- Email: [email protected]
TEM investigations of biogenic magnetite extracted from the human hippocampus
Biochimica et Biophysica Acta 1426 (1999) 212^216
Rapid report
TEM investigations of biogenic magnetite extracted from the human hippocampus Paola P. Schultheiss-Grassi a , Roland Wessiken b , Jon Dobson a
c;
*
Department of Physics, Institute of Geophysics, Swiss Federal Institute of Technology, ETH-Ho«nggerberg, CH-8093 Zurich, Switzerland b Department of Physics, Institute of Solid State Physics, Swiss Federal Institute of Technology, ETH-Ho«nggerberg, CH-8093 Zurich, Switzerland c Department of Physics, Biophysics Programme, University of Western Australia, Nedlands, W.A. 6907, Australia Received 3 September 1998; accepted 4 November 1998
Abstract Modification of chemical and magnetic extraction techniques has yielded biogenic magnetite/maghemite from human hippocampal tissue. Particles were identified using high resolution transmission electron microscopy, electron diffraction and elemental analysis. Though its presence has been inferred from magnetic analyses, this is the first direct observation of magnetic biominerals from the hippocampus. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Magnetite; Maghemite; Hippocampus; Transmission electron microscopy
Biological mineralisation of magnetic iron biominerals is known to occur in a wide variety of organisms, yet studies of the complete process and its relationship to the overall biology of particular organisms are few in number [1^6]. To achieve a complete description requires a multi-disciplinary approach. The general aim of many detailed bioinorganic studies is a better understanding of how mineralisation can be related to the function of the mineralising organism. Biomineralised iron oxides have been reported in a wide variety of living organisms, including humans (e.g. [4,7]), indicating that biomineralisation of iron is a widespread phenomenon. Although considerable research has been done on biomineralisation in bacteria and animals, much less
* Corresponding author. Fax: +61 (8) 9380 1014; E-mail: [email protected]
is known about this phenomenon in humans. The recent discovery of the presence of magnetite (Fe3 O4 ) in tissue samples resected from several human organs has led to much speculation on distribution and role of this material in the central nervous system as well as other parts of the body [7^11]. In order to con¢rm earlier reports of electron microscope investigation of magnetic particles extracted from human brain tissue [7] and to determine whether this material is present in the human hippocampus as suggested by magnetic analyses [12^14], a novel chemical and magnetic extraction method was developed based on earlier studies (e.g. [7]). Magnetic extracts obtained by this method were examined using high resolution transmission electron microscopy (HRTEM), electron di¡raction and energy dispersive X-ray analysis (EDXA). Magnetic particles were extracted from a hippocampal tissue sample resected from a cadaver (96/
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 6 0 - 3
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P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216
213
Fig. 1. Transmission electron micrograph of magnetite and/or maghemite particles extracted from a human hippocampus (96/624). Well de¢ned crystal faces can be seen in some particles.
624) without chemical ¢xation, sealed in an acidwashed vial and submerged in liquid nitrogen to preserve brain chemistry. Previous magnetic analysis of the same sample indicated the presence of low coercivity magnetic material [15]. One of the main di¤culties with magnetic particle extraction from human brain tissue arises due to the ¢ne particle sizes (generally in the order of 100 nm or less). Conventionally, ¢ne particle magnetic mineral extraction techniques rely on the generation of a strong magnetic ¢eld gradient, within which the magnetic grains, obtained by crushing the samples, are trapped [16]. A modi¢cation of this method was adapted for use with this tissue. A solution of one part of ethanol together with one part of ether was prepared in order to remove water and lipid molecules from the tissue samples. The specimen was chopped with a HCl-cleaned, ceramic knife (which is non-magnetic) and placed in a test tube together with the 1:1 ethanol/ether solution. Using a quartz glass pestle, previously cleaned with hydrochloric acid, the tissue was crushed. The tissue was then left in the solution for two hours and subsequently centrifuged at 4000 rpm for 30 min. The water and lipids suspended in the ethanol/
ether solution were removed, along with the solution, using a glass pipette. A fresh solution was added, this time with an ethanol/ether ratio of 1:2. The procedure then was repeated. The third and last time, the solution was prepared with one part of ethanol and 3 parts of ether. The tissue samples then were covered and allowed to dry before being crushed with a quartz glass pestle. Eight rare earth magnets were used to extract magnetite particles adopting the method described by Ariztegui and Dobson [17]. The extracted particles were dispersed ultrasonically in double-distilled water and a drop of the suspension was air-dried onto carbon-coated copper grids. In order to prevent airborne contamination while drying, the grid with the drop of suspension was covered with a petri dish. Several grids were prepared using this method. Transmission electron microscopy (TEM) studies of particles were performed using a high resolution transmission electron microscope (HRTEM) Philips CM30ST with a lanthanum hexaboride (LaB6) cathode operating at an accelerating voltage of 300 keV. This instrument was equipped with an energy dispersive X-ray analyser, capable of detecting all elements with atomic number greater than 5 (Boron).
BBAGEN 20353 18-12-98
214
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216
Fig. 2. Transmission electron micrograph of magnetite and/or maghemite particles extracted from a human hippocampus (96/624) showing some particle dissolution at the edges.
Three copper TEM grids which contained di¡erent concentrations of extracted particles were analysed. Small amounts of electron-dense, crystalline particles were found in each grid. Most of the particles were partially covered with organic material. Particle morphologies and sizes compared well with previously reported TEM observations of magnetic extracts from other regions of the brain [7] and were in the stable, single-magnetic-domain size range (Fig. 1). In some of the observed particles, the edges appeared partially dissolved as illustrated by a variation in electron density (Fig. 2). Elemental composition of the particles was investigated with an energy dispersive X-ray analyser (EDXA). All analysed electron-dense particles exhibited an oxygen peak and an iron peak (Fig. 3a). The copper peak present in the spectrum is due to the grids used for the analyses. The Ca peak is likely due to organics, however, the origin of the Mg peak seen in one of the spectra is not known. Electron di¡raction was performed (after perform-
ing a camera length calibration with gold foil) on the particles and a single crystal di¡raction pattern indicated that the particles are cubic and crystalline (Fig. 3b). The measured d-spacings are consistent with magnetite and/or maghemite. EDX analysis of the extracted particles shows that both iron and oxygen are present and electron diffraction indicates that the particles are crystals of magnetite and/or maghemite. These results are in agreement with magnetic analyses of the same tissue samples, which indicate the likely presence of biogenic magnetite and/or maghemite [15]. Kirschvink et al. ¢rst reported the presence of magnetite and maghemite in human brain tissue in 1992 [7]. The authors also reported what seemed to be dissolution e¡ects along the edges of the crystals and assumed that this implies a yet unknown role in eukaryotic biochemistry. Similar dissolution e¡ects are seen in the particles extracted from the human hippocampus, however, in addition to the partially dissolved particles, several magnetite/maghemite par-
BBAGEN 20353 18-12-98
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216
215
for motor function) were conclusively identi¢ed by electron di¡raction and EDX analysis [7]. This study presents the ¢rst TEM observations of biogenic magnetite and maghemite from the human hippocampus ^ an important structure in the brain responsible for memory, learning and sensory processing which has previously been demonstrated to be sensitive to applied magnetic ¢elds [12,18]. The authors would like to thank Dr. M.A. Klein and Prof. H.G. Wieser for the tissue samples and Prof. William Lowrie for helpful discussion. This work was approved by the University Hospital-Zurich Ethics Commission and was supported by Swiss National Funds Grant No. 3100-042438.94 and The University of Western Australia.
References
Fig. 3. a: EDXA spectrum of particle cluster from Fig. 1; and b: electron di¡raction pattern of particle in Fig. 1 taken along the [0,1,1] axis.
ticles with very well de¢ned crystal faces are observed. In the Kirschvink et al. study [7], TEM micrographs of particles extracted from the human cerebellum (a region of the brain primarily responsible
[1] R.B. Frankel, Magnetic guidance of organisms, Annu. Rev. Biophys. Bioeng. 13 (1984) 85^103. [2] M.M. Walker, M.E. Bitterman, Honeybees can be trained to respond to very small changes in geomagnetic ¢eld intensity, J. Exp. Biol. 145 (1989) 489^494. [3] T.G. St. Pierre, J. Webb, S. Mann, Ferritin and hemosiderin: Structural and magnetic studies of the iron core. In: S. Mann, J. Webb, R.J.P. Williams (Eds.), Biomineralization: Chemical and Biochemical Perspectives, VCH, Weinheim, 1989, pp. 295^344. [4] J. Webb, T.G. St. Pierre, D.J. Macey, Iron biomineralization in invertebrates. In: R.B. Frankel, R. Blakemore (Eds.) Iron Biominerals, Plenum, New York, NY, 1990, pp. 193^ 220. [5] J.L. Kirschvink, S. Padmanabha, C.K. Boyce, J. Oglesby, Measurement of the threshold sensitivity of honeybees to weak, extremely low frequency magnetic ¢elds, J. Exp. Biol. 200 (1997) 1363^1368. [6] M.M. Walker, C.E. Diebel, C.V. Haugh, P.M. Pankhurst, J.C. Montgomery, C.R. Green, Structure and function of the vertebrate magnetic sense, Nature 390 (1997) 371^376. [7] J.L. Kirschvink, A. Kobayashi-Kirschvink, B.J. Woodford, Magnetite biomineralization in the human brain, Proc. Natl. Acad. Sci. USA 89 (1992) 7683^7687. [8] J.L. Kirschvink, Comments on `Constraints on biological e¡ects of weak extremely-low-frequency electromagnetic ¢elds', Phys. Rev. A 46 (1992) 2178^2184. [9] J.L. Kirschvink, Microwave absorption by magnetite: A possible mechanism for coupling non-thermal levels of radiation to biological systems, Bioelectromagnetism 17 (1996) 187^194. [10] J. Dobson, T.G. St. Pierre, Application of the ferromagnetic transduction model to D.C. and pulsed magnetic ¢elds: effects on epileptogenic tissue and implications for cellular
BBAGEN 20353 18-12-98
216
[11]
[12]
[13]
[14]
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216 phone safety, Biochem. Biophys. Res. Commun. 227 (1996) 718^723. P.P. Schultheiss-Grassi, F. Heller, J. Dobson, Analysis of magnetic material in the human heart, spleen and liver, BioMetals 10 (1997) 351^355. J. Dobson, M. Fuller, S. Moser, H.G. Wieser, J.R. Dunn, J. Zoeger, Evocation of epileptiform activity by weak D.C. magnetic ¢elds and iron biomineralization in the human brain. In: C. Baumgartner, L. Deecke, G. Stroink, S.J. Williamson (Eds.) Biomagnetism : Fundamental Research and Applications, Elsevier, Amsterdam, 1995, pp. 16^19. J.R. Dunn, M. Fuller, J. Zoeger, J. Dobson, F. Heller, E. Caine, B.M. Moskowitz, Magnetic material in the human hippocampus, Brain Res. Bull. 36 (1995) 149^153. J. Dobson, P. Grassi, Magnetic properties of human hippo-
[15] [16]
[17]
[18]
campal tissue: Evaluation of artefact and contamination sources, Brain Res. Bull. 39 (1996) 255^259. P.P. Schultheiss-Grassi, J. Dobson, Magnetic analysis of human brain tissue, BioMetals (1998), in press. D.G. Schulze, J.B. Dixon, High gradient magnetic separation of iron oxides and other magnetic minerals from soil clays, Soil Sci. Soc. Am. J. 43 (1979) 795^799. D. Ariztegui, J. Dobson, Magnetic investigations of framboidal greigite formation: A record of anthropogenic environmental changes in eutrophic Lake St Moritz, Switzerland, Holocene 6 (1996) 104^110. M.D. Fuller, J. Dobson, H.G. Wieser, S. Moser, On the sensitivity of the human brain to magnetic ¢elds: Evocation of epileptiform activity, Brain Res. Bull. 36 (1995) 155^ 159.
BBAGEN 20353 18-12-98
Rapid report
TEM investigations of biogenic magnetite extracted from the human hippocampus Paola P. Schultheiss-Grassi a , Roland Wessiken b , Jon Dobson a
c;
*
Department of Physics, Institute of Geophysics, Swiss Federal Institute of Technology, ETH-Ho«nggerberg, CH-8093 Zurich, Switzerland b Department of Physics, Institute of Solid State Physics, Swiss Federal Institute of Technology, ETH-Ho«nggerberg, CH-8093 Zurich, Switzerland c Department of Physics, Biophysics Programme, University of Western Australia, Nedlands, W.A. 6907, Australia Received 3 September 1998; accepted 4 November 1998
Abstract Modification of chemical and magnetic extraction techniques has yielded biogenic magnetite/maghemite from human hippocampal tissue. Particles were identified using high resolution transmission electron microscopy, electron diffraction and elemental analysis. Though its presence has been inferred from magnetic analyses, this is the first direct observation of magnetic biominerals from the hippocampus. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Magnetite; Maghemite; Hippocampus; Transmission electron microscopy
Biological mineralisation of magnetic iron biominerals is known to occur in a wide variety of organisms, yet studies of the complete process and its relationship to the overall biology of particular organisms are few in number [1^6]. To achieve a complete description requires a multi-disciplinary approach. The general aim of many detailed bioinorganic studies is a better understanding of how mineralisation can be related to the function of the mineralising organism. Biomineralised iron oxides have been reported in a wide variety of living organisms, including humans (e.g. [4,7]), indicating that biomineralisation of iron is a widespread phenomenon. Although considerable research has been done on biomineralisation in bacteria and animals, much less
* Corresponding author. Fax: +61 (8) 9380 1014; E-mail: [email protected]
is known about this phenomenon in humans. The recent discovery of the presence of magnetite (Fe3 O4 ) in tissue samples resected from several human organs has led to much speculation on distribution and role of this material in the central nervous system as well as other parts of the body [7^11]. In order to con¢rm earlier reports of electron microscope investigation of magnetic particles extracted from human brain tissue [7] and to determine whether this material is present in the human hippocampus as suggested by magnetic analyses [12^14], a novel chemical and magnetic extraction method was developed based on earlier studies (e.g. [7]). Magnetic extracts obtained by this method were examined using high resolution transmission electron microscopy (HRTEM), electron di¡raction and energy dispersive X-ray analysis (EDXA). Magnetic particles were extracted from a hippocampal tissue sample resected from a cadaver (96/
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 6 0 - 3
BBAGEN 20353 18-12-98
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216
213
Fig. 1. Transmission electron micrograph of magnetite and/or maghemite particles extracted from a human hippocampus (96/624). Well de¢ned crystal faces can be seen in some particles.
624) without chemical ¢xation, sealed in an acidwashed vial and submerged in liquid nitrogen to preserve brain chemistry. Previous magnetic analysis of the same sample indicated the presence of low coercivity magnetic material [15]. One of the main di¤culties with magnetic particle extraction from human brain tissue arises due to the ¢ne particle sizes (generally in the order of 100 nm or less). Conventionally, ¢ne particle magnetic mineral extraction techniques rely on the generation of a strong magnetic ¢eld gradient, within which the magnetic grains, obtained by crushing the samples, are trapped [16]. A modi¢cation of this method was adapted for use with this tissue. A solution of one part of ethanol together with one part of ether was prepared in order to remove water and lipid molecules from the tissue samples. The specimen was chopped with a HCl-cleaned, ceramic knife (which is non-magnetic) and placed in a test tube together with the 1:1 ethanol/ether solution. Using a quartz glass pestle, previously cleaned with hydrochloric acid, the tissue was crushed. The tissue was then left in the solution for two hours and subsequently centrifuged at 4000 rpm for 30 min. The water and lipids suspended in the ethanol/
ether solution were removed, along with the solution, using a glass pipette. A fresh solution was added, this time with an ethanol/ether ratio of 1:2. The procedure then was repeated. The third and last time, the solution was prepared with one part of ethanol and 3 parts of ether. The tissue samples then were covered and allowed to dry before being crushed with a quartz glass pestle. Eight rare earth magnets were used to extract magnetite particles adopting the method described by Ariztegui and Dobson [17]. The extracted particles were dispersed ultrasonically in double-distilled water and a drop of the suspension was air-dried onto carbon-coated copper grids. In order to prevent airborne contamination while drying, the grid with the drop of suspension was covered with a petri dish. Several grids were prepared using this method. Transmission electron microscopy (TEM) studies of particles were performed using a high resolution transmission electron microscope (HRTEM) Philips CM30ST with a lanthanum hexaboride (LaB6) cathode operating at an accelerating voltage of 300 keV. This instrument was equipped with an energy dispersive X-ray analyser, capable of detecting all elements with atomic number greater than 5 (Boron).
BBAGEN 20353 18-12-98
214
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216
Fig. 2. Transmission electron micrograph of magnetite and/or maghemite particles extracted from a human hippocampus (96/624) showing some particle dissolution at the edges.
Three copper TEM grids which contained di¡erent concentrations of extracted particles were analysed. Small amounts of electron-dense, crystalline particles were found in each grid. Most of the particles were partially covered with organic material. Particle morphologies and sizes compared well with previously reported TEM observations of magnetic extracts from other regions of the brain [7] and were in the stable, single-magnetic-domain size range (Fig. 1). In some of the observed particles, the edges appeared partially dissolved as illustrated by a variation in electron density (Fig. 2). Elemental composition of the particles was investigated with an energy dispersive X-ray analyser (EDXA). All analysed electron-dense particles exhibited an oxygen peak and an iron peak (Fig. 3a). The copper peak present in the spectrum is due to the grids used for the analyses. The Ca peak is likely due to organics, however, the origin of the Mg peak seen in one of the spectra is not known. Electron di¡raction was performed (after perform-
ing a camera length calibration with gold foil) on the particles and a single crystal di¡raction pattern indicated that the particles are cubic and crystalline (Fig. 3b). The measured d-spacings are consistent with magnetite and/or maghemite. EDX analysis of the extracted particles shows that both iron and oxygen are present and electron diffraction indicates that the particles are crystals of magnetite and/or maghemite. These results are in agreement with magnetic analyses of the same tissue samples, which indicate the likely presence of biogenic magnetite and/or maghemite [15]. Kirschvink et al. ¢rst reported the presence of magnetite and maghemite in human brain tissue in 1992 [7]. The authors also reported what seemed to be dissolution e¡ects along the edges of the crystals and assumed that this implies a yet unknown role in eukaryotic biochemistry. Similar dissolution e¡ects are seen in the particles extracted from the human hippocampus, however, in addition to the partially dissolved particles, several magnetite/maghemite par-
BBAGEN 20353 18-12-98
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216
215
for motor function) were conclusively identi¢ed by electron di¡raction and EDX analysis [7]. This study presents the ¢rst TEM observations of biogenic magnetite and maghemite from the human hippocampus ^ an important structure in the brain responsible for memory, learning and sensory processing which has previously been demonstrated to be sensitive to applied magnetic ¢elds [12,18]. The authors would like to thank Dr. M.A. Klein and Prof. H.G. Wieser for the tissue samples and Prof. William Lowrie for helpful discussion. This work was approved by the University Hospital-Zurich Ethics Commission and was supported by Swiss National Funds Grant No. 3100-042438.94 and The University of Western Australia.
References
Fig. 3. a: EDXA spectrum of particle cluster from Fig. 1; and b: electron di¡raction pattern of particle in Fig. 1 taken along the [0,1,1] axis.
ticles with very well de¢ned crystal faces are observed. In the Kirschvink et al. study [7], TEM micrographs of particles extracted from the human cerebellum (a region of the brain primarily responsible
[1] R.B. Frankel, Magnetic guidance of organisms, Annu. Rev. Biophys. Bioeng. 13 (1984) 85^103. [2] M.M. Walker, M.E. Bitterman, Honeybees can be trained to respond to very small changes in geomagnetic ¢eld intensity, J. Exp. Biol. 145 (1989) 489^494. [3] T.G. St. Pierre, J. Webb, S. Mann, Ferritin and hemosiderin: Structural and magnetic studies of the iron core. In: S. Mann, J. Webb, R.J.P. Williams (Eds.), Biomineralization: Chemical and Biochemical Perspectives, VCH, Weinheim, 1989, pp. 295^344. [4] J. Webb, T.G. St. Pierre, D.J. Macey, Iron biomineralization in invertebrates. In: R.B. Frankel, R. Blakemore (Eds.) Iron Biominerals, Plenum, New York, NY, 1990, pp. 193^ 220. [5] J.L. Kirschvink, S. Padmanabha, C.K. Boyce, J. Oglesby, Measurement of the threshold sensitivity of honeybees to weak, extremely low frequency magnetic ¢elds, J. Exp. Biol. 200 (1997) 1363^1368. [6] M.M. Walker, C.E. Diebel, C.V. Haugh, P.M. Pankhurst, J.C. Montgomery, C.R. Green, Structure and function of the vertebrate magnetic sense, Nature 390 (1997) 371^376. [7] J.L. Kirschvink, A. Kobayashi-Kirschvink, B.J. Woodford, Magnetite biomineralization in the human brain, Proc. Natl. Acad. Sci. USA 89 (1992) 7683^7687. [8] J.L. Kirschvink, Comments on `Constraints on biological e¡ects of weak extremely-low-frequency electromagnetic ¢elds', Phys. Rev. A 46 (1992) 2178^2184. [9] J.L. Kirschvink, Microwave absorption by magnetite: A possible mechanism for coupling non-thermal levels of radiation to biological systems, Bioelectromagnetism 17 (1996) 187^194. [10] J. Dobson, T.G. St. Pierre, Application of the ferromagnetic transduction model to D.C. and pulsed magnetic ¢elds: effects on epileptogenic tissue and implications for cellular
BBAGEN 20353 18-12-98
216
[11]
[12]
[13]
[14]
P.P. Schultheiss-Grassi et al. / Biochimica et Biophysica Acta 1426 (1999) 212^216 phone safety, Biochem. Biophys. Res. Commun. 227 (1996) 718^723. P.P. Schultheiss-Grassi, F. Heller, J. Dobson, Analysis of magnetic material in the human heart, spleen and liver, BioMetals 10 (1997) 351^355. J. Dobson, M. Fuller, S. Moser, H.G. Wieser, J.R. Dunn, J. Zoeger, Evocation of epileptiform activity by weak D.C. magnetic ¢elds and iron biomineralization in the human brain. In: C. Baumgartner, L. Deecke, G. Stroink, S.J. Williamson (Eds.) Biomagnetism : Fundamental Research and Applications, Elsevier, Amsterdam, 1995, pp. 16^19. J.R. Dunn, M. Fuller, J. Zoeger, J. Dobson, F. Heller, E. Caine, B.M. Moskowitz, Magnetic material in the human hippocampus, Brain Res. Bull. 36 (1995) 149^153. J. Dobson, P. Grassi, Magnetic properties of human hippo-
[15] [16]
[17]
[18]
campal tissue: Evaluation of artefact and contamination sources, Brain Res. Bull. 39 (1996) 255^259. P.P. Schultheiss-Grassi, J. Dobson, Magnetic analysis of human brain tissue, BioMetals (1998), in press. D.G. Schulze, J.B. Dixon, High gradient magnetic separation of iron oxides and other magnetic minerals from soil clays, Soil Sci. Soc. Am. J. 43 (1979) 795^799. D. Ariztegui, J. Dobson, Magnetic investigations of framboidal greigite formation: A record of anthropogenic environmental changes in eutrophic Lake St Moritz, Switzerland, Holocene 6 (1996) 104^110. M.D. Fuller, J. Dobson, H.G. Wieser, S. Moser, On the sensitivity of the human brain to magnetic ¢elds: Evocation of epileptiform activity, Brain Res. Bull. 36 (1995) 155^ 159.
BBAGEN 20353 18-12-98