First published online December 16, 2008
Journal of Experimental Biology 212, 126-136 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.020412
Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands
Cathleen Rotte,
Jeannine Witte,
Wolfgang Blenau,
Otto Baumann and
Bernd Walz*
Institute of Biochemistry and Biology, Department of Animal Physiology,
University of Potsdam Karl-Liebknecht-Str. 24–25, 14476 Potsdam,
Germany
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Fig. 1. Anti-GABA labeling of a subesophageal ganglion (SOG, whole-mount) that was
backfilled via the salivary duct nerve (SDN) with TMR-dextran. (A) A
SOG (outline indicated by gray broken lines). The nerve (indicated by gray
dotted lines) is partially out of view because it leaves the plane of the
optical section and was turned over the ganglion during the embedding
procedure. Upon entering the ganglion, the pathways (indicated by white dotted
lines) of the two thick axons diverge. One axon extends to a soma with
contralateral, anterioventral position (SN1) and the other originates at a
soma with ipsilateral, midventral position (SN2). (B–D) Anti-GABA
labeling (green) of a SOG that was backfilled with TMR-dextran (red). The
right panel shows the composite images. SN1 is labeled only with TMR-dextran
(B) whereas SN2 is labeled with both TMR-dextran and anti-GABA (C). Other
GABA-positive neurons contain no TMR-dextran (D). Bar A, 250 µm, bar for
B,C in D, 50 µm.
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Fig. 2. Distribution of GABA-positive fibers on the various components of the
salivary gland complex. Whole-mount preparations of salivary glands were
stained with anti-GABA (green) and AlexaFluor488-phalloidin (blue), and imaged
by confocal microscopy. (A) A simplified schematic illustration of the
organization of the salivary gland complex. The paired salivary glands consist
of lobules of acinar tissue. The salivary ducts unite to an efferent salivary
duct (1) from each gland, and the two efferent salivary ducts unite to a
single main salivary duct (2). The paired reservoirs open into reservoir ducts
(3) that unite to the main reservoir duct (4). Red rectangles in A outline the
areas shown in B–E. (B) The salivary duct nerve (SDN) contains two thick
axons, one of them labeled by anti-GABA. (C) Anti-GABA-immunoreactive fibers
(arrowheads) in the reservoir muscle. (D) A nerve that interlinks an acinar
lobule (lower right) with the reservoir (asterisks). Anti-GABA-positive fibers
(arrowheads) branch and have numerous varicosities within the nerve. (E) An
acinar lobule and the associated salivary duct (arrow). P-cells are arranged
in pairs with their microvilli intensely stained with phalloidin (blue),
providing the appearance of `bow ties'. A loose network of anti-GABA-reactive
fibers is associated with the acinar tissue but not the salivary duct (arrow).
(F–H,J–L) Two series of confocal sections through acinar lobules.
Each image shows the sum of 17 consecutive optical sections, representing a
total thickness of 6.2 µm. The labelling in the upper right indicates the
plane of the optical section. (I,M) The sum of all images. P-cells are
indicated by asterisks. C-cells in the interior of the acinar lobules are
identified by short phalloidin-stained microvilli (open arrowheads). In the
lobule shown in (F–I), GABA-positive fibers remain and terminate
(arrowhead in G) on the surface of the lobule. In (J–M), GABA-positive
fibers extend deep into the acinar lobules (arrows). All bars, 50 µm; bar
in M is for F–M.
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Fig. 3. Distribution of putative release sites for GABA on the salivary gland
complex. Whole-mount preparations of salivary glands were stained with
anti-GABA (green), anti-synapsin (red) and AlexaFluor488-phalloidin (blue),
and imaged by confocal microscopy. Broken-lined rectangles outline the areas
that are shown at higher magnification in the insets on the upper right of
each image. Putative release sites for GABA are labeled by anti-GABA and
anti-synapsin and are indicated by yellow staining in the composite images on
the right. Red foci on the composite images may represent release sites for
other neurotransmitters. (A–C) Putative release sites for GABA on an
acinar lobule. Some putative GABA release sites (open arrowheads) are
juxtaposed, others (arrowheads) reside at distance to release sites for other
neurotransmitters. (D–F) Putative release sites for GABA in a nerve that
interlinks acinar lobules. Most putative release sites for GABA (arrowheads)
keep some distance to release sites for other neurotransmitters. Bars
A–F, 25 µm.
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Fig. 4. Changes in the membrane potential (Vm) recorded from
acinar cells during bath application of GABA, dopamine and 5-HT. (A)
Superfusion of isolated salivary glands with PS containing 1 mmol
l–1 GABA does not affect the resting Vm
of acinar cells. Control application of 100 nmol l–1 dopamine
evokes multiphasic changes in the Vm of the acinar cell,
showing that the isolated gland is functionally intact. (B,D) Co-application
of GABA and either dopamine or serotonin (5-HT). 1 mmol l–1
GABA has no measurable effect on the amplitudes and kinetics of dopamine- or
5-HT-induced Vm changes in acinar cells. (C) Quantitative
summary (means ± s.e.m.) of the experiments illustrated in B and D.
Response amplitudes induced by co-application of dopamine or 5-HT and GABA,
normalized to control amplitudes with 5-HT or dopamine, respectively, prior to
co-application. The amplitude was taken as the range from the
hyperpolarization peak to the depolarization peak. 1 mmol l–1
GABA did not significantly alter the dopamine-(N=5) and 5-HT-induced
(N=6) changes in Vm. Duration of dopamine, 5-HT
and GABA applications are indicated by bars.
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Fig. 5. Membrane potential (Vm) changes in acinar cells induced
by electrical salivary duct nerve (SDN)-stimulation (A,C). Asterisks mark the
timepoints of electrical stimulation (5 V, 0.2 ms, 5 Hz, 2 s trains). The
black arrow in A indicates the hyperpolarization, the open arrow the
depolarization induced by one train of electrical stimuli. The box in C
indicates the response shown in A. (B) Dose-response relations determined from
the changes in Vm induced by bath application of
increasing dopamine (black line, means ± s.e.m., N=7) and
serotonin (broken line, means ± s.e.m., N=7) concentrations.
The mean change in Vm recorded from acinar cells upon
electrical SDN-stimulation (13 mV; black line, N=21) is within the
dynamic range of the dose-response relations for both neurotransmitters. (D)
Bath application of GABA (5 µmol l–1) during
SDN-stimulation leads to enhanced amplitudes of the electrical responses
recorded from acinar cells. The response amplitudes recover after GABA
washout. (E) Comparison of the amplitudes of changes in Vm
induced by SDN-stimulation in the absence (control) and in the presence of
GABA (N=21, means ± s.e.m.). The mean amplitude of at least
two or more consecutive control stimulations was compared by a paired
t-test to the mean amplitude of at least two or more stimulations
during bath application of GABA. ***P<0.0001.
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Fig. 6. Effects of agonists and antagonists for various GABA receptor subtypes on
the membrane potential (Vm) changes evoked by salivary
duct nerve (SDN)-stimulation. Asterisks mark the timepoints of stimulation (5
V, 0.2 ms, 5 Hz, 2 s trains). The duration of drug application is indicated by
bars. (A,B) Application of SKF97541 or baclofen mimic the GABA-induced
enhancement in the electrical responses. (C,D) CGP52432 and CGP54626 suppress
the GABA-induced enhancement. (E) The GABABR antagonist CGP52432
suppresses the SKF97541-induced enhancement. (F) Bicuculline-induced
augmentation of electrical responses of acinar cells to SDN-stimulation. (G)
Bar chart for the effects of all tested drugs. The gray dotted line indicates
the control amplitude (=100%) induced by electrical SDN-stimulation in the
absence of GABA and other drugs. The number of replicates for each bar is
stated in parentheses. The bars show means±s.e.m. Muscimol
(GABAAR agonist), picrotoxin (GABAAR antagonist), THIP
(GABACR antagonist and partial GABAAR agonist) and TPMPA
(GABACR antagonist) did not affect the control amplitudes or the
GABA-induced enhancement. The GABABR-specific agonists SKF97541 and
baclofen enhanced the acinar cell response amplitudes. The GABABR
antagonists CGP54626 and CGP52432 suppressed the GABA-induced enhancement.
Bicuculline also enhanced the cell responses to SDN-stimulation. The mean
amplitude of at least two stimulations during GABA-bath application and/or
drug treatment was normalized to the mean amplitude of at least two
consecutive control stimulations for each experiment. The data were analyzed
by applying one-way analysis of variance and Tukey's post test.
*P<0.05, **P<0.001.
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Fig. 7. Effects of GABA on the rates of fluid and protein secretion induced by
electrical salivary duct nerve (SDN)-stimulation. (A) Experimental regime and
fluid secretion induced by electrical SDN-stimulation in the absence (8 V, 0.2
ms stimuli applied at 10 Hz) and presence (8 V, 0.2 ms stimuli applied at 2
Hz) of GABA. 5 µmol l–1 GABA increased the rate of fluid
secretion induced by a low frequency of electrical stimuli. After GABA washout
the gland was stimulated at a frequency of 10 Hz again to obtain a control for
gland functionality. The open rectangle indicates the five saliva samples/time
periods that served as control for the protein assay in C. The light gray
rectangle labels the five saliva samples/time periods that served as a control
for the comparison of the secretion rates in B. The dark gray rectangle
indicates the saliva samples/time period when GABA was applied. Application
times are indicated by horizontal bars. (B) 5 µmol l–1
GABA increases the rate of fluid secretion 2.5-fold from 6.8±1.1 to
16.7±1.7 nl min–1 (N=7,
*P<0,0171). (C) 5 µmol l–1 GABA
increases the rate of protein secretion from 2.9±0.7 µg
min–1 4-fold to 12±3.8 µg min–1
(N=7, *P=0.03).
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