Cellular distribution of a high-affinity glutamate transporter in the nervous system of the cabbage looper Trichoplusia ni
Richard B. Gardiner1,
Kyrre Ullensvang2,
Niels C. Danbolt2,
Stanley Caveney3 and
B. Cameron Donly1,*
1 Southern Crop Protection and Food Research Centre, Agriculture and
Agri-Food Canada, London, Ontario, Canada N5V 4T3
2 Anatomical Institute, IMBA, University of Oslo, N-0317 Oslo,
Norway
3 Department of Zoology, The University of Western Ontario, London, Ontario,
Canada N6A 5B7
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Fig. 1. Immunoblots (western blots) of crude protein extracts of High Five (H5)
cells expressing TrnEAAT1 and non-expressing control cells probed with
antibodies to the C-terminal (A) and N-terminal (B) peptide. The two
antibodies produced similar patterns in the membrane fractions and failed to
detect bands in the lanes containing the water-soluble fractions. Lanes: 1,
H5-infected soluble fraction; 2, H5-infected membrane fraction; 3, H5 control
water-soluble fraction; 4, H5 control membrane fraction. The positions of
marker proteins (kDa) are shown.
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Fig. 2. Immunoblots (western blots) of caterpillar and moth tissue samples probed
with the N- and C-terminal-specific TrnEAAT1 antibodies. (A) Tissue extracts
probed with the antibody to the C-terminal peptide; (B) extracts probed with
antibody to the N-terminal peptide. Lanes: 1, moth head membrane fraction; 2,
caterpillar skeletal muscle membrane fraction; 3, moth head water-soluble
fraction; 4, caterpillar skeletal muscle water-soluble fraction. The
transporter bands are detected in the membrane fractions as in
Fig. 1, and the positions of
marker proteins (kDa) are shown.
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Fig. 3. Whole-mount view of an abdominal segment of a caterpillar stained with the
TrnEAAT1 C-terminal antibody. A branched network of nerves found on the
surface of the skeletal muscle fibres labelled strongly with this antibody.
The stained projections of these nerves do not appear to cross the segmental
borders. The only other structures labelled by the antibody were the ganglia.
FB, fat body; G, ganglion; NC, nerve cord; SB, segmental boundary; SM,
skeletal muscle. The tissue was probed with antibody directed to the
C-terminal region of the transporter protein. A matching pattern was produced
by the N-terminal antibody in separate preparations (not shown). Bound
antibodies were visualized with the alkaline phosphatase substrates BCIP/NBT.
Bar, 1 mm.
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Fig. 4. Isolated larval skeletal muscle fibre stained with anti-C-terminal TrnEAAT1
antibody showing a complex pattern of neuronal innervation. A portion of
trachea (T) is filled with air, with axon branches running over and under it.
The semi-transparent nature of the preparation reveals that the branching
continues around the entire surface of the muscle fibre. The fibre was stained
with the alkaline phosphatase substrate Vector Blue. N, motor neuron; SB,
segmental boundary. Bar, 500 µm.
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Fig. 5. (A) Methylene Blue stained preparation showing the pattern of nerve
innervation on a portion of caterpillar skeletal muscle. (B) Portion of larval
muscle, probed with anti-C-terminal antibody and Vector Red staining, showing
a motor neuron running alongside and passing under a tracheal segment on the
muscle surface. Note the similarity to the Methylene Blue preparation. A,
axon; M, muscle; MN, motor neuron; T, trachea. Bar, 100 µm.
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Fig. 6. High magnification view of the surface of a muscle fibre showing an
unstained axon that consequently appears light in density, surrounded by
heavily stained glial cells. Darkly stained plaques can be seen along the
axon. The outer perimeter (arrow, G) of the glial cells is lightly stained.
The stain is Vector Red. A, axon; M, muscle fibre; N, muscle nucleus; P,
plaque. Bar, 10 µm.
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Fig. 7. Electron micrograph of a portion of a glial cell on the surface of a muscle
fibre following TrnEAAT1 immunogold labelling. The gold particles are on the
cytoplasmic side of the cell membrane where both the N- and C-terminal regions
of the transporter are located. G, glial cell; RS, rete synapticum. Bar, 1
µm. (Inset) Cross-section (light micrograph) through a muscle fibre showing
darkly stained glial cells (arrowheads) that insulate the axons (unstained)
from the haemolymph (note similarity to electron micrograph). Tissue was
probed with the glutamate transporter C-terminal antibody and visualized with
secondary antibody-gold enhanced with silver (tissue stained with Toluidine
Blue). MF, muscle fibre. Bar, 100 µm.
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Fig. 8. Electron micrograph of an axon associated with glial cells on a muscle
fibre. The glial cell membranes are gold-labelled on cytoplasmic faces. The
axon plasma membrane is unlabelled. A, axon; G, glial cell. Bar, 1 µm.
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Fig. 9. A caterpillar abdominal ganglion showing dense staining of the inner
neuropile (Np). The pattern produced was of a diffuse staining with no cell
bodies evident. No staining is evident in the connectives (C), cortex (Co), or
nerve branches (N). (BCIP/NBT staining). Bar, 100 µm.
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Fig. 10. Electron micrograph of ganglionic neuropile stained with gold-labelled
secondary antibody to TrnEAAT1 C-terminal antibody. The glial membranes
(arrows) are labeled with gold, with only background labeling evident in nerve
cells. N, nerve. Bar, 0.5 µm.
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© The Company of Biologists Ltd 2002
