First published online 16 September 2003
doi: 10.1242/jcs.00762
Inhibitory neurons from fetal rat cerebral cortex exert delayed axon formation and active migration in vitro
Kensuke Hayashi1,*,
Rika Kawai-Hirai1,
Akihiro Harada1 and
Kuniaki Takata2
1 Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showamachi, Maebashi, Gunma 371-8512, Japan
2 Department of Anatomy and Cell Biology, Gunma University School of Medicine, 3-39-22 Showamachi, Maebashi, Gunma 371-8512, Japan
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Fig. 1. Properties of cortical neurons in a glia-free culture for 3 days. (A-C) Neurons were stained with antibodies to GABA, to phosphorylated neurofilaments (P-Nf) and to class III ß-tubulin (TUJ1), respectively. A GABA-positive neuron is indicated by the arrow in (A). GABA-negative neurons, as indicated by the arrowheads in (A), extended phosphorylated neurofilament-positive axons, as indicated by the arrowheads in (B). All neurons were reactive with the TUJ1 antibody. Bar, 100 µm. (D) Distribution of standardized values for GABA immunoreactivity of the cultured cells after 3 DIV. Striped bars represent neurons bearing a phosphorylated neurofilament-positive axon, open bars represent neurons without axons and solid bars represent nonneuronal cells. Most excitatory neurons (standardized GABA intensity <0.4) bore an axon, whereas most inhibitory neurons (standardized GABA intensity  0.4; shaded area) did not.
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Fig. 3. Time-lapse video microscopic analysis of neuronal behavior in glia-free culture. Neurons were monitored every 6 minutes for 6 days and then stained with antibodies to identify neuronal type. (A) An excitatory neuron extended several minor processes, one of which (arrow) began to elongate rapidly and to form an axon. (B) Two processes of an inhibitory neuron exhibited alternate growth and retraction without giving rise to an axon during the 6 days of recording. Note the movement of the cell body towards the growing process. (C) Two processes of an inhibitory neuron exhibited alternate growth and retraction without axon formation, but movement of the cell body was not detected. (D) An inhibitory neuron for which extensive movement of the cell body was apparent. The long process indicated by the arrows was revealed to be an axon by staining with antiphosphorylated neurofilaments (not shown). Time is shown in hours:minutes. Bar, 50 µm.
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Fig. 2. Properties of cortical neurons in a glia-free culture for 6 days (A and B) or after exposure to BDNF for 3 days (C-E). (A,B) Two neurons immunoreactive with anti-GABA (A) and TUJ1 (not shown) each possess a phosphorylated neurofilament-positive axon (B). (C,D) Cortical neurons in a glia-free culture in the presence of BDNF (5 µg/ml) for 3 days. Neurons were stained with antibodies to GABA (C) and to phosphorylated neurofilaments (D). The arrow in (C) indicates a GABA-positive neuron without an axon. The arrowhead in (D) indicates the highly branched axons of GABA-negative neurons. Bar, 100 µm. (E) Effect of BDNF on the intensity of GABA immunoreactivity in inhibitory neurons after 3 DIV. Data are means±s.d. for 36 neurons cultured with BDNF and 32 neurons cultured in its absence. *P<0.001 (Student's t-test).
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Fig. 4. Properties of cortical neurons cultured on a glial cell layer for 2 days. (A) An inhibitory neuron (arrow) revealed by anti-GABA staining. (B) Two excitatory neurons (arrows), each bearing an axon (arrowheads), were revealed by antiphosphorylated neurofilament staining. The antibodies to phosphorylated neurofilaments also stain nonspecifically the nuclei of cells including those of glia. Bar, 100 µm.
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Fig. 5. Time-lapse analysis of the migration of cortical neurons after culture on a glial cell layer for 3 days. (A) Histogram of the distance migrated by inhibitory (solid bars) and excitatory (open bars) neurons over a 10 hour period. (B) Images collected at 30 minute intervals of migrating inhibitory neurons. The neurons each possessed a leading process with a growth cone at the tip as well as a trailing process. Reversal of the direction of migration was accompanied by the disappearance of the growth cone from the original leading process and the appearance of a new growth cone at the tip of the previous trailing process. Bar, 20 µm.
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Fig. 6. Translocation of the centrosome and the Golgi apparatus during the reversal of migration in inhibitory neurons. Cortical neurons were cultured for 1 day on a glial cell layer. The migration of inhibitory neurons was then monitored for 120 minutes before staining of the centrosome with anti-pericentrin (green), of the Golgi apparatus with anti-Golgi 58K protein (red) and of the nucleus with DAPI (blue). (A) A neuron that migrated without reversal of direction for 120 minutes. The centrosome and Golgi apparatus were located at the base of the leading process (arrowhead). The arrow indicates the direction of migration. (B) A neuron that reversed direction during the last 60 minutes of recording. The centrosome and the Golgi apparatus were located at the rear side of the nucleus (arrowhead). Bars, 10 µm. (C) The position of the centrosome relative to that of the leading process in individual migrating inhibitory neurons is indicated by dots. The nucleus is represented by the large open circle and the site of origin of the leading process is indicated by the arrow. In about 75% of neurons, the centrosome was located at the front side of the nucleus. (D-F) The data shown in (C) were divided into three groups based on the time of reversal of the direction of migration. (D) Neurons that reversed their direction of migration during the last 60 minutes of recording. (E) Neurons that reversed direction during the first 60 minutes of recording but not thereafter. (F) Neurons that did not reverse the direction of movement during recording. The centrosome thus translocated to the base of the leading process after the reversal of migration.
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Fig. 7. Localization of the centrosome in axon-extending neurons. (A) Neurons cultured on a glial cell layer for 1 day were stained with antiphosphorylated neurofilaments (red), anti-pericentrin (green) and DAPI (blue). Centrosomes were located either at the base of the extending axon (left panel), at the side of the nucleus relative to the position of the axon (middle panel) or on the opposite side of the nucleus from the axon (right panel). Arrows indicate axons. Bar, 10 µm. (B) Summary of the position of the centrosome (dots) in 51 axon-extending neurons. The arrow represents the site of origin of the extending axon. There was no relation between the position of the centrosome and the site of axon extension.
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Fig. 8. Movement of intracellular organelles within the leading process of migrating neurons cultured on a glial cell layer. (A) The leading process of an inhibitory neuron (asterisk) moved beneath a process of an adjacent neuron (arrowheads). The images were collected at 10 minute intervals. The nucleus of the migrating neuron was not able to pass beneath the adjacent process; by contrast, cytoplasmic components moved into the leading process towards the growth cone, resulting in swelling (arrow) of the middle portion of the leading process. Bar, 20 µm. (B,C) Additional examples of neurons with a swelling (arrows) of the leading process. Immunostaining revealed the presence of the Golgi apparatus and the centrosome (arrowheads) within the swellings. Right-hand panels are merged images of staining with anti-pericentrin (green), anti-Golgi 58K protein (red) and DAPI (blue). Bar, 10 µm.
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Fig. 9. The proposed intrinsic developmental programs of excitatory and inhibitory neurons. In excitatory neurons, axon formation occurs soon after the emergence of minor processes. By contrast, inhibitory neurons undergo a migratory stage before axon formation. During this migratory stage, the centrosome is tethered to the leading process, whereas the centrosome is dissociated from the extending axon.
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© The Company of Biologists Ltd 2003
