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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
Institute of Biochemistry and Biology, Department of Animal Physiology, University of Potsdam Karl-Liebknecht-Str. 24–25, 14476 Potsdam, Germany
* Author for correspondence (e-mail: walz{at}uni-potsdam.de)
Accepted 3 November 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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-aminobutyric acid (GABA)
and their physiological role. Immunofluorescence revealed a
GABA-immunoreactive axon that originates within the subesophageal ganglion at
the salivary neuron 2 (SN2) and this extends within the salivary duct nerve
towards the salivary gland. GABA-positive fibers form a network on most acinar
lobules and a dense plexus in the interior of a minor fraction of acinar
lobules. Co-staining with anti-synapsin revealed that some putative GABAergic
terminals seem to make pre-synaptic contacts with GABA-negative release sites.
Many putative GABAergic release sites are at some distance from other synapses
and at distance from the acinar tissue. Intracellular recordings from isolated
salivary glands have revealed that GABA does not affect the basolateral
membrane potential of the acinar cells directly. When applied during salivary
duct nerve stimulation, GABA enhances the electrical response of the acinar
cells and increases the rates of fluid and protein secretion. The effect on
electrical cell responses is mimicked by the GABAB receptor
agonists baclofen and SKF97541, and blocked by the GABAB receptor
antagonists CGP52432 and CGP54626. These findings indicate that GABA has a
modulatory role in the control of salivation, acting presynaptically on
serotonergic and/or dopaminergic neurotransmission.
Key words: GABA, salivary gland, innervation, insect, cockroach, Periplaneta americana
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Walz et al., 2006). In
cockroaches, saliva production by the acinar-type salivary glands is
controlled by the nervous system. Two paired neurons within the subesophageal
ganglion (SOG), salivary neuron 1 (SN1) and salivary neuron 2 (SN2), send
their axons by way of the salivary duct nerve (SDN) towards the salivary gland
(Whitehead, 1971;
Ali, 1997;
Walz et al., 2006;
Watanabe and Mizunami, 2006).
SN1 contains dopamine as neurotransmitter
(Elia et al., 1994) but the
neurotransmitter of SN2 is unknown. Besides the SN1 and SN2 axons with a
thickness of several micrometers, the SDN contains thinner axons that also
project from the SOG towards the salivary gland. These thin axons contain
serotonin (5-hydroxytryptamine; 5-HT) as neurotransmitter
(Davis, 1985;
Baumann et al., 2002;
Baumann et al., 2004). An
additional source of serotonergic innervation of the salivary gland is
provided by the stomatogastric nervous system
(Whitehead, 1971;
Bowser-Riley et al., 1978;
Baumann et al., 2002).
Serotonergic and dopaminergic fibers innervate the secretory tissue of the
cockroach salivary glands in different patterns. The salivary glands are
composed of three main cell types with different functions
(Just and Walz, 1994;
Just and Walz, 1996): the
grape-like acini consist of a pair of ion-secreting peripheral cells (P-cells)
and several protein-secreting central cells (C-cells). The P-cells are
innervated by both dopaminergic and serotonergic fibers, and are responsible
for ion and water transport. The C-cells are innervated solely by serotonergic
fibers and are responsible for protein secretion
(Baumann et al., 2002;
Baumann et al., 2004). Thus,
with dopamine stimulation, a protein-free saliva is produced in the acini
whereas 5-HT stimulates the secretion of protein-containing saliva
(Just and Walz, 1996;
Marg et al., 2004;
Rietdorf et al., 2005;
Troppmann et al., 2007). The
NaCl-rich primary saliva is modified when it passes the extensive duct system.
As a result, the final saliva is hypo-osmotic
(Gupta and Hall, 1983;
Lang and Walz, 2001;
Rietdorf et al., 2003;
Hille and Walz, 2007).
Although the innervation pattern of cockroach salivary glands has been
studied in detail, the neurotransmitter content of SN2 is still unknown.
Watkins and Burrows have provided evidence that the SN2 soma in the locust
Schistocerca gregaria contains GABA
(Watkins and Burrows, 1989).
Moreover, one thick axon within the SDN of the locust shows GABA
immunoreactivity, supporting the view that SN2 is GABAergic
(Watkins and Burrows, 1989).
The function of GABA in locust salivary glands is still undisclosed. As locust
salivary glands are very similar to those of cockroaches with regard to
morphology and innervation (Ali,
1997), in the present study, we examined whether there is also
GABAergic innervation of the salivary glands in the cockroach Periplaneta
americana. We determined the source of GABAergic innervation and the
spatial relationship of GABA-positive nerve fibers to the various cellular
components of the salivary gland complex, and we located putative sites of
GABA release on the salivary gland. By intracellular recordings from a
nerve–gland preparation and by measuring secretion rates, we obtained
information on the possible function of the GABAergic innervation and examined
the pharmacological profile of GABA action in this system.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Antibodies
The following primary antibodies were used: rabbit polyclonal antibody
(pAb) anti-GABA (A2052; Sigma, Taufkirchen, Germany), guinea pig pAb anti-GABA
(GZ1081; Biotrend, Cologne, Germany), rat monoclonal antibody (mAb)
anti-serotonin (MAB352; Chemicon, Hampshire, UK), mouse mAb SYNORF1 against
Drosophila synapsin (kindly provided by E. Buchner, University of
Würzburg, Germany) (Klagges et al.,
1996). Secondary antibodies conjugated to Cy3 or Cy5 were obtained
from Rockland (Gilbertsville, PA, USA) and Dianova (Hamburg, Germany).
Labeling specificity has been demonstrated previously for the antibodies
against serotonin and synapsin (Baumann et
al., 2002; Baumann et al.,
2004). To determine whether labeling for GABA is also specific,
cryostat sections of cockroach salivary glands were co-stained with both
antisera against GABA; the antisera, although made against different
GABA-conjugates (rabbit anti-GABA, GABA conjugated to albumin; guinea pig
anti-GABA, GABA conjugated to keyhole limpet hemocyanin) labeled identical
structures (data not depicted). Moreover, staining was almost completely
abolished by pre-absorption of anti-GABA with 10 mmol l–1
GABA but not with 10 mmol l–1 glutamate, the substrate of
GABA synthesis.
Immunofluorescence labeling
Salivary glands were isolated, fixed for 2 h at room temperature (RT) in 3%
paraformaldehyde, 75 mmol l–1 lysine-HCl, 10 mmol
l–1 Na-perjodate, 0.2 mol l–1 sucrose, 0.1
mol l–1 Na-phosphate buffer (pH 7.0), and washed 3x10
min with phosphate-buffered saline (PBS). Cryosections or entire salivary
glands were processed for immunofluorescence labeling as described previously
(Baumann et al., 2002;
Baumann et al., 2004).
Anti-synapsin was applied at a dilution of 1:25, anti-serotonin at a dilution
of 1:200, rabbit anti-GABA at a dilution of 1:20,000 or 1:40,000, and guinea
pig anti-GABA at a dilution of 1:1000. In order to identify the various cell
types and to provide a spatial reference for the position of nerve fibers,
specimens were co-labeled with the F-actin probe AlexaFluor488-phalloidin
(Invitrogen, Karlsruhe, Germany). Fluorescence images were recorded with a
Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).
Backfill labeling
The SOG with the SDN was isolated in physiological saline (PS). The nerve
was severed and inserted into a droplet of tetramethylrhodamine dextran
(TMR-dextran; product number D3308; Invitrogen). After incubation for 48 h at
4°C, specimens were fixed for 2 h at RT as described above, washed
6x20 min with 0.5% Triton-X100 in PBS, dehydrated in a graded ethanol
series, cleared with methylsalicylate, rehydrated and the entire ganglia were
processed for immunofluorescence labeling with anti-GABA. After dehydration,
specimens were embedded in methylsalicylate.
Electrophysiology
The dissected glands were placed in a recording chamber and continuously
superfused with PS at a flow rate of 
2 ml min–1. Sharp
microelectrodes, used for impaling acinar cells, were filled with 3 mol
l–1 KCl and had tip resistances of 70–100
M
when inserted into the bath with PS. After impalement the basolateral
membrane potential (Vm) of acinar cells was recorded by
using a BRAMP 01 bridge amplifier (npi electronic GmbH, Tamm, Germany). The
output of the amplifier was displayed on an oscilloscope (Philips PM3331
Combiscope, Kassel, Germany), continuously recorded on a chart recorder (Kipp
and Zonen, DELFT BV BD122, Gengenbach Messtechnik, Reichenbach/Fils, Germany)
and digitalized at a sampling rate of 1 Hz with a KUSB-3102 A/D-converter
(Keithley, Germering, Germany). A/D conversion, data display, storage and
output were controlled by TestPoint software (Keithley, Germering,
Germany).
Although the microelectrodes were positioned close to an acinus for
impalement under optical control through a stereomicroscope, we never knew
whether the microelectrode had impaled a P- or a C-cell. We made no attempts
in this study to label the cells from which we recorded because P- and C-cells
are dye-coupled (Lang and Walz,
1999). For SDN-stimulation, the salivary gland main duct and the
attached SDN were taken up into a suction electrode that was coupled to a
stimulus isolation unit (SIU 5, Grass Technologies Product Group, Astro-Med
GmbH, Rodgau, Germany) and a Grass S48 stimulator. The SDN was stimulated
electrically for 2 or 5 s at 5Hz (resulting in trains of either 10 or 25
stimuli) with each stimulus lasting 0.2 ms in a sufficient strength
(5–10 V) to induce acinar cell responses. Prism 4.0 (GraphPad Software,
San Diego, CA, USA) was used for statistical analysis. Data were analyzed
statistically by using either a paired t-test or an analysis of
variance (ANOVA) with one-way ANOVA followed by a Tukey post test.
Measurements of fluid and protein secretion
Measurements of fluid secretion were carried out as described previously
(Just and Walz, 1996).
Briefly, the paired salivary glands were dissected and immediately transferred
into the Ringer-filled perfusion bath of a double-chamber having a perfusion
bath and a paraffin oil-filled bath, separated by a narrow Vaseline® gap.
The perfusion bath was continuously perfused with oxygenated PS. During
dissection, the main salivary duct was separated from the adherent reservoir
duct and only the salivary duct was drawn through the Vaseline® gap into
the oil bath, where droplets of secreted saliva were collected every minute
and then transferred into a storage paraffin pool for volume determinations.
The volume of the spherical saliva droplets was calculated from their
diameter. For electrical stimulation of the salivary nerve, the reservoir duct
and the attached SDN were drawn into a suction electrode (in the perfusion
chamber), and the glands were stimulated electrically (as described above)
sufficiently strong to induce saliva secretion (0.2 ms pulses, 5–10 V,
10 Hz). For statistical analysis, a paired t-test was applied to
compare rates of fluid secretion in the presence and absence of GABA.
After volume determination, the saliva droplets were transferred to an
Eppendorf tube filled with liquid paraffin and stored at –20°C.
Protein content of the saliva samples was determined as described previously
(Rietdorf et al., 2005) using
a modified Bradford assay (Bradford,
1976). For each experiment, saliva droplets from 5 min
experimental periods were pooled and diluted with 50 µl H2O and
200 µl Roti Nanoquant (Roth, Karlsruhe, Germany) working solution.
Standards with eight bovine serum albumin concentrations between 0 and 100
µgml–1 were used for calibration. To account for the small
amounts of liquid paraffin, transferred together with the saliva droplets, 5
µl liquid paraffin was added to each standard. The absorption of standards
and saliva samples was measured using a GeneQuantTM 1300
spectrophotometer (GE Healthcare, Munich, Germany) to determine protein
content. A linear regression was calculated from the data obtained from
standard solutions and used for quantification of the protein content in the
saliva droplets. A paired t-test was used to compare the rates of
protein secretion in the absence and presence of GABA.
Chemicals
The GABA receptor ligands (RS)-baclofen, SKF97541 (3-APPA) (both
GABABR agonists), CGP52432, CGP54626 hydrochloride (both
GABABR antagonists), muscimol (GABAAR agonist),
picrotoxin, (–)-bicuculline methochloride (both GABAAR
antagonists), (1,2,5,6-tetrahydropyridin-4-yl)-methylphosphinic acid (TPMPA)
and 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) (both
GABACR antagonists, the latter is also a partial GABAAR
agonist) were obtained from Biotrend. GABA, dopamine hydrochloride and 5-HT
were obtained from Sigma.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
To examine whether cockroach SN2 is also GABA-positive, the SOG was stained
with anti-GABA. To identify SN2, the SDN was cut and backfilled with
TMR-dextran. Fig. 1A–D
shows the results of backfilling. In the SDN, two axons were stained
intensively by TMR-dextran (Fig.
1A, lower right). Upon entering the SOG, the pathways of these
axons diverged (Fig. 1A). One
axon extended to a soma on the contralateral, anterioventral side of the
ganglion, thus representing SN1 (Gifford
et al., 1991; Watanabe and
Mizunami, 2006). The other TMR-dextran-filled axon could be traced
to a soma of ipsilateral, midventral position within the ganglion and
represents SN2 (Gifford et al.,
1991; Watanabe and Mizunami,
2006). When ganglia were stained with anti-GABA after backfilling,
the SN1 soma was labeled only with TMR-dextran whereas the SN2 soma contained
TMR-dextran and anti-GABA immunofluorescence
(Fig. 1B,C). Other
GABA-positive neurons in the SOG were without TMR-dextran signal
(Fig. 1D). These results
suggest that the SN2 neuron in the SOG of the cockroach contains GABA.
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Distribution of GABA-positive fibers over and within the salivary gland complex
Fig. 2A presents a schematic
view of the organization of the cockroach salivary gland complex
(Just and Walz, 1994) in order
to give the reader some landmarks. The paired salivary glands consist of many
lobules, each with numerous secretory acini. Every acinus has two
fluid-secreting P-cells and several protein-secreting C-cells. The primary
saliva produced by the acini passes through an extensive duct system. The
salivary ducts of each of the paired glands merge into a single efferent
salivary duct. One reservoir is associated with each of the paired glands. The
reservoirs open into the reservoir ducts that extend alongside of the efferent
salivary ducts and finally fuse to form the main reservoir duct. A reservoir
muscle is attached to the orifice of each reservoir.
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;
Baumann et al., 2004), but only
few lobules contained GABA-positive fibers (data not presented). Moreover, a
similar difference in anti-GABA labeling pattern between acinar lobules was
observed on cryostat sections that provide equal access for antibodies,
independent of the location of the acinar lobule within a salivary gland (data
not presented). We thus conclude that acinar lobules differ in their
innervation pattern with GABA-positive fibers; most lobules have GABA-positive
fibers only on their outer surface, and few lobules are penetrated by these
fibers.
Position of putative release sites for GABA
By double-labeling with anti-synapsin and anti-GABA, we examined the
distribution of putative release sites for GABA in whole-mount preparations of
salivary gland complexes (Fig.
3A–F). Foci with anti-synapsin immunoreactivity were
detected along thin GABA-positive fibers and usually correlated with the
presence of varicosities (Fig.
3A–F, insets). We term such synapsin foci `putative release
sites for neurotransmitters'. Thicker GABA-positive fibers with uniform
diameter, including the GABA-positive axon in the SDN, displayed only weak
homogeneous staining for synapsin (Fig.
3D–F). Putative release sites were present along
GABA-positive fibers on the outer surface of the efferent reservoir duct, the
outer surface of the reservoir, and within the reservoir muscle (data not
depicted). Moreover, thin varicose GABA-positive fibers in association with
the acinar lobules (Fig.
3A–C) and in nerves that interlinked the acinar lobules
contained putative release sites (Fig.
3D–F). It may thus be concluded that GABA release occurs at
all structures of the salivary gland complex that are associated with varicose
GABA-positive fibers.
In principle, GABA could act on two different target sites in the salivary gland complex. First, it may directly affect some of the various cell types in the salivary gland complex. Second, it could act presynaptically on release sites of other neurons that innervate the salivary gland complex. If the latter were the case, it may be expected that release sites for GABA have a close spatial relationship to release sites for other neurotransmitters. Thus, in whole-mount preparations double-labeled with anti-GABA and anti-synapsin, GABA-positive synapsin foci should be localized next to GABA-negative synapsin foci. Fig. 3A–C demonstrates that this is in fact the case for many but not all of the putative release sites for GABA.
Does GABA affect the acinar cells directly?
In order to obtain information about the possible functions of the
GABAergic innervation, we studied the effects of GABA on isolated salivary
glands by intracellular recordings from acinar cells. When isolated salivary
glands were superfused with PS containing dopamine or 5-HT, either transmitter
induced multiphasic changes in the basolateral Vm of the
acinar cells (Fig. 4A,B,D).
These electrical responses were composed of a brief initial hyperpolarization
(up to 7 mV upon application of 100 nmol l–1 dopamine or
5-HT), followed by a large and long-lasting depolarization (up to 25 mV) and
sometimes an afterhyperpolarization, irrespectively of whether dopamine or
5-HT was applied (Fig. 4A,B,D).
Dose-response curves for dopamine and 5-HT
(Fig. 5B) revealed
EC50 values of 30 nmol l–1 and 47 nmol
l–1, respectively, when the neurotransmitters were applied in
the bathing solution. Bath application of GABA at concentrations up to 1 mmol
l–1, however, had no measurable effect on the resting
Vm of the acinar cells
(Fig. 4A). To determine whether
GABA modulates the effects of dopamine and/or 5-HT on the acinar cells, GABA
was co-applied with the latter neurotransmitters.
Fig. 4B–D illustrates
that GABA has no obvious effect on the amplitude or kinetics of the electrical
responses induced by sub-saturating concentrations (100 nmol
l–1) of dopamine or 5-HT. These results suggest that GABA
does not directly affect the basolateral Vm of the acinar
cells.
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Does GABA act presynaptically?
To examine whether GABA affects the dopaminergic and/or serotonergic
neurotransmission, we developed a nerve–gland preparation that permitted
electrical stimulation of the SDN via a suction electrode and
simultaneous intracellular recordings of the basolateral
Vm of the acinar cells.
Fig. 5A shows the typical
changes in the Vm recorded from an acinar cell that was
induced by electrical SDN-stimulation. A train of 10 stimuli (0.2 ms, 5 V)
applied at a frequency of 5 Hz caused responses that consisted of a
hyperpolarization (Fig. 5A,
black arrow) of approx. 5±1 mV that was followed by a transient
depolarization of approx. 8±1 mV (N=21; means±s.e.m.)
(Fig. 5A, open arrow). A
comparison of Fig. 5A,C and
Fig. 4B,D reveals that the
amplitudes and kinetics of stimulus-induced changes in acinar cell
Vm were very similar, irrespectively of whether the SDN
was electrically stimulated, or whether dopamine or 5-HT were applied in the
bath. In fact, the amplitudes of the changes in Vm induced
by SDN-stimulation fell into the dynamic range of the response amplitudes
generated by bath application of increasing dopamine or 5-HT concentrations
(dose-response relationship in Fig.
5B). However, the shape of the electrically induced responses was
variable in that the size of the hyperpolarization and the depolarization
phase contributed in different amounts to the overall amplitude of the voltage
changes (compare Fig. 5A,C,
equal size of hyperpolarization and depolarization;
Fig. 6C, small
hyperpolarization, large depolarization;
Fig. 6E, large
hyperpolarization, small depolarization). This variability was observed
between different preparations, but not during an experiment with one
preparation. The factors determining the size of the electrical response
components remain unknown but could depend on the cell type that was impaled
or on co-release of other neurotransmitters with dopamine and 5-HT.
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These data demonstrate that GABA augments the electrical responses of acinar cells only when these are induced by SDN-stimulation, suggesting presynaptic effects on dopaminergic and/or serotonergic neurotransmission.
Pharmacological properties of the GABA effects
To obtain information about the receptor types that are involved in the
GABA-mediated effect, we performed pharmacological experiments with agonists
and/or antagonists of the various subtypes of GABA receptors. Baclofen (1 mmol
l–1) and SKF97541 (5 µmol l–1), agonists
of GABAB receptors (GABABR) (for reviews, see
Bowery et al., 2002;
Bettler et al., 2004), mimicked
the GABA-induced enhancement of the electrical responses of the acinar cells
to SDN-stimulation, with SKF97541 being the more effective agonist
(Fig. 6A,B,G). The
GABABR antagonists CGP54626 (5µmoll–1) and
CGP52432 (5µmoll–1) suppressed the enhancement of the
acinar cell response amplitudes when co-applied with GABA
(Fig. 6C,D,G). Likewise,
CGP52432 suppressed the SKF97541-induced enhancement of the electrical
responses elicited by SDN-stimulation (Fig.
6E,G). These data suggest the involvement of a GABABR
in the GABA-induced augmentation of the response amplitudes of salivary gland
acinar cells that are induced by electrical SDN-stimulation.
With respect to the GABAA receptor (GABAAR), neither the agonist muscimol (100 µmol l–1) nor the antagonist picrotoxin (100 µmol l–1) affected the Vm changes induced by SDN-stimulation (Fig. 6G). Co-application of picrotoxin and GABA led to enhanced cell responses that did not differ significantly from those induced by GABA alone (Fig. 6G). However, the GABAAR antagonist bicuculline (5 µmol l–1) enhanced the cell responses significantly when applied alone or when co-applied with GABA, whereby co-application tended to further enhance the response amplitudes, although this effect was not statistically significant (Fig. 6F,G). These data may indicate an additional involvement of an ionotropic GABA receptor.
The GABAC receptor (GABACR) specific antagonists
TPMPA and THIP (100 µmol l–1) had no significant effect on
the response amplitudes when applied alone. When THIP and TPMPA were applied
together with GABA, the response amplitudes were not significantly enhanced
when compared with the control stimulations but were also not significantly
reduced when compared with the GABA effect.
(Fig. 6G, first bar) As THIP is
also acting as an agonist on GABAARs
(Waszczak et al., 1980), the
tendency to reduce the GABA-induced augmentation of Vm
changes induced by SDN-stimulation may be due to an activation of
GABAARs. However, we do not suggest ionotropic receptors of the
GABAC type to be involved in the GABAergic neurotransmission in
cockroach salivary glands, as both agonists had no effect when they were
applied alone.
Does GABA affect the rates of fluid and protein secretion?
As GABA augmented the electrical changes in membrane potential induced by
SDN-stimulation, it was to be expected that it also affects the rate of fluid
and protein secretion. In order to test this hypothesis, we measured the rates
of fluid and protein secretion during SDN-stimulation in the absence and
presence of GABA in the bathing solution.
Unstimulated glands did not secrete measurable amounts of saliva, and bath application of 5 µmol l–1 GABA did not elicit saliva secretion (not shown). Electrical SDN-stimulation (0.2 ms pulses, 8 V, 10 Hz) induced saliva secretion that reached its maximal rate within 3 min (Fig. 7A). The rate of secretion during this first stimulation period served as a control for gland function, and an identical stimulation regime was repeated at the end of each experiment. In order to test whether GABA affects the rate of fluid secretion, we reduced the stimulation frequency (1–2 Hz) close to threshold for fluid secretion because electrical SDN-stimulation does not only stimulate the thick dopaminergic axon and, perhaps, also thin serotonergic axons in the SDN but probably also stimulates the thick GABAergic axon. After 10 min, 5µmol l–1 GABA was applied in the bath for 5 min (Fig. 7A). During GABA application, the rate of fluid secretion induced by SDN-stimulation increased 2.5-fold from 6.8±2.4 to 16.7±2.4 nl min–1 (Fig. 7B). After GABA washout, the rate of secretion decreased (Fig. 7B) to 3.7±0.9 nl min–1 (N=7). Thus, GABA does not only augment neurotransmission but also the rate of fluid secretion.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Gifford et al., 1991;
Elia et al., 1994;
Baumann et al., 2002;
Baumann et al., 2004), and that
the release of dopamine and 5-HT by these neurons elicits salivation
(Bowser-Riley and House, 1976;
Just and Walz, 1996). However,
based on morphological data, it had to be assumed that additional
neurotransmitters and/or neurohormones are released at the cockroach salivary
gland. First, the SDN contains two thick axons and several thin ones
(Whitehead, 1971;
Baumann et al., 2004;
Watanabe and Mizunami, 2006).
One thick axon (SN1) is dopaminergic whereas the thin axons are serotonergic
(Davis, 1985;
Baumann et al., 2002;
Baumann et al., 2004), leaving
the neurotransmitter content of the second thick axon (SN2) unidentified.
Second, there are at least two different types of nerve terminals on the
salivary gland, as judged by the morphology of their synaptic vesicles, and
each terminal contains clear vesicles and dense-core vesicles
(Maxwell, 1978;
Baumann et al., 2004). Thus,
co-transmitters may be released along with dopamine and serotonin at the
salivary gland. In accordance with the above hypothesis, the present study provides evidence for the presence of an additional neurotransmitter in nerve fibers that innervate the salivary glands of the cockroach, viz. GABA. The question of whether or not GABA is a co-transmitter of serotonergic and/or dopaminergic neurons could be unequivocally resolved by back-tracing the GABAergic neuron along the SDN to its soma in the SOG. GABA is present only in one thick axon within the SDN and this axon originates from the SN2 soma. Moreover, the distribution of GABA-positive nerve fibers over the salivary gland complex displays differences to both the dopaminergic and the serotonergic innervation pattern (Fig. 7). GABA is thus not a co-transmitter of dopaminergic or serotonergic neurons but GABAergic fibers provide a third source of innervation for the salivary gland.
The GABAergic innervation pattern of the acinar tissue parallels neither
the serotonergic nor the dopaminergic innervation pattern. Serotonergic fibers
form a dense network over and within each acinar lobule whereas the salivary
duct system, except for short segments next to acinar lobules, lacks
serotonergic innervation (Baumann et al.,
2002; Baumann et al.,
2004). Dopaminergic fibers form a loose network on the outer
surface of acinar lobules and ramify within nerves that interlink acinar
lobules. Moreover, dopaminergic fibers are present on the salivary duct system
over its entire length (Davis,
1985; Baumann et al.,
2002; Baumann et al.,
2004). GABAergic fibers also form a loose meshwork on the outer
surface of most acinar lobules. In contrast to the dopaminergic system,
however, GABAergic fibers extend into a few lobules to terminate between
C-cells. GABAergic fibers also ramify within nerves that interlink acinar
lobules, although not as extensively as the dopaminergic fibers, and the
entire salivary duct system lacks GABA-positive fibers.
The finding that acinar lobules differ in their anti-GABA labeling pattern
was quite unexpected because serotonergic and dopaminergic innervation does
not display such variability (Baumann et
al., 2002; Baumann et al.,
2004). This raises the question of whether these lobules also
differ in other attributes. So far, evidence in favour or against this
possibility is lacking.
What are the targets of GABA action on the salivary gland? In theory, there
are two possibilities that do not exclude each other: GABA may act (1)
directly on P-cells and/or C-cells, or (2) presynaptically on release sites
for serotonin and/or dopamine. To distinguish between these possibilities, we
have localized putative sites of GABA release. Using anti-synapsin as a
marker, we have shown that some of the putative GABA release sites on the
secretory tissue reside next to GABA-negative release sites, suggesting that
these are presynaptic to release sites for other neurotransmitters. However,
we cannot exclude the possibility that the close apposition is coincidental
because direct presynaptic contacts have not been detected on electron
micrographs (Maxwell, 1978;
Baumann et al., 2004).
Conversely, numerous putative GABA release sites have no other putative
release sites close by. This especially concerns putative GABA release sites
in nerves that interlink acinar lobules, providing further evidence that these
structures serve as neurohemal organs
(Baumann et al., 2004).
Support for a presynaptic GABA action is provided by: (1) the observation
that GABA does not induce fluid secretion and (2) the results of our
intracellular recordings from acinar cells. GABA had no direct effect on the
resting Vm of acinar cells and it did not affect their
electrical responses induced by bath application of sub-saturating
concentrations of dopamine or 5-HT. These results agree with earlier
observations made on isolated salivary glands of the cockroach Nauphoeta
cinerea, showing that GABA does not affect the resting
Vm of acinar cells
(Bowser-Riley and House, 1976).
In this study, we found that superfusion of Periplaneta salivary
glands with GABA augments the electrical responses of acinar cells when these
are induced by electrical stimulation of the SDN. It may thus be concluded
that GABA does not act directly on the acinar cells but rather modulates
dopaminergic and/or serotonergic neurotransmission in the salivary gland.
The GABA-induced augmentation of the electrical responses of the acinar
cells and of the rates of fluid and protein secretion induced by electrical
stimulation of the SDN was rather unexpected, as GABA is best known for its
inhibitory function. It has been reported, however, that GABA can also exert
excitatory action in some systems (Beg and
Jorgensen, 2003; Gulledge and
Stuart, 2003; Stein and
Nicoll, 2003; Gisselmann et
al., 2004). We noted the narrow range of GABA concentrations that
affect electrical responses induced by SDN-stimulation. One has to be aware,
however, that SDN-stimulation may lead not only to the release of 5-HT and
dopamine but also to the release of GABA. Thus, our assays are not suitable to
show or measure the entire dynamic range for GABA action.
In Nauphoeta, GABA did not augment but slightly reduced the
electrical responses induced in secretory cells by electrical stimulation of
the SDN (Bowser-Riley and House,
1976). However, this inhibitory effect in Nauphoeta
required GABA concentrations of >100 µmol. In our Periplaneta
nerve-gland preparation, 5 µmol GABA was sufficient for the enhancement of
the responses induced by SDN-stimulation.
Which receptors mediate the GABA action in the salivary gland? The effects
of GABA on the acinar cell response amplitudes were sensitive to a number of
GABABR-specific ligands and recovered slowly. This indicates the
involvement of metabotropic GABA receptors (GABABRs). Both tested
GABABR agonists enhanced the acinar cell response amplitudes
induced by electrical SDN-stimulation and, thus, mimicked the GABA-induced
augmentation. Moreover, the GABABR antagonists suppressed the
GABA-induced augmentation of the electrical responses to SDN-stimulation. As
GABA has potentiating effects in our preparation, we suppose an increased
transmitter release from dopaminergic and/or serotonergic release sites.
GABABRs are described to couple to the cAMP pathway by increasing
or decreasing adenylyl cyclase acitivity
(Hill, 1985;
Olianas and Onali, 1999;
Mezler et al., 2001). Elevated
presynaptic cAMP can increase transmitter release
(Yoshihara et al., 2000;
Kaneko and Takahashi, 2004;
Cheung et al., 2006). GABA
could increase transmitter release from dopaminergic and/or serotonergic
release sites on Periplaneta salivary glands in a similar way,
resulting in enhanced responses of the acinar cells upon SDN-stimulation.
The bicuculline sensitivity of the GABA effect indicates that an ionotropic
GABA receptor (GABAAR) may be involved in addition to the
GABABR. GABAARs are known to inhibit neurotransmission
by increasing chloride currents that hyperpolarize the pre- or postsynaptic
membrane and thus reduce transmitter release and/or excitability (for reviews,
see MacDermott et al., 1999;
Engelmann and MacDermott, 2004). The enhancement of acinar cell responses by
the GABAAR antagonist bicuculline supports this view.
The salivary glands in Periplaneta are innervated by serotonergic
and dopaminergic neurons (Davis,
1985; Elia et al.,
1994; Baumann et al.,
2002; Baumann et al.,
2004), and stimulation by dopamine or serotonin results in the
secretion of saliva without or with proteins
(Just and Walz, 1996;
Marg et al., 2004;
Rietdorf et al., 2005;
Troppmann et al., 2007). Thus,
it must be envisaged that the involved GABA receptor subtypes are located on
different neurons so that the release of dopamine and serotonin is modulated
by GABA via an ionotropic or a metabotropic GABA receptor
differentially. Because GABA enhanced the rate of protein secretion 4-fold, it
seems likely that GABABR are located at least on the serotonergic
neurons. The unequivocal identification of the target neuron(s) for GABA
action will require the localization of the GABA receptors.
Besides the acinar tissue, GABAergic fibers and putative GABA release sites
are also associated with the reservoir system including the reservoir muscle.
The functions of the reservoir system are still enigmatic. It may be a storage
compartment for saliva but it could also have osmoregulatory function
(Raychaudhuri and Gosh, 1963;
Sutherland and Chillseyzn,
1968). The reservoir muscle may act as an occlusor of the
reservoir orifice and, as the muscle relaxes, hemolymph pressure may cause
compression of the reservoir walls and emptying of the reservoir
(Sutherland and Chillseyzn,
1968). In view of this scenario and in view of the confinement of
GABAergic fibers to the orifice region of the reservoir and of the reservoir
muscle, this fiber system could also be involved in the regulation of
reservoir emptying.
In view of these findings, it may be concluded that GABA acts on cockroach
salivary glands as a neuromodulator rather than as a classical
neurotransmitter. This GABA action is very likely to be functionally important
for the regulation of salivation and saliva composition because SN2 displays
spontaneous spike activity that increases during feeding
(Watanabe and Mizunami,
2006).
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Ali, D. (1997). The aminergic and peptidergic innervation of insect salivary glands. J. Exp. Biol. 200,1941 -1949.[Abstract]
Baumann, O., Dames, P., Kühnel, D. and Walz, B. (2002). Distribution of serotonergic and dopaminergic nerve fibers in the salivary gland complex of the cockroach Periplaneta americana. BMC Physiol. 2,1 -15.[Medline]
Baumann, O., Kühnel, D., Dames, P. and Walz, B.
(2004). Dopaminergic and serotonergic innervation of cockroach
salivary glands: distribution and morphology of synapses and release sites.
J. Exp. Biol. 207,2565
-2575.
Beg, A. A. and Jorgensen, E. M. (2003). EXP-1 is an excitatory GABA-gated cation channel. Nat. Neurosci. 6,1145 -1152.[CrossRef][Medline]
Bettler, B., Kaupmann, K., Mosbacher, J. and Gassmann, M.
(2004). Molecular structure and physiological functions of
GABAB. receptors. Physiol. Rev.
84,835
-867.
Bowery, N. G., Bettler, B., Froestl, W., Gallagher, J. P.,
Marshall, F., Raiteri, M., Bonner, T. I. and Enna, S. J.
(2002). International Union of Pharmacology. XXXIII. Mammalian
-aminobutyric acidB. receptors: structure and function.
Pharmacol. Rev. 54,247
-264.
Bowser-Riley, F. and House, C. R. (1976). The
actions of some putative neurotransmitters on the cockroach salivary gland.
J. Exp. Biol. 64,665
-676.
Bowser-Riley, F., House, C. R. and Smith, R. K.
(1978). Competitive antagonism by phentolamine of responses to
biogenic amines and the transmitter at a neuroglandular junction.
J. Physiol. (Lond.) 279,473
-489.
Bradford, M. M. (1976). A rapid and sensitive methof for the quantification of microgramm quantities of protein utilizing the principle of protein-dye-binding. Anal. Biochem. 72, 15-23.
Cheung, U., Atwood, H. L. and Zucker, R. S. (2006). Presynaptic effectors contributing to cAMP-induced synaptic potentiation in Drosophila. J. Neurobiol. 66,273 -280.[CrossRef][Medline]
Davis, N. T. (1985). Serotonin-immunoreactive visceral nerves and neurohemal system in the cockroach Periplaneta americana (L.). Cell Tissue Res. 240,593 -600.[CrossRef]
Elia, A. J., Ali, D. W. and Orchard, I. (1994). Immunochemical staining of tyrosine hydroxylase(TH)-like material in the salivary glands and ventral nerve cord of the cockroach, Periplaneta americana (L.). J. Insect Physiol. 40,671 -683.[CrossRef]
Engelman, H. S. and MacDermott, A. B. (2004). Presynaptic ionotropic receptors and control of transmitter release. Nat. Rev. Neurosci. 5,135 -145.[CrossRef][Medline]
Gifford, A. N., Nicholson, R. A. and Pitman, R. M.
(1991). The dopamine and 5-hydroxytryptamine content of locust
and cockroach salivary neurones. J. Exp. Biol.
161,405
-414.
Gisselmann, G., Plonka, J., Pusch, H. and Hatt, H. (2004). Drosophila melanogaster GRD and LCCH3 subunits form heteromultimeric GABA-gated cation channels. Br. J. Pharmacol. 142,409 -413.[CrossRef][Medline]
Gulledge, A. T. and Stuart, G. J. (2003). Excitatory actions of GABA in the cortex. Neuron 37,299 -309.[CrossRef][Medline]
Gupta, B. L. and Hall, T. A. (1983). Ionic distribution in dopamine-stimulated NaCl fluid-secreting cockroach salivary glands. Am. J. Physiol. 244,R176 -R186.[Medline]
Hill, D. R. (1985). GABAB receptor modulation of adenylate cyclase activity in rat brain slices. Br. J. Pharmacol. 84,249 -257.[Medline]
Hille, C. and Walz, B. (2007). A vacuolar-type
H+-ATPase and a Na+/H+ exchanger contribute
to intracellular pH regulation in cockroach salivary ducts. J. Exp.
Biol. 210,1463
-1471.
Just, F. and Walz, B. (1994). Salivary glands of the cockroach, Periplaneta americana: new data from light and electron microscopy. J. Morphol. 220, 35-46.[CrossRef][Medline]
Just, F. and Walz, B. (1996). The effects of serotonin and dopamine on salivary secretion by isolated cockroach salivary glands. J. Exp. Biol. 199,407 -413.[Abstract]
Kaneko, M. and Takahashi, T. (2004).
Presynaptic mechanism underlying cAMP-dependent synaptic potentiation.
J. Neurosci. 24,5202
-5208.
Klagges, B. R., Heimbeck, G., Godenschwege, T. A., Hofbauer, A.,
Pflugfelder, G. O., Reifegerste, R., Reisch, D., Schaupp, M., Buchner, S. and
Buchner, E. (1996). Invertebrate synapsins: a single gene
codes for several isoforms in Drosophila. J. Neurosci.
16,3154
-3165.
Lang, I. and Walz, B. (1999). Dopamine stimulates salivary duct cells in the cockroach, Periplaneta americana.J. Exp. Biol. 202,729 -738.[Abstract]
Lang, I. and Walz, B. (2001). Dopamine-induced epithelial K+ and Na+ movements in the salivary ducts of Periplaneta americana. J. Insect Physiol. 47,465 -474.[CrossRef][Medline]
MacDermott, A. B., Role, L. W. and Siegelbaum, S. A. (1999). Presynaptic ionotropic receptors and the control of transmitter release. Annu. Rev. Neurosci. 22,443 -485.[CrossRef][Medline]
Marg, S., Walz, B. and Blenau, W. (2004). The effects of dopamine receptor agonists and antagonists on the secretory rate of cockroach (Periplaneta americana) salivary glands. J. Insect Physiol. 50,821 -830.[CrossRef][Medline]
Maxwell, D. J. (1978). Fine structure of the axons associated with the salivary apparatus of the cockroach, Nauphoeta cinerea. Tissue Cell 10,699 -706.[CrossRef][Medline]
Mezler, M., Müller, T. and Raming, K. (2001). Cloning and functional expression of GABAB receptors from Drosophila. Eur. J. Neurosci. 13,477 -486.[CrossRef][Medline]
Olianas, M. C. and Onali, P. (1999). GABAB receptor-mediated stimulation of adenylyl cyclase activity in membranes of rat olfactory bulb. Br. J. Pharmacol. 126,657 -664.[CrossRef][Medline]
Raychaudhuri, D. N. and Ghosh, S. K. (1963). Study of the histophysiology of the salivary apparatus of Periplaneta americana Linn. Zool. Anz. 173,227 -237.
Rietdorf, K., Lang, I. and Walz, B. (2003). Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation, and effects of ouabain and bumetanide. J. Insect Physiol. 49,205 -215.[CrossRef][Medline]
Rietdorf, K., Blenau, W. and Walz, B. (2005). Protein secretion in cockroach salivary glands requires both, an increase in Ca2+ and cAMP concentrations. J. Insect Physiol. 51,1083 -1091.[CrossRef][Medline]
Stein, V. and Nicoll, R. A. (2003). GABA generates excitement. Neuron 37,375 -378.[CrossRef][Medline]
Sutherland, D. J. and Chillseyzn, J. M. (1968). Function and operation of cockroach salivary reservoir. J. Insect Physiol. 14,21 -31.[CrossRef][Medline]
Troppmann, B., Walz, B. and Blenau, W. (2007). Pharmacology of serotonin-induced salivary secretion in Periplaneta americana. J. Insect Physiol. 53,774 -781.[CrossRef][Medline]
Walz, B., Baumann, O., Krach, C., Baumann, A. and Blenau, W. (2006). The aminergic control of cockroach salivary glands. Arch. Insect Biochem. Physiol. 62,141 -152.[CrossRef][Medline]
Waszczak, B. L., Hruska, R. E. and Walters, J. R. (1980). GABAergic actions of THIP in vivo and vitro: a comparison with muscimol and GABA. Eur. J. Pharmacol. 65,21 -29.[CrossRef][Medline]
Watanabe, H. and Mizunami, M. (2006). Classical
conditioning of activities of salivary neurones in the cockroach.
J. Exp. Biol. 209,766
-779.
Watkins, B. L. and Burrows, M. (1989). GABA-like immunoreactivity in the suboesophageal ganglion of the locust Schistocerca gregaria. Cell Tissue Res. 258, 53-63.
Whitehead, A. T. (1971). The innervation of the salivary gland in the American cockroach: light and electron microscopic observations. J. Morphol. 135,483 -506.[CrossRef]
Yoshihara, M., Suzuki, K. and Kidokoro, Y.
(2000). Two independent pathways mediated by cAMP and protein
kinase A enhance spontaneous transmitter release at Drosophila
neuromuscular junctions. J. Neurosci.
20,8315
-8322.
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