholine: control by dietary choline, Science, 191 (1976) 561-562.
385 3 Dudar, J. D., The effect of septal nuclei stimulation on the release of acetylcholine from the rabbit hippocampus, Brain Research, 83 (1975) 123-133. 4 Fonnum, F., Topographical and subcellular localization of choline acetyltransferase in rat hippocampal formation, J. Neurochem., 17 (1970) 1029-1037. 5 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain - - I, J. Neurochem., 13 (1966) 655-669. 6 Kuhar, M. J., Sethy, V. H., Roth, R. H. and Aghajanian, G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 7 Mellgren, S. I. and Srebro, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesion in the rat, Brain Research, 52 (1973) 19-36. 8 Sethy, V. H., Roth, R. H., Kuhar, M. J. and Van Woert, M. H., Choline and acetylcholine: regional distribution and effect of degeneration of cholinergic nerve terminals in the rat hippocampus, Neuropharmacology, 12 (1973) 819-823. 9 Shea, P. A. and Aprison, M. H., An enzymatic method for measuring picomole quantities of acetylcholine and choline in CNS tissue, ,4nalyt. Biochem., 56 (1973) 165-177. 10 Shute, C. C. D. and Lewis, P. R., Electron microscopy of cholinergic terminals and acetylcholinesterase-containing neurons in the hippocampal formation of the rat, Z. Zellforsch., 69 (1966) 334-343. 11 Smith, C. M., Acetylcholine release from the cholinergic septohippocampal pathway, Life ScL, 14 (1974) 2159-2166. 12 Stavinoha, W. B., Weintraub, S. T. and Modak, A. T., The use of microwave heating to inactivate cholinesterase in the rat brain prior to analysis for acetylcholine, J. Neurochem., 20 (1973) 361-371. 13 Ulus, I. H., Hirsch, M. J. and Wurtman, R. J., Trans-synaptic induction of adrenomedullary tyrosine hydroxylase activity by choline: evidence that choline administration increases cholinergic transmission, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 798-800. 14 Ulus, I. H. and Wurtman, R. J., Choline administration: activation of tyrosine hydroxylase in dopaminergic neurons of rat brain, Science, 194 (1976) 1060-1061.