GABA-like immunoreactivity in nonspiking interneurons of the locust metathoracic ganglion
M. Wildman,
S. R. Ott
and
M. Burrows*
Department of Zoology, University of Cambridge, Cambridge CB2 3EJ,
UK
Present address: School of Biological Sciences, Queen Mary, University of
London, Mile End Road, London E1 4NS, UK
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Fig. 1. Location of the cell bodies of nonspiking interneurons in the metathoracic
ganglion analysed in this study. The interneurons were first characterized
physiologically by intracellular recordings from their neurites and were then
stained by intracellular injection of Lucifer Yellow. The ganglion is viewed
ventrally and the lateral nerves innervating the left hind leg and left hind
wing are numbered. All the stained interneurons affected motor neurons
innervating muscles in the left hind leg. The filled circles represent the 15
interneurons that were GABA-immunopositive, and the open circles represent the
two interneurons that were not GABA-immunopositive. The numbering of the cell
bodies refers to the figures in which these interneurons are illustrated.
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Fig. 2. Colocalisation of Lucifer-Yellow-fluorescence and
anti-GABA-immunofluorescence in a nonspiking local interneuron in the
metathoracic ganglion, demonstrated by confocal microscopy (all images are
from whole mounts of ganglia, viewed ventrally). (A) A single cell body
(arrow) in the anterior lateral region of the ganglion shows intense
fluorescence for Lucifer Yellow (in green pseudocolour in this and following
figures; slight background is due to tissue autofluorescence). (B) The same
cell body (arrow) shows intense fluorescence for Cy3 in the same optical
section, indicating binding of the GABA antibody (Cy3/GABA in red
pseudocolour). A number of other small cell bodies are also
GABA-immunopositive. A and B each show a composite of two confocal planes
separated by 3.1 µm. (C) A merging of the two images in A and B results in
a single yellow cell body, indicating colocalisation of the two fluorophores
in the interneuron. The surrounding large cell bodies belong to glutamatergic
motor neurons (five are indicated with asterisks) that showed no
immunoreactivity with the GABA antibody. (D) The morphology of the neuron as
revealed by the Lucifer Yellow dye (branches reconstructed from 29 confocal
planes, each separated by 5.4 µm; cell body from a single confocal plane 56
µm more ventral than the ventralmost plane used in the reconstruction of
the branches). A single primary neurite (arrow) emerges from the cell body to
give rise to a profusion of fine branches in the neuropil. The ganglion is
viewed ventrally with anterior to the top. Scale bars, 100 µm.
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Fig. 3. A GABA-positive nonspiking interneuron in the metathoracic ganglion whose
morphology can be directly related to previous descriptions; ventral view. (A)
The cell body of the interneuron (arrow) appears yellow when the
Lucifer-Yellow-fluorescence (green) and the Cy3/GABA-immunofluorescence (red)
are merged, indicating colocalization of the dyes. Combined from three
confocal planes each separated by 5.4 µm. (B) Morphology of the interneuron
as revealed by the Lucifer Yellow dye (reconstructed from 41 confocal planes
each separated by 5.4 µm). The primary neurite crosses the midline and then
forms an extensive array of fine branches in neuropil close to the
contralateral edge of the ganglion. A tracheole anterior to the cell body is
strongly autofluorescent. Scale bars, 100 µm.
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Fig. 4. Two nonspiking interneurons that do not show GABA-immunofluorescence. (A) A
nonspiking interneuron, viewed ventrally, with its cell body in the posterior
lateral group. The cell body (arrow) appears green after merging the
Lucifer-Yellow-fluorescence (green) and the Cy3/GABA-immunofluorescence (red),
indicating the absence of GABA immunoreactivity. A few small neighbouring cell
bodies are GABA-immunopositive (red). Two confocal planes each separated by
8.8 µm were combined to show the cell body, and seven further planes with
the same spacing of the green alone were combined to show the fine primary
neurite and neuropilar branches. Lateral nerve 5 (N5) is on the right. (B) The
green cell body of an anterior lateral interneuron, indicating that it is not
GABA-immunopositive (arrow); some other small cell bodies are red, indicating
that they are immunopositive. Combined from two confocal planes separated by
4.5 µm. (C) Injecting a pulse of hyperpolarizing current into the
interneuron shown in B causes an increase in the frequency of motor spikes
recorded in the flexor tibiae muscle but has little effect on spikes in the
slow extensor tibiae motor neuron or in an unidentified motor neuron in the
coxa. Scale bars in A and B, 100 µm.
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© The Company of Biologists Ltd 2002
