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Localization in rat brain of the trace metals, zinc and manganese, after intracerebroventricular injection
BRAIN
RESEARCH ELSEVIER
Brain Research 658 (1994) 252-254
Short communication
Localization in rat brain of the trace metals, zinc and manganese, after intracerebroventricular injection Atsushi Takeda, Jinko Sawashita, Shoji Okada
*
Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan
Accepted 28 June 1994
Abstract
Autoradiographic studies of rat brain, after 65ZnCIz or 54MnCIz injection into the lateral ventricle, revealed that 65Zn and 54 Mn were transferred freely through the cerebrospinal and extracellular fluid compartments; both tracers appeared in all the ventricles 1 h after injection. At 6 days after injection, 65Zn was concentrated in the hippocampal formation and hypothalamic nuclei but not in the cerebral cortex and superior colliculus of the ipsilateral hemisphere, with a lower uptake into contralateral hemisphere except for the hypothalamic nuclei. 54 Mn was concentrated in some brainstem nuclei, such as the red and pontine reticular nuclei, to about the same extent in both hemispheres. These results suggest that both metals were taken up gradually into brain mainlyvia the cerebrospinal fluid. Relatively high uptake of 65Zn into the cerebral cortex and superior colliculus after intravenous injection suggests uptake in those regions is through two blood/brain barriers. Keywords: Cerebrospinal fluid; Choroid plexus; Blood-brain barrier; Zinc; Manganese; Essential trace metal
The route of brain uptake of essential trace metals generally remains unknown and needs to be solved for the clarification of their roles in brain function. Recently, as a result of a study using radioactive zinc (Zn) and manganese (Mn) we suggested that they were gradually taken up by brain via cerebrospinal fluid (CSF) from the choroid plexus [13]. The present study shows that brain uptake of both metals occurred via the CSF after intracerebroventricular (i.c.v.) injection. Male rats (n = 4 per group) of the Wistar strain (110-140 g) were anesthetized with' pentobarbital and placed in a stereotaxic instrument. 65ZnCIz (92.5 MBq (2.5 mCi)/mg, Du Pont/NEN Research Products, USA), diluted with 0.1 M acetate buffer (pH 4.0) (37 kBq/lO J,LI), or 54MnCIz (3.19 GBq (86.23 mCi)/mg, Du Pont/NEN Research Products), diluted with 0.1 M Tris-HCI buffer (pH 7.6) (148 kBq/10 j.d), was injected at 1 J,Ll/min into the left lateral ventricle of rats at the coordinates of - 0.92 mm posterior to bregma, + 1.6 mm lateral to midline suture and - 3.4 mm from dura [10] via a microdialysis probe without a dialyzing membrane and using a microinjection pump (CMA/100, Carnegie Medicine Sweden). As controls, 65ZnCIz (740
* Corresponding author.
Fax: (81) (54) 264-5705.
0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00789-6
kBq/0.2 ml) or 54MnCIz (1.48 MBQ/0.2 ml) was injected into the tail vein of rats. After given times, the brains were excised and treated as reported previously [13]. The autoradiograms were obtained after exposure to the imaging plates for 1-2 days (ventricular injection) or 7 days (intravenous (i.v.) injection). By 1 h after left lateral ventricular injection, both 65 Zn and 54 Mn had penetrated into the interventricular foramen of Monroe, the third ventricle, the cerebral aqueduct (Fig. 1) and the fourth ventricle (data not shown), but not into the right lateral ventricle (Fig. 1). At this time, brain uptake of both the tracers via CSF was very slight but increased gradually thereafter. 65Zn was relatively concentrated in the hippocampal formation, hypothalamic nuclei and globus pallidus, but not in the cerebral cortex and superior colliculus, of the ipsilateral hemisphere, and in hypothalamic nuclei of the contralateral hemisphere (Fig. 1a and Table 1). 54 Mn was concentrated in some brainstem nuclei in both hemispheres; these included the thalamus, hypothalamic nuclei, red nucleus and pontine reticular nuclei. (Fig. 1b and Table 2). When 65 Zn and 54 Mn were incubated for 30 min with serum or CSF obtained from rats and subjected to HPLC using a TSK-G3000SW column (0.75 x 60 em) with 0.1 M sodium sulfate/0.05 M sodium acetate (pH
A Takeda et al. / Brain Research 658 (1994) 252-254
253
6d
1h
a
•
I.
, ..
,
b
•
Fig. 1. 65 Zn_ and 54Mn-imaging of rat brain . The radio imaging at 1 h and 6 days afte r i.v, (left-hand side) and i.c.v. (right-h and side) injection of 65ZnCl z (a) or 54 MnClz (b) was performed on selected coronal slices of rat brain from the rostral pole of the cerebral cortex (top) to the spinal cord (bottom). Th e experim ent s performed in quadruplicate gave similar results.
254
A. Takeda et al. / Brain Research 658 (1994) 252-254
Table 1 distribution in rat brain
65 Z n
Region
i.c.v. Contra.
Cerebral cortex Amygdaloid nuclei Caudate putamen Globus pallid us Hippocampal formation CAI+CA2 CA3+DG Thalamus Hypothalamic nuclei Superior colliculus
i.v. Ipsi.
64± 9 86±21 131 ±38 249±44
230± 52 239± 28 506± 21 846± 141
267 283 270 293
247±49 300±53 198 ± 17 639±30 173±33
748 ± 126 1061± 191 405± 28 733± 23 249± 41
246 280 242 222 270
Each value (mean ± S.EJ represents the radioactivity (PSL/mm 2 ) in the brain of rats 6 days after i.c.v. injection. In the case of i.v, injection, each value represents a typical one (PSL/mm 2 ) . The radioactivity of each region indicated with surrounding lines in Fig. la was measured with a Bio-imaging Analyzer [13]. The radioactivities of the caudate putamen and globus pallidus, which cannot see in Fig. l a, were measured by using coronal slices containing these regions in the same way. In the case of i.c.v. injection, Contra. and Ipsi. represent contralateral and ipsilateral hemispheres, respectively.
5.0» as eluent, both the tracers were eluted at the positions where the tracers themselves were eluted (data not shown). 65Zn and 54 Mn are concentrated in the choroid plexus. But, the transfer of both tracers into CSF is very slow. It is, therefore, uncertain whether Zn [2,5,7,11] and Mn [1,8,9,12] are taken up through the blood-brain or blood-CSF barriers, although both the barriers for Zn and Mn uptake may not necessarily function in the same way in different brain region. In the present study, as an approach to understanding the Table 2 54 Mn distribution in rat brain Region
Cerebral cortex Hippocampal formation CAl+CA2 CA3+DG Thalamus Hypothalamic nuclei Red nucleus Interpeduncular nuclei Inferior colliculus Pontine reticular nuclei Olivary nuclei
i.c.v,
i.v.
Contra.
Ipsi.
191 ± 20
390 ± 35
177
371± 53 535± 48 489± 63 71O± 62 492 ± 59 764 ± 77 645 ± 85 747 ± 114 750 ± 102 833 ± 113 771 ± 98 604 ± 27 678 ± 22 819± 82 874± 65 61O± 81 654± 76
211 267 273 241 322 293 322 360 374
Each value (mean ± S.E.) represents the radioactivity (PSL/mm 2 ) in the brain of rats 6 days after i.c.v. injection. In the case of i.v. injection, each value represents a typical one (PSL/mm 2 ) . The radioactivity of each region indicated with surrounding lines in Fig. Ib was measured with a Bio-imaging Analyzer [13]. In the case of i.c.v. injection, Contra. and Ipsi. represent contralateral and ipsilateral hemispheres, respectively.
contribution of both barriers to the uptake of these trace metals, we used high resolution autoradiography for determining the localization of 65 Zn and 54 Mn in brain. The brain images of rats after injection into unilateral ventricle showed that both the tracers could enter extracellular fluid through the pial or ependymal surface and were relatively concentrated in circumventricular regions. There was, however, a difference in the regions which concentrated the two tracers. These results indicate that Zn and Mn are taken up gradually via CSF from the choroid plexus. In particular, the blood-CSF barrier may be involved in the uptake of both metals into circumventricular regions such as the hippocampal formation and hypothalamus. On the other hand, the finding that the uptake of 65 Zn into the cerebral cortex and superior colliculus after i.c.v. injection was relatively low in comparison with i.v. injection suggests that the blood-brain barrier may be relatively important for Zn uptake into those regions. Although the chemical forms of Zn and Mn in transport across both the barriers have not been clarified [4,6], the result of in vitro incubation with serum or CSF suggests that protein-unbound forms may be involved in the transport of both Zn [3,6] and Mn [9,12] as previously suggested by several researchers. [1] Aschner, M. and Aschner, J.L., Manganese transport across the blood-brain barrier: relationship to iron homeostasis, Brain Res. Bull., 24 (1990) 857-860. [2] Blair-West, J.R., Denton, D.A., Gibson, A.P. and McKinley, MJ., Opening the blood-brain barrier to zinc, Brain Res., 507 (1990) 6-10. [3] Buxani, S. and Adu, J., Histidine-stimulated 65 Zn transport at the BBB of the anesthetized rat, J Physiol., 438 (1991) 121. [4] Fishman, J.B., Rubin, J.B., Handrahan, J.V., Connor, J.R. and Fine, R.E., Receptor-mediated transcytosis of transferrin across the blood-brain barrier, J. Neurosci. Res., 18 (1987) 299-304. (5) Franklin, PA, Pullen, R.G.L. and Hall, G.H., Blood-brain exchange routes and distribution of 65 Z n in rat brain, Neurochem. Res., 17 (1992) 767-771. [6] Frederickson, CJ., Neurobiology of zinc and zinc-containing neurons, Int. Rev. Neurobiol., 31 (1990) 145-238. (7) Kasarskis, E.J., Zinc metabolism in normal and zinc-deficient rat brain, Exp. Neurol., 85 (1984) 114-127. (8) Kaur, G., Hasan, S.K. and Srivastava, R.C, The distribution of manganese-54 in fetal, young and adult rats, Toxieol. Leu., 5 (1980) 423-426. [9] Murphy, VA, Wadhwani, K.C, Smith, Q.R. and Rapoport, S.l., Saturable transport of manganese across the rat blood-brain barrier, J Neurochem., 57 (1991) 948-954. (10) Paxinos, G. and Watson, C, The Rat Brain, Academic Press, Inc., 1986. [11] Pullen, R.G.L., Franklin, PA and Hall, G.H. 65Z inc uptake from blood into brain and other tissues in the rat, Neurochem. Res., 10 (1990) 1003-1008. (12) Rabin, 0., Hegedus, L., Bourre, J.-M. and Smith, Q.R., Rapid brain uptake of manganesetll) across the blood-brain barrier, J. Neurochem., 61 (1993) 509-517. [13] Takeda, A., Akiyama, T., Sawashita, J. and Okada, S. Brain uptake of trace metals, zinc and manganese, in rats, Brain Res., 640 (1994) 341-344.
RESEARCH ELSEVIER
Brain Research 658 (1994) 252-254
Short communication
Localization in rat brain of the trace metals, zinc and manganese, after intracerebroventricular injection Atsushi Takeda, Jinko Sawashita, Shoji Okada
*
Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422, Japan
Accepted 28 June 1994
Abstract
Autoradiographic studies of rat brain, after 65ZnCIz or 54MnCIz injection into the lateral ventricle, revealed that 65Zn and 54 Mn were transferred freely through the cerebrospinal and extracellular fluid compartments; both tracers appeared in all the ventricles 1 h after injection. At 6 days after injection, 65Zn was concentrated in the hippocampal formation and hypothalamic nuclei but not in the cerebral cortex and superior colliculus of the ipsilateral hemisphere, with a lower uptake into contralateral hemisphere except for the hypothalamic nuclei. 54 Mn was concentrated in some brainstem nuclei, such as the red and pontine reticular nuclei, to about the same extent in both hemispheres. These results suggest that both metals were taken up gradually into brain mainlyvia the cerebrospinal fluid. Relatively high uptake of 65Zn into the cerebral cortex and superior colliculus after intravenous injection suggests uptake in those regions is through two blood/brain barriers. Keywords: Cerebrospinal fluid; Choroid plexus; Blood-brain barrier; Zinc; Manganese; Essential trace metal
The route of brain uptake of essential trace metals generally remains unknown and needs to be solved for the clarification of their roles in brain function. Recently, as a result of a study using radioactive zinc (Zn) and manganese (Mn) we suggested that they were gradually taken up by brain via cerebrospinal fluid (CSF) from the choroid plexus [13]. The present study shows that brain uptake of both metals occurred via the CSF after intracerebroventricular (i.c.v.) injection. Male rats (n = 4 per group) of the Wistar strain (110-140 g) were anesthetized with' pentobarbital and placed in a stereotaxic instrument. 65ZnCIz (92.5 MBq (2.5 mCi)/mg, Du Pont/NEN Research Products, USA), diluted with 0.1 M acetate buffer (pH 4.0) (37 kBq/lO J,LI), or 54MnCIz (3.19 GBq (86.23 mCi)/mg, Du Pont/NEN Research Products), diluted with 0.1 M Tris-HCI buffer (pH 7.6) (148 kBq/10 j.d), was injected at 1 J,Ll/min into the left lateral ventricle of rats at the coordinates of - 0.92 mm posterior to bregma, + 1.6 mm lateral to midline suture and - 3.4 mm from dura [10] via a microdialysis probe without a dialyzing membrane and using a microinjection pump (CMA/100, Carnegie Medicine Sweden). As controls, 65ZnCIz (740
* Corresponding author.
Fax: (81) (54) 264-5705.
0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00789-6
kBq/0.2 ml) or 54MnCIz (1.48 MBQ/0.2 ml) was injected into the tail vein of rats. After given times, the brains were excised and treated as reported previously [13]. The autoradiograms were obtained after exposure to the imaging plates for 1-2 days (ventricular injection) or 7 days (intravenous (i.v.) injection). By 1 h after left lateral ventricular injection, both 65 Zn and 54 Mn had penetrated into the interventricular foramen of Monroe, the third ventricle, the cerebral aqueduct (Fig. 1) and the fourth ventricle (data not shown), but not into the right lateral ventricle (Fig. 1). At this time, brain uptake of both the tracers via CSF was very slight but increased gradually thereafter. 65Zn was relatively concentrated in the hippocampal formation, hypothalamic nuclei and globus pallidus, but not in the cerebral cortex and superior colliculus, of the ipsilateral hemisphere, and in hypothalamic nuclei of the contralateral hemisphere (Fig. 1a and Table 1). 54 Mn was concentrated in some brainstem nuclei in both hemispheres; these included the thalamus, hypothalamic nuclei, red nucleus and pontine reticular nuclei. (Fig. 1b and Table 2). When 65 Zn and 54 Mn were incubated for 30 min with serum or CSF obtained from rats and subjected to HPLC using a TSK-G3000SW column (0.75 x 60 em) with 0.1 M sodium sulfate/0.05 M sodium acetate (pH
A Takeda et al. / Brain Research 658 (1994) 252-254
253
6d
1h
a
•
I.
, ..
,
b
•
Fig. 1. 65 Zn_ and 54Mn-imaging of rat brain . The radio imaging at 1 h and 6 days afte r i.v, (left-hand side) and i.c.v. (right-h and side) injection of 65ZnCl z (a) or 54 MnClz (b) was performed on selected coronal slices of rat brain from the rostral pole of the cerebral cortex (top) to the spinal cord (bottom). Th e experim ent s performed in quadruplicate gave similar results.
254
A. Takeda et al. / Brain Research 658 (1994) 252-254
Table 1 distribution in rat brain
65 Z n
Region
i.c.v. Contra.
Cerebral cortex Amygdaloid nuclei Caudate putamen Globus pallid us Hippocampal formation CAI+CA2 CA3+DG Thalamus Hypothalamic nuclei Superior colliculus
i.v. Ipsi.
64± 9 86±21 131 ±38 249±44
230± 52 239± 28 506± 21 846± 141
267 283 270 293
247±49 300±53 198 ± 17 639±30 173±33
748 ± 126 1061± 191 405± 28 733± 23 249± 41
246 280 242 222 270
Each value (mean ± S.EJ represents the radioactivity (PSL/mm 2 ) in the brain of rats 6 days after i.c.v. injection. In the case of i.v, injection, each value represents a typical one (PSL/mm 2 ) . The radioactivity of each region indicated with surrounding lines in Fig. la was measured with a Bio-imaging Analyzer [13]. The radioactivities of the caudate putamen and globus pallidus, which cannot see in Fig. l a, were measured by using coronal slices containing these regions in the same way. In the case of i.c.v. injection, Contra. and Ipsi. represent contralateral and ipsilateral hemispheres, respectively.
5.0» as eluent, both the tracers were eluted at the positions where the tracers themselves were eluted (data not shown). 65Zn and 54 Mn are concentrated in the choroid plexus. But, the transfer of both tracers into CSF is very slow. It is, therefore, uncertain whether Zn [2,5,7,11] and Mn [1,8,9,12] are taken up through the blood-brain or blood-CSF barriers, although both the barriers for Zn and Mn uptake may not necessarily function in the same way in different brain region. In the present study, as an approach to understanding the Table 2 54 Mn distribution in rat brain Region
Cerebral cortex Hippocampal formation CAl+CA2 CA3+DG Thalamus Hypothalamic nuclei Red nucleus Interpeduncular nuclei Inferior colliculus Pontine reticular nuclei Olivary nuclei
i.c.v,
i.v.
Contra.
Ipsi.
191 ± 20
390 ± 35
177
371± 53 535± 48 489± 63 71O± 62 492 ± 59 764 ± 77 645 ± 85 747 ± 114 750 ± 102 833 ± 113 771 ± 98 604 ± 27 678 ± 22 819± 82 874± 65 61O± 81 654± 76
211 267 273 241 322 293 322 360 374
Each value (mean ± S.E.) represents the radioactivity (PSL/mm 2 ) in the brain of rats 6 days after i.c.v. injection. In the case of i.v. injection, each value represents a typical one (PSL/mm 2 ) . The radioactivity of each region indicated with surrounding lines in Fig. Ib was measured with a Bio-imaging Analyzer [13]. In the case of i.c.v. injection, Contra. and Ipsi. represent contralateral and ipsilateral hemispheres, respectively.
contribution of both barriers to the uptake of these trace metals, we used high resolution autoradiography for determining the localization of 65 Zn and 54 Mn in brain. The brain images of rats after injection into unilateral ventricle showed that both the tracers could enter extracellular fluid through the pial or ependymal surface and were relatively concentrated in circumventricular regions. There was, however, a difference in the regions which concentrated the two tracers. These results indicate that Zn and Mn are taken up gradually via CSF from the choroid plexus. In particular, the blood-CSF barrier may be involved in the uptake of both metals into circumventricular regions such as the hippocampal formation and hypothalamus. On the other hand, the finding that the uptake of 65 Zn into the cerebral cortex and superior colliculus after i.c.v. injection was relatively low in comparison with i.v. injection suggests that the blood-brain barrier may be relatively important for Zn uptake into those regions. Although the chemical forms of Zn and Mn in transport across both the barriers have not been clarified [4,6], the result of in vitro incubation with serum or CSF suggests that protein-unbound forms may be involved in the transport of both Zn [3,6] and Mn [9,12] as previously suggested by several researchers. [1] Aschner, M. and Aschner, J.L., Manganese transport across the blood-brain barrier: relationship to iron homeostasis, Brain Res. Bull., 24 (1990) 857-860. [2] Blair-West, J.R., Denton, D.A., Gibson, A.P. and McKinley, MJ., Opening the blood-brain barrier to zinc, Brain Res., 507 (1990) 6-10. [3] Buxani, S. and Adu, J., Histidine-stimulated 65 Zn transport at the BBB of the anesthetized rat, J Physiol., 438 (1991) 121. [4] Fishman, J.B., Rubin, J.B., Handrahan, J.V., Connor, J.R. and Fine, R.E., Receptor-mediated transcytosis of transferrin across the blood-brain barrier, J. Neurosci. Res., 18 (1987) 299-304. (5) Franklin, PA, Pullen, R.G.L. and Hall, G.H., Blood-brain exchange routes and distribution of 65 Z n in rat brain, Neurochem. Res., 17 (1992) 767-771. [6] Frederickson, CJ., Neurobiology of zinc and zinc-containing neurons, Int. Rev. Neurobiol., 31 (1990) 145-238. (7) Kasarskis, E.J., Zinc metabolism in normal and zinc-deficient rat brain, Exp. Neurol., 85 (1984) 114-127. (8) Kaur, G., Hasan, S.K. and Srivastava, R.C, The distribution of manganese-54 in fetal, young and adult rats, Toxieol. Leu., 5 (1980) 423-426. [9] Murphy, VA, Wadhwani, K.C, Smith, Q.R. and Rapoport, S.l., Saturable transport of manganese across the rat blood-brain barrier, J Neurochem., 57 (1991) 948-954. (10) Paxinos, G. and Watson, C, The Rat Brain, Academic Press, Inc., 1986. [11] Pullen, R.G.L., Franklin, PA and Hall, G.H. 65Z inc uptake from blood into brain and other tissues in the rat, Neurochem. Res., 10 (1990) 1003-1008. (12) Rabin, 0., Hegedus, L., Bourre, J.-M. and Smith, Q.R., Rapid brain uptake of manganesetll) across the blood-brain barrier, J. Neurochem., 61 (1993) 509-517. [13] Takeda, A., Akiyama, T., Sawashita, J. and Okada, S. Brain uptake of trace metals, zinc and manganese, in rats, Brain Res., 640 (1994) 341-344.