GABA-like immunoreactivity in nonspiking interneurons of the locust metathoracic ganglion
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
* Author for correspondence (e-mail: mb135{at}hermes.cam.ac.uk)
Accepted 22 August 2002
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: GABA, immunohistochemistry, premotor interneurons, motor control, locust, Schistocerca gregaria
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the thoracic motor networks of insects, nonspiking interneurons mediate
powerful effects on postsynaptic neurons by the graded release of chemical
transmitter (Burrows and Siegler,
1978). Both inhibitory and excitatory effects on motor neurons
occur (Burrows and Siegler,
1976
,
1978
), but all connections
between interneurons appear to be inhibitory
(Burrows, 1979a
;
Burrows, 1987
). Many nonspiking
neurons release transmitter tonically, so that single synaptic potentials are
sufficient to modulate this graded release
(Burrows, 1979b
). Despite the
extensive studies on nonspiking interneurons in these networks, the identity
of their transmitter(s) remains unknown. GABA (
-aminobutyric acid) is
extensively associated with inhibition and is widely distributed in insect
thoracic ganglia (Watson,
1986
; Watson and Pflüger,
1987
). Three common inhibitory motor neurons that innervate leg
muscles (Hale and Burrows,
1985
; Hoyle, 1966
;
Pearson and Bergman, 1969
)
show GABA-like immunoreactivity (Watson,
1986
), and GABA is used by these neurons as an inhibitory
transmitter in the muscles (Usherwood and
Grundfest, 1965
). GABA is also present in one population of
spiking local interneurons that have inhibitory actions on nonspiking
interneurons and motor neurons (Burrows,
1987
; Burrows and Siegler,
1982
; Watson and Burrows,
1987
), in a population of intersegmental interneurons
(Watson and Laurent, 1990
) and
in unidentified interneurons that mediate presynaptic inhibition of the
central terminals of proprioceptors
(Burrows and Laurent, 1993
;
Watson et al., 1993
) and
exteroceptors (Watson and Pflüger,
1994
). In the terminal abdominal ganglion of the crayfish, an
identified nonspiking interneuron that inhibits other spiking interneurons
(Nagayama et al., 1994
;
Reichert et al., 1983
) is
immunoreactive for GABA (Nagayama et al.,
1996
), as are some other nonspiking interneurons
(Nagayama et al., 1997
).
In the present paper, we show, by intracellular dye injection into physiologically characterized nonspiking interneurons in a locust thoracic ganglion and subsequent immunohistochemistry with an antibody raised against GABA, that many of these interneurons are GABA-immunopositive. The diversity of these local nonspiking interneurons is, however, emphasized by the finding that not all of them show GABA immunoreactivity.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrophysiology and intracellular dye injection
Intracellular recordings from the neuropilar processes of nonspiking local
interneurons were made in the left half of the metathoracic ganglion with
thin-walled glass microelectrodes, filled at their tips with a 5% aqueous
solution of Lucifer Yellow and in their shanks with 0.5 mol l-1
lithium chloride so that they had resistances of approximately 25 M.
Interneurons were identified as nonspiking according to established criteria
(Burrows and Siegler, 1978
). In
short, an interneuron was considered to be nonspiking if (1) it was never
observed to spike in response to any injury inflicted by the initial
penetration of the microelectrode, to mechanosensory stimulation of
exteroceptors or proprioceptors of the legs, to injection of depolarizing
current, or on rebound from a hyperpolarizing current pulse and (2) if
depolarizing and/or hyperpolarizing injection of current produced motor
effects without the occurrence of spikes in the interneuron. Motor effects
were monitored by observing movements of the left hind leg and by recording
from selected leg muscles with implanted pairs of fine steel pins or 50
µm-diameter silver wire, insulated except for the tips.
Following physiological characterization, an interneuron was filled with
Lucifer Yellow by the application of hyperpolarizing current pulses (-7 nA,
500 ms duration at 1 Hz) for 20-30 min, superimposed on a constant current of
-2 nA. The mesothoracic and metathoracic ganglia were then removed from the
locust and fixed for 30 min at 5°C in 4% paraformaldehyde and 0.1%
gluteraldehyde in 0.1 mol l-1 phosphate buffer (PB; pH 7.2)
(Nagayama et al., 1996). They
were postfixed overnight at 5°C in 2% paraformaldehyde and 15% picric acid
in PB and then washed in two changes of PB, each for 10 min.
GABA immunohistochemistry in intact ganglia
To improve penetration of the antibody, the ganglia were dehydrated through
a graded ethanol series (10 min each in 30%, 50% and 70% ethanol) and stored
for 1-5 days in 70% ethanol. They were then placed in 90% ethanol for 1 h,
rehydrated through the same series of ethanol and washed three times, each for
10 min, in PB at 37°C. The permeability of the ganglionic sheath was
improved by treatment with 0.1% collagenase (Sigma type IV) and 0.1%
hyaluronidase (Sigma type I-S) in PB for 1 h at 37°C, followed by three
washes, each for 10 min, in PB and a further 10 min wash in PB containing 0.3%
Triton X-100 (PB-Tx). Non-specific antibody binding was blocked by
pre-incubation with 5% normal goat serum in PB-Tx (PB-Tx-NGS) for 6 h. The
ganglia were then incubated with a polyclonal anti-GABA primary antibody
(Sigma, Poole, UK; product number A2052; diluted 1:750 in PB-Tx-NGS) for
approximately 90 h at 5°C on a rotator. The antibody was raised in rabbit
using a GABABSA (bovine serum albumin) conjugate as the immunogen. To
remove immunoglobulins that do not specifically bind to GABA the antibody was
affinity-purified. Dot blot assays show positive binding with GABA and
GABA-keyhole limpet haemocyanin conjugate but not with BSA. This antibody has
been used extensively in previous studies on locust (Schistocerca
gregaria; Judge and Leitch,
1999; Seidel and Bicker,
1997
), bee (Apis mellifera;
Ganeshina and Menzel, 2001
),
crayfish (Procambarus clarkii;
Pearlstein et al., 1998
;
Watson et al., 2000
) and crab
(Cancer borealis; Kilman and
Marder, 1996
; Swensen et al.,
2000
), in each of which the specificity of the antibody was
established. Following three washes in PB-Tx, each for 2 h, the ganglia were
incubated for approximately 42 h at 5°C on a rotator in Cy3-conjugated
secondary antibody [`affinity-purified, cyanine fluorophore Cy3-conjugated
goat anti-rabbit immunoglobulin G (IgG) (H+L)', where H+L refers to whole
antibody molecules with full-length heavy and light chains; Jackson
ImmunoResearch Laboratories, West Grove, PA, USA] diluted 1:200 in PB-Tx-NGS.
Finally, ganglia were washed in three changes of PB-Tx, each for 2 h,
dehydrated through an ethanol series (30%, 50%, 70%, 90% and two changes of
100%, each for 10 min), and cleared and mounted in methyl salicylate.
The ganglia were viewed under a Leica DMR confocal microscope running Leica TCS NT software (Leica, Nussloch, Germany). Observation conditions that avoid cross-talk between the Lucifer Yellow and the Cy3 fluorescence signal were established in control preparations (see below). The excitation wavelength was set to 488 nm for Lucifer Yellow and 568 nm for Cy3, and the detector range was set to 500-530 nm for Lucifer Yellow and 550-600 nm for Cy3. All images presented are from optical confocal sections of whole ganglia. Images from different focal planes were stacked as layers and combined in Photoshop 5.5 (Adobe Systems Inc., Mountain View, CA, USA) or in the public domain software NIH-Image (U.S. National Institutes of Health; http://rsb.info.nih.gov/nih-image/index.html).
GABA immunohistochemistry in frozen sections
Eight ganglia containing a Lucifer-Yellow-injected interneuron were
sectioned rather than treated as whole mounts. After fixation as above,
ganglia were cryoprotected overnight at 5°C in 20% sucrose in PB, embedded
in 20% gelatine and frozen. Cryosections were cut at 20 µm, collected on
chrome alum-gelatine-coated slides and air-dried for approximately 30 min. The
embedding gelatine that surrounded the sections was removed by dipping the
slides in warm PB (approximately 40°C). After pre-incubation in PB-Tx-NGS
for 1-2 h at room temperature, the sections were incubated in the primary
anti-GABA antibody (1:750 in PB-Tx-NGS) overnight at 5°C, washed three
times for 15 min each in PB-Tx, and incubated in the Cy3-conjugated
anti-rabbit-IgG antibody (1:200 in PB-Tx-NGS) for 1 h at room temperature.
Finally, the sections were washed three times in PB-Tx, each for 10 min,
mounted in buffered glycerol and viewed with a Zeiss Axiophot compound
microscope (Zeiss, Oberkochen, Germany).
Controls
The following control experiments were performed. First, to rule out the
possibility that Cy3 labelling was due to binding of the secondary antibody to
endogenous epitopes, the primary antibody was omitted in negative controls. No
positive staining was observed under these conditions. Second, as a positive
control for GABA immunodetection, we used the GABAergic common inhibitor motor
neurons (Hale and Burrows,
1985; Watson,
1986
; Wolf and Lang,
1994
). A common inhibitor was identified by intracellular
recording and subsequent injection of Lucifer Yellow. After processing for
GABA immunohistochemistry, Lucifer-Yellow-fluorescence and
GABA-immunofluorescence were consistently colocalized in the common
inhibitors. Third, to exclude possible crossreactivity of the antibody with
glutamate, identified flexor tibiae motor neurons that are glutamatergic
(Bicker et al., 1988
;
Usherwood, 1994
;
Watson and Seymour-Laurent,
1993
) but do not contain GABA
(Watson, 1986
;
Watson et al., 1985
) were
injected with Lucifer Yellow and then processed for GABA immunohistochemistry.
No fluorescence signal was detected in the flexor tibiae neurons with the
excitation/emission filter settings used for Cy3 detection. These conditions
were also used to exclude the possibility that Lucifer-Yellow-fluorescence
gave cross-talk with the filter settings used for Cy3 or that the primary
antibody was binding non-specifically to neurons that do not contain GABA.
The results are based on successful identification and staining of 17 nonspiking interneurons in 14 locusts.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
GABA immunostaining in nonspiking interneurons
The metathoracic ganglion treated with either our wholemount or frozen
section immunostaining procedures revealed a large number of
GABA-immunoreactive cell bodies, the distribution of which corresponded to
that previously described with a different antibody
(Watson, 1986). The three
common inhibitory motor neurons to each hind leg, which have been identified
as GABAergic in previous studies, were consistently labelled by our GABA
immunohistochemistry. The excitatory motor neurons that contain glutamate
were, however, not labelled, demonstrating that the GABA antiserum did not
crossreact with glutamate (see Fig.
2A-C and below).
|
Fifteen out of a total of 17 nonspiking interneurons that were injected intracellularly with Lucifer Yellow showed strong GABA immunoreactivity. In several preparations, the Lucifer Yellow dye diffused throughout the recorded nonspiking interneuron to reveal the shape and distribution of its fine neurites. This staining was retained during the immunohistochemical processing, and, by confocal microscopy, the morphology of the neurones could be reconstructed from a series of optical sections (Figs 2D, 3B, 4A). Fig. 2 shows a GABA-immunopositive nonspiking interneuron in the anterior lateral group, injected with Lucifer Yellow following physiological characterization. A single cell body (Fig. 2A, arrow) showed intense fluorescence with the excitation/emission filter setting for Lucifer Yellow detection (shown in green pseudocolour in this and all following figures; the background is due to the autofluorescence of locust nervous tissue at these shorter wavelengths). The same cell body showed intense fluorescence when the same optical section was viewed with the excitation/emission filter setting that detects the Cy3 fluorophore tagged to the GABA antibody (Fig. 2B, red pseudocolour in this and all following figures). The cell body (Fig. 2B, arrow) was a member of a group of similar sized somata that were also GABA-immunopositive. When the two images were merged, the cell body of the nonspiking interneuron was yellow, indicating colocalization of the Lucifer Yellow and Cy3/GABA fluorophores (Fig. 2C, arrow). By contrast, the cell bodies of neighbouring immunopositive neurons remained red. The larger and more peripherally located neurites of these neurons were also GABA-immunopositive, but deeper neurites were not labelled presumably because, in the whole ganglia used, the antibody failed to penetrate. Importantly, the large cell bodies of glutamatergic motor neurons (five are indicated by asterisks in Fig. 2C) were not stained, demonstrating that the antiserum did not bind to glutamate. Confocal reconstruction of the Lucifer Yellow staining in this interneuron showed a single primary neurite emerging anteriorly from the cell body and then turning dorsally to give rise to a profusion of fine branches in the neuropil (Fig. 2D).
|
|
Fig. 3 shows an interneuron
among a different group of neurons, just contralateral to the midline, which
was also found to be GABA-immunopositive. Colocalization of Lucifer Yellow
(green) and Cy3/GABA (red) are indicated by the cell body appearing yellow
(Fig. 3A, arrow). When
hyperpolarizing current was injected into this interneuron, the tibia of the
left hind leg extended slowly and the spike frequency of the slow extensor
tibiae motor neuron was increased, but no effect of depolarizing current could
be discerned in tibial muscles. The cell body was contralateral to the leg
muscles that were affected. Confocal reconstruction of the interneuron based
on the Lucifer Yellow staining showed that the primary neurite crossed the
midline in a dorsal commissure (Siegler
and Burrows, 1979; Watkins et
al., 1985
; Wilson,
1981
) and then gave rise to a profusion of fine branches in the
neuropil in the left half of the ganglion
(Fig. 3B; ventral view,
arborizations are hence in the right half of the image). Interneurons with
this morphology were encountered in five of the 14 locusts.
Examples of other GABA-positive interneurons with differing physiological effects were also found in both the anterior lateral and posterior lateral groups (Fig. 1). For instance, when depolarizing current was injected into an interneuron in the anterior lateral group, the tibia of the left hind leg was slowly extended and muscle recordings showed that the slow extensor tibiae motor neuron increased its spike rate. Similarly, when depolarizing current was injected into an interneuron with a cell body in the posterior lateral group of nonspiking interneurons, the tibia of the left hind leg was flexed and muscle recordings showed that flexor tibiae motor neurons increased their spike rate. The cell bodies of both of these interneurons showed colocalisation of Lucifer Yellow and GABA staining (not illustrated).
Some nonspiking interneurons do not stain for GABA
Two of the 17 interneurons did not show GABA immunoreactivity in our
double-labelling experiments. The cell body of one of these interneurons in
the posterior lateral group was clearly labelled with Lucifer Yellow but not
with the GABA antibody and therefore appeared green when the two pseudocolour
images were merged (Fig. 4A).
Morphological reconstruction of the Lucifer-Yellow-filled interneuron revealed
the primary neurite running anteriorly before giving rise to a profuse array
of fine branches in the neuropil. A few nearby cell bodies of similar diameter
stained red and were clearly GABA-immunopositive.
The second interneuron was in the anterior lateral group (Fig. 4B). Its cell body again appeared green when the Lucifer-Yellow- and Cy3/GABA-fluorescence was combined, indicating that it was not GABA-immunoreactive. In the same optical section, a large group of small-diameter cell bodies stained red and were therefore GABA-immunopositive. When hyperpolarizing current was injected into the Lucifer-Yellow-labelled nonspiking interneuron, the frequency of spikes in flexor tibiae motor neurons was increased, whereas there was little effect on the spikes in the slow extensor tibiae motor neuron and in an unidentified motor neuron to a coxal muscle (Fig. 4C).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nonspiking interneurons analysed in this study do not represent the
sampling of a particular population of nonspiking interneurons. The recordings
were made from the neuropil and therefore different types of nonspiking
interneurons were encountered largely at random. This is reflected in the fact
that their cell bodies were in four different locations within the ganglion
where cell bodies of nonspiking neurons are known to reside
(Siegler and Burrows, 1979).
On the basis of their morphology and physiological action, some of the
interneurons encountered in the present study can be directly related to
previous and more detailed descriptions
(Siegler and Burrows, 1979
;
Watkins et al., 1985
). For
example, the interneuron in Fig.
3 is one in a group of three nonspiking interneurons described
first in locusts (Siegler and Burrows,
1979
; Wilson,
1981
) and then in stick insects (Carausius morosus;
Büschges, 1990
). Two of
the nonspiking interneurons with cell bodies in this position have the pattern
of branches shown here, whereas the third has an additional ipsilateral field
of branches. In stick insects, the two former interneurons were originally
called E4 neurons (E for excitatory) but the name was subsequently revised so
that one was called E4 and the other I4 (I for inhibitory) in both locusts and
stick insects (Büschges and Wolf,
1995
) to reflect the finding that there were two neurons with
similar shapes (Wilson, 1981
)
but different actions on the same motor neurons
(Wolf and Büschges,
1995
). Interneurons with cell bodies in this position that we
stained in five locusts always showed GABA immunoreactivity, suggesting that
at least one of them has an inhibitory action mediated by GABA but leaving
open the question of whether the other mediates its effects by disinhibition
or is really excitatory.
The GABA immunoreactivity in some nonspiking interneurons suggests that
they may use GABA as their transmitter, while its absence in others suggests
that they must use a different transmitter(s). This further emphasizes the
diversity of nonspiking interneurons already revealed by their morphology and
physiological actions. All of the known interconnections between nonspiking
interneurons involve inhibition (Burrows,
1979a), but both inhibitory and excitatory effects of nonspiking
interneurons on motor neurons have been described (Burrows and Siegler,
1976
,
1978
). The inhibitory actions
of the nonspiking interneurons at these synapses involve conductance increases
in postsynaptic neurons with a time course that suggests the use of
conventional transmitters such as GABA. Some of the excitatory effects on
motor neurons may be explicable by disinhibition through the web of
connections that a particular nonspiking interneuron makes with other
nonspiking interneurons. Alternatively, GABA itself may exert a direct
depolarizing effect on postsynaptic neurons as it does in some crustacean
networks (Swensen et al.,
2000
). The finding that a few of the nonspiking interneurons do
not show GABA-like immunoreactivity does, however, suggest that some
interneurons might mediate direct excitation of postsynaptic neurons by the
release of transmitters other than GABA.
If the properties of the nonspiking interneurons in our sample are
representative of the overall thoracic population, then this suggests that
GABAergic inhibition is a predominant feature of the processing by these
interneurons in the local circuits that control leg movements. The exclusively
inhibitory nature of the local interactions between nonspiking interneurons
(Burrows, 1979a) supports this
interpretation. While the orchestration of motor output by nonspiking
interneurons seems to be predominantly mediated by inhibition, the nature and
postsynaptic action of the transmitter used by the GABA-negative nonspiking
interneurons now needs to be determined to decide between two possibilities;
either the transmitter used by the nonspiking interneurons that do not show
GABA immunoreactivity may only exert inhibitory effects, so that any
excitatory effects are due to disinhibition, or some nonspiking interneurons
may use a transmitter that has direct excitatory effects on motor neurons.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bicker, G., Schafer, S., Ottersen, O. P. and Storm-Mathisen, J. (1988). Glutamate-like immunoreactivity in identified neuronal populations of insect nervous systems. J. Neurosci. 8,2108 -2122.[Abstract]
Burrows, M. (1979a). Graded synaptic
transmission between local pre-motor interneurons of the locust. J.
Neurophysiol. 42,1108
-1123.
Burrows, M. (1979b). Synaptic potentials effect the release of transmitter from locust nonspiking interneurons. Science 204,81 -83.[Medline]
Burrows, M. (1980). The control of sets of motoneurones by local interneurones in the locust. J. Physiol. 298,213 -233.[Abstract]
Burrows, M. (1987). Inhibitory interactions between spiking and nonspiking local interneurones in the locust. J. Neurosci. 7,3282 -3292.[Abstract]
Burrows, M. and Laurent, G. (1993). Synaptic potentials in the central terminals of locust proprioceptive afferents generated by other afferents from the same sense organ. J. Neurosci. 13,808 -819.[Abstract]
Burrows, M. and Siegler, M. V. S. (1976). Transmission without spikes between locust interneurones and motoneurones. Nature 262,222 -224.[Medline]
Burrows, M. and Siegler, M. V. S. (1978). Graded synaptic transmission between local interneurones and motoneurones in the metathoracic ganglion of the locust. J. Physiol. 285,231 -255.[Abstract]
Burrows, M. and Siegler, M. V. S. (1982). Spiking local interneurons mediate local reflexes. Science 217,650 -652.[Medline]
Büschges, A. (1990). Nonspiking pathways in a joint-control loop of the stick insect Carausius morosus. J. Exp. Biol. 151,133 -160.
Büschges, A. and Wolf, H. (1995).
Nonspiking local interneurons in insect leg motor control. I. Common layout
and species-specific response properties of femur-tibia joint control pathways
in stick insect and locust. J. Neurophysiol.
73,1843
-1860.
Dicaprio, R. A. (1989). Nonspiking interneurons in the ventilatory central pattern generator of the shore crab, Carcinus maenas. J. Comp. Neurol. 285,83 -106.[Medline]
Ganeshina, O. and Menzel, R. (2001). GABA-immunoreactive neurons in the mushroom bodies of the honeybee: an electron microscopy study. J. Comp. Neurol. 349,335 -349.
Graubard, K. (1978). Synaptic transmission
without action potentials: input-output properties of a non-spiking
presynaptic neuron. J. Neurophysiol.
41,1014
-1025.
Hale, J. P. and Burrows, M. (1985). Innervation patterns of inhibitory motor neurones in the thorax of the locust. J. Exp. Biol. 117,401 -413.[Abstract]
Hoyle, G. (1966). Functioning of the inhibitory conditioning axon innervating insect muscles. J. Exp. Biol. 44,429 -453.[Medline]
Judge, S. and Leitch, B. (1999). GABA immunoreactivity in processes presynaptic to the locust wing. J. Comp. Neurol. 407,103 -114.[Medline]
Kilman, V. L. and Marder, E. (1996). Ultrastructure of the stomatogastric ganglion neuropil of the crab, Cancer borealis. J. Comp. Neurol. 374,362 -375.[Medline]
Kondoh, Y., Morishita, H., Arima, T., Okuma, J. and Hasegawa, Y. (1991). White noise analysis of graded response in a wind-sensitive, nonspiking interneuron of the cockroach. J. Comp. Physiol. A 168,429 -443.[Medline]
Laughlin, S. B., de Ruyter van Steveninck, R. R. and Anderson, J. C. (1998). The metabolic cost of neural information. Nat. Neurosci. 1,36 -41.[Medline]
Mendelson, M. (1971). Oscillator neurons in crustacean ganglion. Science 171,1170 -1173.[Medline]
Nagayama, T. (1997). Organization of exteroceptive inputs onto nonspiking local interneurones in the crayfish terminal abdominal ganglion. J. Exp. Zool. 279, 29-42.
Nagayama, T., Aonuma, H. and Miyata, H. (1996).
GABA-like immunoreactivity of an identified nonspiking local interneurone in
the crayfish terminal abdominal ganglion. J. Exp.
Biol. 199,2447
-2450.
Nagayama, T. and Hisada, M. (1987). Opposing parallel connections through crayfish local nonspiking interneurons. J. Comp. Neurol. 257,347 -358.[Medline]
Nagayama, T., Namba, H. and Aonuma, H. (1994). Morphological and physiological bases of crayfish local circuit neurones. Histol. Histopath. 9,791 -805.[Medline]
Nagayama, T., Namba, H. and Aonuma, H. (1997). Distribution of GABAergic premotor nonspiking local interneurones in the terminal abdominal ganglion of the crayfish. J. Comp. Neurol. 389,139 -148.[Medline]
Nagayama, T., Takahata, M. Y. and Hisada, M. (1984). Functional characteristics of local non-spiking interneurons as the pre-motor elements in crayfish. J. Comp. Physiol. A 154,499 -510.
Pearlstein, E., Watson, A. H. D., Bevengut, M. and Cattaert, D. (1998). Inhibitory connections between antagonistic motor neurones of the crayfish walking legs. J. Comp. Neurol. 399,241 -254.[Medline]
Pearson, K. G. and Bergman, S. J. (1969). Common inhibitory motorneurones in insects. J. Exp. Biol. 50,445 -471.[Medline]
Pearson, K. G. and Fourtner, C. R. (1975).
Nonspiking interneurons in walking system of the cockroach. J.
Neurophysiol. 38,33
-52.
Reichert, H., Plummer, M. R., Hagiwara, G., Roth, R. L. and Wine, J. J. (1982). Local interneurons in the terminal abdominal ganglion of the crayfish. J. Comp. Physiol. A 149,145 -162.
Reichert, H., Plummer, M. R. and Wine, J. J. (1983). Identified nonspiking local interneurons mediate nonrecurrent, lateral inhibition of crayfish mechanosensory interneurons. J. Comp. Physiol. A 151,261 -276.
Seidel, C. and Bicker, G. (1997). Colocalization of NADPH-diaphorase and GABA-immunoreactivity in the olfactory and visual system of the locust. Brain Res. 769,273 -280.[Medline]
Shaw, S. R. (1968). Organisation of the locust retina. Symp. Zool. Soc. Lond. 23,135 -163.
Siegler, M. V. S. and Burrows, M. (1979). The morphology of local non-spiking interneurones in the metathoracic ganglion of the locust. J. Comp. Neurol. 183,121 -148.[Medline]
Swensen, A. M., Golowasch, J., Christie, A. E., Coleman, M. J.,
Nusbaum, M. P. and Marder, E. (2000). GABA and responses to
GABA in the stomatogastric ganglion of the crab Cancer borealis. J.
Exp. Biol. 203,2075
-2092.
Usherwood, P. N. R. (1994). Insect glutamate receptors. Adv. Insect Physiol. 24,309 -341.
Usherwood, P. N. R. and Grundfest, H. (1965).
Peripheral inhibition in skeletal muscle of insects. J.
Neurophysiol. 28,497
-518.
Watkins, B. L., Burrows, M. and Siegler, M. V. S. (1985). The structure of locust non-spiking interneurones in relation to the anatomy of their segmental ganglion. J. Comp. Neurol. 240,233 -255.[Medline]
Watson, A. H. D. (1986). The distribution of GABA-like immunoreactivity in the thoracic nervous system of the locust Schistocerca gregaria. Cell Tissue Res. 246,331 -341.
Watson, A. H. D., Bevengut, M., Pearlstein, E. and Cattaert, D. (2000). GABA and glutamate-like immunoreactivity at synapses on depressor motorneurones of the leg of the crayfish, Procambarus clarkii. J. Comp. Neurol. 422,510 -520.[Medline]
Watson, A. H. D. and Burrows, M. (1987). Immunocytochemical and pharmacological evidence for GABAergic spiking local interneurones in the locust. J. Neurosci. 7,1741 -1751.[Abstract]
Watson, A. H. D., Burrows, M. and Hale, J. P. (1985). The morphology and ultrastructure of common inhibitory motor neurones in the thorax of the locust. J. Comp. Neurol. 239,341 -359.[Medline]
Watson, A. H. D., Burrows, M. and Leitch, B. (1993). GABA-immunoreactivity in processes presynaptic to the terminals of afferents from a locust leg proprioceptor. J. Neurocytol. 22,547 -557.[Medline]
Watson, A. H. D. and Laurent, G. (1990). GABA-like immunoreactivity in a population of locust intersegmental interneurones and their inputs. J. Comp. Neurol. 302,761 -767.[Medline]
Watson, A. H. D. and Pflüger, H. J. (1987). The distribution of GABA-like immunoreactivity in relation to ganglion structure in the abdominal nerve cord of the locust (Schistocerca gregaria). Cell Tissue Res. 249,391 -402.
Watson, A. H. D. and Pflüger, H. J. (1994). Distribution of input synapses from processes exhibiting GABA- or glutamate-like immunoreactivity onto terminals of prosternal filiform afferents in the locust. J. Comp. Neurol. 343,617 -629.[Medline]
Watson, A. H. D. and Seymour-Laurent, K. J. (1993). The distribution of glutamate-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria). Cell Tissue Res. 273,557 -570.
Wilson, J. A. (1981). Unique, identifiable nonspiking interneurons in the locust mesothoracic ganglion. J. Neurobiol. 12,353 -366.[Medline]
Wolf, H. and Büschges, A. (1995).
Nonspiking local interneurons in insect leg motor control. II. Role of
nonspiking local interneurons in the control of leg swing during walking.
J. Neurophysiol. 73,1861
-1875.
Wolf, H. and Lang, D. M. (1994). Origin and clonal relationship of common inhibitory motoneurons CI1 and CI3 in the locust CNS. J. Neurobiol. 25,846 -864.[Medline]
Zettler, F. and Jarvilehto, M. (1971). Decrement free conduction of graded potentials along the axon of a monopolar neuron. Z. Vergl. Physiol. 75,402 -421.