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The Journal of Neuroscience, October 15, 1999, 19(20):8808-8817
Presynaptic Inhibition of Primary Olfactory Afferents Mediated by
Different Mechanisms in Lobster and Turtle
Matt
Wachowiak and
Lawrence B.
Cohen
Department of Cellular and Molecular Physiology, Yale University
School of Medicine, New Haven, Connecticut 06520, and Marine Biological
Laboratory, Woods Hole, Massachusetts 02543
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ABSTRACT |
Presynaptic regulation of transmission at the first olfactory
synapse was investigated by selectively imaging axon terminals of
receptor neurons in the lobster olfactory lobe and turtle olfactory bulb. In both species, action potential propagation into axon terminals
after olfactory nerve stimulation was measured using voltage-sensitive
dyes. In addition, in the turtle, calcium influx into terminals was
measured by selectively labeling receptor neurons with
dextran-conjugated calcium indicator dyes. In the lobster, application
of the inhibitory transmitters GABA or histamine suppressed action
potentials in the terminals. The suppression was blocked by picrotoxin
and cimetidine, respective antagonists to lobster GABA and histamine
receptors. These results suggest that previously characterized GABA and
histaminergic interneurons regulate olfactory input by suppressing
action potential propagation into axon terminals of olfactory
afferents. In contrast, in the turtle olfactory bulb, neither GABA nor
dopamine had any effect on receptor cell action potentials as measured
with voltage-sensitive dyes. However, calcium influx into axon
terminals was reduced by the GABAB agonist baclofen and the
dopamine D2 agonist quinpirole, and paired-pulse
suppression of calcium influx was reduced by the GABAB
antagonist saclofen. These results indicate that in the turtle, GABA
and dopamine mediate presynaptic inhibition not by affecting action
potentials directly, as in the lobster, but by reducing calcium influx
via GABAB and dopamine D2 receptors. Thus,
although mediated by different cellular mechanisms, presynaptic
regulation of olfactory input to the CNS, via dual synaptic pathways,
is a feature common to vertebrates and invertebrates. This inhibition
may be important in the processing of olfactory information.
Key words:
olfactory bulb; presynaptic inhibition; imaging; voltage-sensitive dye; calcium dye; GABA; dopamine; histamine
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INTRODUCTION |
Presynaptic inhibition of primary
afferent fibers is an important feature of many vertebrate and
invertebrate sensory systems, including cutaneous and muscle stretch
receptor afferents in mammals (for review, see Rudomin, 1990 ; Watson,
1992) and stretch receptor, proprioreceptor, and mechanoreceptor
afferents of arthropods (Blagburn and Sattelle, 1987; Burrows and
Laurent, 1993; Clarac and Cattaert, 1996). Functional roles of
presynaptic afferent inhibition include preventing habituation,
sharpening the receptive fields of afferent fibers, and serving as a
mechanism for gain control of sensory input to the CNS (Watson, 1992).
In the olfactory system, studies in both invertebrates and vertebrates
have suggested that presynaptic inhibition may play a role in
processing olfactory input. In the cockroach, GABAergic interneurons
form presynaptic contacts with olfactory receptor axon terminals
(Distler and Boeckh, 1997). In the vertebrate olfactory bulb,
physiological evidence for presynaptic inhibition comes from
measurements of postsynaptic activity elicited by olfactory nerve shock
and recorded with field potentials, intracellular mitral cell
recordings, or optical imaging, all of which show that nerve shock
causes a long-lasting suppression of responses to subsequent shocks
that cannot be explained by inhibition of postsynaptic olfactory bulb
neurons (Orbach and Cohen, 1983; Mori et al., 1984; Nickell et al.,
1994; Potapov, 1985; Keller et al., 1998). In the latter three
studies, the suppression was mimicked by the
GABAB receptor agonist baclofen, suggesting that
GABAB receptors may mediate presynaptic
inhibition. In addition, D2 dopamine receptors
are present in the olfactory nerve layer of the rat olfactory bulb
(Nickell et al., 1991), leading to the hypothesis that
D2 receptors may also regulate transmission at the first olfactory synapse. However, a direct physiological
demonstration of the events underlying presynaptic inhibition was
hampered by the inaccessibility of presynaptic terminals or their
postsynaptic sites to conventional electrode-based recording techniques.
In a recent study (Wachowiak and Cohen, 1998), we used
voltage-sensitive dyes to record action potentials selectively from olfactory receptor axon terminals in the spiny lobster and demonstrated that presynaptic afferent inhibition is present in this system. The
present study was designed to characterize the role of inhibitory transmitters in mediating presynaptic afferent inhibition in the lobster and to test whether presynaptic inhibition also regulates olfactory input to the olfactory bulb of a vertebrate, the box turtle
Terapene carolina. In the turtle, we measured action
potential invasion of receptor cell axon terminals as well as calcium
influx into the terminals after anterograde labeling with a
dextran-conjugated calcium indicator (Friedrich and Korsching, 1997).
Our findings indicate that in the lobster, GABA and histamine can each
suppress action potential invasion of olfactory axon terminals, whereas in the turtle the inhibitory transmitters GABA and dopamine cause no
such suppression. However, GABA and dopamine do appear to regulate calcium influx into the terminals of turtle olfactory afferents via
GABAB and D2 receptor
activation, respectively.
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MATERIALS AND METHODS |
Preparations. Optical signals were recorded from
three in vitro preparations: the olfactory lobe of the spiny
lobster Panulirus argus, using a perfused, isolated brain
preparation; the lobster antennular nerve, using a cut section of
nerve; and the olfactory bulb of the turtle T. carolina,
using a superfused hemisected bulb preparation. The lobster brain and
turtle olfactory bulb preparations have been described previously
(Berkowicz et al., 1994; Wachowiak and Ache, 1994).
In the lobster brain preparation, the animal was chilled on ice and
decapitated, and the brain was cannulated via the medial artery and
perfused with oxygenated Panulirus saline. The brain was
then removed from the head, along with associated blood vessels, by
cutting all nerve roots and the optic tracts leading to the protocerebrum. The brain was pinned in a Sylgard recording chamber with
the olfactory lobes facing upward and was desheathed, and the left
antennular nerve was placed in a glass suction electrode for stimulation.
In the nerve preparation, a 5-7 mm length of nerve was excised just
distal to the point where the nerve entered the brain and secured in a
recording chamber containing two suction electrodes. One suction
electrode was used to stimulate from one cut end, and the other
electrode was used to record the compound action potential from the
other end. The nerve was continuously superfused with oxygenated saline
as in the isolated brain preparation.
In the turtle preparation, animals were anesthetized by chilling on ice
for 2-3 hr and decapitated, and the forebrain including the intact
olfactory bulbs was removed and placed in chilled turtle Ringer's
solution bubbled with 95% O2/5%
CO2. The olfactory bulb from one side was
isolated by making a coronal cut through the forebrain just caudal to
the bulb. The bulb was then hemisected in the horizontal plane, and one
hemisection was pinned down in the recording chamber with the outer
surface facing upward. The olfactory nerve, which is large and distinct
in turtles, was drawn into a suction electrode for stimulation. The
hemisection was kept continuously superfused with turtle Ringer's
solution (1-2 ml/min) via the same system as for the lobster, with the
perfusion cannula secured in the bath.
Drug delivery and solution replacement. A three-way solenoid
valve (Cole-Parmer, Chicago, IL) allowed switching between normal and
Ca2+-free saline solutions in the
perfusion system. Trials with a marker dye in the solution showed that
complete replacement of the solution occurred at the tip of the
perfusion cannula within 2 min at a flow rate of 0.5 ml/min.
Replacement of saline in the tissue of the lobster brain or turtle bulb
was presumably slower. In the lobster the brain was perfused with
Ca2+-free solution for 5-10 min before
further trials, whereas in the turtle bulb 15-20 min was allowed for
solution replacement.
Transmitter agonists and antagonists were introduced into the
background perfusion via a manual four-way loop injector (Rheodyne, Rohnert Park, CA). Injected solutions reached the cannula tip within 30 sec, and the size of the injection loop was large enough to allow a
sustained peak concentration for up to 4 min. Normally, trials were
performed 2 min after drug injection. The drug concentrations reported
here are those injected into the perfusion system. No attempt was made
to correct for possible dilution in the brain or in the bath.
Optical recordings. For the voltage-sensitive dye
experiments, lobster brain tissue was stained with di-2-ANEPHQ
(JPW-2081; synthesized by J. P. Wuskell and L. M. Loew,
University of Connecticut Health Center, Farmington, CT) by introducing
2 ml dye (0.06 mg/ml) into the perfusion line or by applying dye (0.36 mg/ml) in the bath for 20 min. Dye perfusion resulted in uniform
staining of the brain, whereas bath application stained only the
outermost 50-100 µm of tissue. Turtle olfactory bulb tissue was
stained by bath application of di-4-ANEPPS (0.05 mg/ml) or RH-414 (0.05 mg/ml) (Grinvald et al., 1994) (both from Molecular Probes, Eugene, OR)
for 30 min.
For the turtle calcium-dye experiments, olfactory receptor axon
terminals were selectively labeled by anterograde transport of the
dextran-conjugated calcium indicator Calcium Green-1 or Calcium Crimson
(both 10 kDa; Molecular Probes) applied to the olfactory epithelium
using a protocol adapted from Friedrich and Korsching (1997). Turtles
were first chilled on ice for 1-2 hr, then placed upside down with the
mouth held open; 25-60 µl of a 2% dye solution dissolved in 0.1 M NaCl plus 0.5% Triton X-100 was injected into each
naris. The pharyngeal opening of each naris was plugged to prevent
leakage, and the epithelium was stained for 10-20 min, after which the
plugs were removed and the mouth closed, allowing the animal to expel
the dye from the nose. The turtles were then held at room temperature
for 4-12 d before recording. Sections through the nares and olfactory
bulb of experimental animals revealed fluorescence in the olfactory
cell body layer of the epithelium and the olfactory nerve and
glomerular layers of the olfactory bulb. Although glomeruli could be
clearly distinguished by the labeled fibers, no fluorescence was
visible in subglomerular layers (data not shown).
To record voltage-sensitive dye and Calcium Crimson signals, the
preparation was illuminated with light from a tungsten halogen lamp
passed through a 520 ± 45 nm interference filter, reflected from
a 590 nm long-pass dichroic mirror, and focused on the preparation using a 25 mm, 0.95 f, video lens (Kleinfeld and Delaney, 1996). For Calcium Green signals, a 460 ± 30 nm interference filter and a 515 nm dichroic mirror were used. In all experiments, a
3.5-mm-diameter field was imaged with a 0.4 NA onto an array of 464 photodiodes. Each photodiode received light from a square region of the
object plane ~150 µm on a side. The photocurrents from each diode
were amplified separately, bandpass-filtered by the amplifiers
(0.07-500 Hz), and digitized at 1 kHz under the control of NeuroPlex
software (OptImaging, LLC, Fairfield, CT) on an IBM-PC computer.
Additional digital filtering and spatial averaging were performed
off-line using NeuroPlex. All recordings were from single trials. The
mean photocurrent from the diodes was ~8 nA for the voltage-sensitive dye recordings (average from five representative preparations) and 0.4 nA for the calcium dye recordings. Because we were interested in
measuring population responses from a large fraction of the neurons in
a particular region (e.g., olfactory nerve, olfactory lobe, olfactory
bulb), spatial averages of signals from four to nine adjacent
photodiodes were used in measuring all responses. The photodiodes were
chosen such that they covered 10-50% of the region of interest.
However, measurements made with single photodiodes consistently showed
the same effects as reported from the spatial averages. To
determine the detector locations relative to the image of the
preparation, a high-resolution image of the acquisition area was
obtained by deflecting the light path to a 768 × 494 pixel CCD
camera (Dage MTI, Michigan City, IN).
Experimental protocol and data analysis. The experimental
protocol was to record responses to a pair of electrical shocks to the
olfactory nerve [300-400 msec interstimulus interval (ISI)] with the lobster brain or turtle bulb in a series of different solutions (for example, normal saline,
Ca2+-free saline,
Ca2+-free saline with agonist or
antagonist, recovery in Ca2+-free, and
recovery normal salines). A single series of solution changes could
take up to 40 min. For the voltage-sensitive dye experiments, the
overall signal amplitude often decreased during this time, probably
attributable to dye washout (we did not detect a significant
amount of photobleaching), especially in the lobster preparations
because of the relatively low hydrophobicity of JPW-2081P. To correct
for this effect, the rate of washout was assumed to be linear and was
calculated based on the amplitudes of test responses in normal saline
at the beginning and end of each experiment. Amplitude measurements
were then standardized using this time-dependent correction factor. The
time-corrected data were used to generate the numbers reported in the
text and shown in the summary figures. Because these corrections were
not large, uncorrected data are shown in the traces of Figures 1, 2, 4,
6, 7, and 8. No correction was used for the calcium dye experiments
because we saw no sign of dye washout or photobleaching. In all traces
in the figures, fluorescence changes corresponding to a depolarization
and an increase in Ca2+ are plotted
upward, regardless of the direction of change in fluorescence.
Solutions. Normal Panulirus saline consisted of
(in mM): 460 NaCl, 13 KCl, 13 CaCl2, 10 MgCl2, 14 Na2SO4, 1.7 glucose, 3 HEPES, pH 7.4. Normal turtle Ringer's solution consisted of (in
mM): 96.5 NaCl, 2.6 KCl, 4 CaCl2, 2 MgCl2, 10 glucose,
31.5 NaHCO3, pH 7.4. For
Ca2+-free,
high-Mg2+ solutions (lobster and turtle
salines), the CaCl2 was replaced with
MgCl2. In addition, 1 mM
EGTA was added to the Ca2+-free turtle
Ringer's solution. GABA, histamine, quinpirole, ( )-baclofen, ()-saclofen, DL-2-amino-5-phosphonovaleric acid
(AP5), and 6,7-dinitroquinoxaline-2,3-dione (DNQX) were prepared from
frozen aliquots of stock solution (1-100 mM).
Dopamine was prepared fresh daily in turtle Ringer's solution plus
0.1% sodium metabisulfate to prevent oxidation. Picrotoxin, cimetidine, and bicuculline methiodide were prepared fresh daily. All
solutions were at pH 7.4. Agonist and antagonists were obtained from
RBI (Natick, MA), and all other chemicals were obtained from Sigma (St.
Louis, MO) or Fisher (Fair Lawn, NJ).
Animals. Spiny lobsters, obtained in the Florida Keys by the
Keys Marine Laboratory, were kept in flow-through seawater (20-22°C) at the Marine Biological Laboratory (Woods Hole, MA). Turtles were
obtained from Charles D. Sullivan Co. (Nashville TN) and kept at 14°C
until ready for use. All experiments were performed at room temperature
(22-24°C). Experimental protocols were approved by the Yale Animal
Care and Use Committee and by the Marine Biological Laboratory
Institutional Animal Care and Use Committee.
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RESULTS |
Evoked optical responses in lobster olfactory lobe
Optical signals arising selectively from lobster olfactory
receptor cell axon terminals were isolated by delivering paired electrical shocks to the olfactory (antennular) nerve and by perfusing the brain with
Ca2+-free/high-Mg2+
saline. Figure 1 shows responses evoked
by paired-pulse nerve stimulation as recorded from three sites in a
preparation stained by perfusion of voltage-sensitive dye: the
antennular nerve (A), the olfactory lobe
(B), and a second-order olfactory neuropile, the
accessory lobe (C). In normal saline (Fig. 1,
top traces) the typical response patterns were as follows.
In the antennular nerve, we observed short-latency spikes reflecting
directly evoked action potentials in response to both the conditioning
and test pulses. In contrast, the olfactory lobe signal evoked by a
conditioning pulse consisted of a large, rapid transient followed by a
smaller component consisting of a slow depolarization and,
occasionally, a series of two to four small-amplitude spikes (not
apparent in Fig. 1). This complex response presumably reflects primary
afferent as well as postsynaptic activity. The test pulse evoked only a simple small-amplitude spike with no late component. Finally, in the
accessory lobe, the conditioning pulse evoked a short-latency, very
small-amplitude depolarization followed by a complex long-latency response, also consisting of a depolarization underlying a burst of
spikes or oscillations. The test pulse elicited only the small, short-latency response.
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Figure 1.
Voltage-sensitive dye signals evoked by
paired-pulse stimulation in normal (top traces) and zero
Ca2+/high-Mg2+ saline
(bottom traces), recorded from three different locations
in the isolated lobster brain. Paired-pulse suppression is not seen in
the olfactory nerve (A) but is dramatic in the
olfactory and accessory lobes (B, C). A
schematic diagram of the brain, bottom right, shows the
lateral olfactory lobes, innervated by primary olfactory afferents in
the antennular nerves, and the medial accessory lobes, which receive no
primary afferent input. Arrows indicate the conditioning
and test stimuli (2 msec duration). Each trace is the spatial average
of five to seven photodetectors. In addition to analog filtering before
data acquisition (0.07-500 Hz), the traces were digitally filtered
using a low-pass Gaussian filter and a high-pass RC filter
(A, 2.8-100 Hz; B, 1.4-40 Hz;
C, 1.4-100 Hz). The undershoot after the large spiking
responses (both traces in A, top
traces in B and C) is an artifact
of the digital high-pass RC filter. See Results for description
of evoked responses.
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The latencies, overall response patterns, and severe paired-pulse
depression of the complex responses in the olfactory and accessory
lobes agree closely with previous intracellular recordings from
individual interneurons in these lobes. Those recordings demonstrated
that all known olfactory and accessory lobe interneuron types show no
response to a test pulse at the 300-400 msec interstimulus intervals
used here (Wachowiak and Ache, 1994; Wachowiak et al., 1995, 1997).
Thus, as in our previous study (Wachowiak and Cohen, 1998), we conclude
that the olfactory lobe signal evoked by the test pulse to the
antennular nerve selectively reflects action potential activity in the
incoming olfactory receptor cell axons.
Presynaptic inhibition revealed by synaptic blockade
The bottom traces in Figure 1C show that perfusion of
the brain with Ca2+-free saline blocked or
severely attenuated synaptic transmission, as seen by the elimination
of the complex, long-latency component of the accessory lobe signal,
which receives only second-order inputs via olfactory lobe interneurons
(Wachowiak et al., 1995). The remaining small, short-latency component
likely reflects activation of nonolfactory afferents from the
antennular nerve passing underneath the accessory lobe. This component
has a latency similar to that of the antennular nerve response and was
not seen in the bath-stained preparations in which the deep tissue
underlying the accessory lobe was unstained. In the olfactory lobe,
perfusion with Ca2+-free saline initially
resulted in a delayed and prolonged depolarization after the
conditioning pulse that eventually disappeared after 5-10 min of
continued perfusion (data not shown). As reported previously (Wachowiak
and Cohen, 1998), intracellular recordings from olfactory lobe
projection neurons confirm that Ca2+-free
saline blocks or severely attenuates synaptic transmission from
receptor cells to interneurons. The final response pattern in the
olfactory lobe (Fig. 1B, bottom trace),
which persisted for as long as Ca2+-free
saline was present, consisted of a simple short-duration spike, similar
in latency to the evoked antennular nerve signal. In perfusion-stained
preparations (n = 8), where the optical signal is
dominated by activity in interneurons, the conditioning pulse response
in Ca2+-free saline was 36 ± 9% of
its amplitude in normal saline, with a latency to peak 2-3 msec
shorter in Ca2+-free saline. This decrease
was smaller in the two bath-stained preparations (72 ± 3% of
normal), where the optical signal is dominated by activity in receptor
cells and superficially branching interneurons (data not shown). In
subsequent analyses, we group the data from both bath- and
perfusion-stained preparations.
In contrast, the amplitude of the test response showed a significant
increase in the presence of
Ca2+-free saline (Fig.
1B, bottom trace). In 10 preparations, the amplitude of the test response increased to 149 ± 15% (mean ± SEM) of its value in normal saline (p = 0.009; one-sample t test, hypothesized mean = 100),
whereas the amplitude of the conditioning response decreased to 38 ± 6% of its control value (p < 0.0001). These
results agree with those of our preliminary study, in which 1 mM Ca2+ was used to
reduce synaptic transmission (Wachowiak and Cohen, 1998). The increase
in test response amplitude reflects an increase in the action potential
signal in the primary olfactory afferents under conditions of synaptic
blockade. The simplest explanation of this result is the elimination or
reduction of the normal, synaptically mediated suppression of action
potential propagation into the terminals that occurs in normal saline.
Although the conditioning and test pulse responses had identical
waveforms in Ca2+-free saline, we observed
that the test response was slightly, but significantly, greater in
amplitude than the conditioning response (12 ± 3%;
n = 10; p = 0.001) (Figs.
1B, 2). This difference was much less than the amount by which the test response increased in
Ca2+-free saline. Because of its
persistence and uncertain importance, we propose to name this
enhancement of the test response the Cohen Effect.
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Figure 2.
Effect of Ca2+-free
saline, GABA, histamine, and antagonists on the olfactory lobe response
evoked by paired pulses. A, Ca2+-free
saline eliminates postsynaptic components of the conditioning
response and increases the amplitude of the test response. GABA (100 µM) nearly eliminates both evoked responses, and the
effect of GABA is blocked in the presence of 100 µM
picrotoxin. Finally, the evoked response recovers to its initial
pattern after washout in normal saline. B, A different
preparation than in A. Again,
Ca2+-free saline eliminates postsynaptic activity
and increases the test response amplitude. Histamine (30 µM) nearly eliminates the evoked response, and this
effect is blocked by 5 mM cimetidine, followed by recovery
in normal saline. In A and B, a reduction
in absolute response amplitude attributable to dye washout can be seen
by comparing the top and bottom traces.
The change in test response amplitude at these two time points was used
to estimate the rate of dye washout, which was used as a correction
factor when normalizing response amplitudes for computing the values
shown in Figures 3 and 5. The undershoot in the top and
bottom traces is attributable to the digital high-pass
RC filter. Digital filtering was performed as described in Figure 1
using low- and high-pass cutoffs of 5.7 and 60 Hz.
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Transmitters mediating presynaptic afferent inhibition in
the lobster
The transmitters GABA and histamine both mediate inhibition in the
lobster olfactory lobe via distinct receptors (Wachowiak and Ache,
1997). The histamine receptor is weakly antagonized by cimetidine,
whereas the GABA receptor is antagonized by picrotoxin but not
bicuculline (Zhainazarov et al., 1997). We tested the ability of the
two transmitters to mediate inhibition of olfactory afferents by
perfusion-application in the presence of
Ca2+-free saline. Both transmitters
significantly and reversibly reduced the amplitude of the olfactory
lobe spike signal in Ca2+-free saline
(Fig. 2). The effects of histamine and GABA appeared dose-dependent,
with histamine more potent than GABA in reducing the spike amplitude.
Histamine (30 µM) reduced the evoked response amplitude
by 61 ± 8% (n = 3), whereas the same
concentration of GABA did not reduce the response (0 ± 1%
reduction; n = 2). Likewise, 100 µM histamine caused a significantly greater
reduction (82 ± 7%, n = 4) than did 100 µM GABA (45 ± 10%, n = 5; p = 0.025).
The effects of GABA and histamine were selectively blocked by their
respective receptor antagonists (Fig. 2). In two experiments, one of
which is shown in Figure 3, the effect of
GABA on the olfactory lobe response was antagonized by picrotoxin,
after which histamine, still in a picrotoxin background, was applied.
In both cases, histamine greatly reduced the response amplitude,
indicating that GABA and histamine mediate suppression of receptor cell
activity via distinct receptor pathways. As summarized in Figure
4, across all preparations that were
tested, picrotoxin (100 µM) reduced the effect of 100 µM GABA by 96 ± 2% (n = 4;
p < 0.0001) but did not change the effect of
histamine. Likewise, cimetidine (5 mM) reduced
the effect of 30 µM histamine by 59 ± 6% (n = 3; p < 0.001) but did not
change the effect of GABA. In three experiments, bicuculline methiodide
(0.1 and 1 mM) failed to reduce the suppression
mediated by 60 µM GABA.
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Figure 3.
GABA and histamine mediate suppression of primary
afferent activation via distinct receptor pathways.
Traces shown are from one experiment in which
suppression of the afferent signal was caused by GABA (100 µM) perfusion, followed by a block of the suppression by
picrotoxin (100 µM). Histamine (100 µM) and picrotoxin were then copresented, resulting in a
strong suppression. The afferent signal recovered on washout (data not
shown). Digital filter cutoffs were 2.8 and 60 Hz.
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Figure 4.
Effect of antagonists on suppression mediated by
histamine and GABA in the lobster. The vertical axis represents the
percentage of suppression, originally elicited by the agonist, that
persists in the presence of the antagonist. Thus, 100% means that the
suppression was unchanged in the presence of antagonist. Suppression
caused by histamine (30 µM) was reduced by cimetidine (5 mM) but not picrotoxin (100 µM), whereas
suppression caused by GABA (100 µM) was blocked by
picrotoxin but not cimetidine or bicuculline. Data from the bicuculline
experiments include different concentrations of GABA and bicuculline
(100 and 60 µM GABA vs 100 µM bicuculline,
and 60 µM GABA vs 1 mM bicuculline). Error
bars indicate standard error; numbers indicate number of
experiments. Asterisks indicate significance of
difference from agonist alone (100%); *p < 0.001;
**p < 0.0001.
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Axonal sensitivity to GABA and histamine-mediated suppression
We tested the possibility that lobster olfactory receptor axons
express GABA and histamine receptors at sites other than their terminal
branches and their peripheral somata by optically recording compound
action potentials in an isolated section of the antennular nerve. The
length of nerve was taken just distal to its entrance into the brain,
and so contained sections of olfactory receptor axons between 2 and 10 mm from their terminal regions. We measured the effect of GABA and
histamine on the amplitude of the compound action potential. GABA and
histamine were each capable of suppressing action potentials in
antennular nerve axons: 300 µM GABA was tested in four
preparations and caused a 49 ± 11% (p = 0.013) reduction in compound action potential amplitude, whereas 300 µM histamine was tested in two preparations and
caused reductions of 31 and 58%. In the two preparations tested, 100 µM picrotoxin greatly reduced the effect of
GABA (100 and 73% recovery of amplitude in normal saline).
In addition to containing the small-diameter (<1-5 µm) olfactory
receptor axons, the antennular nerve also contains a smaller number of
large-diameter (10-50 µm) afferents originating from other sensory
cells on the antennule (Schmidt and Ache, 1992). In two preparations
(data not shown), a small peak could be seen in addition to the large,
primary peak of the compound action potential. The smaller peak
preceded the major peak and, in the one preparation where this was
measured, had a lower threshold stimulus intensity. The smaller peak
presumably reflects the activation of the larger-diameter fibers, which
would have a faster conduction velocity and lower threshold and produce
a smaller optical signal because of the relatively small number present
in the nerve. GABA was applied to the nerve in both of these
preparations, and in both cases the early peak appeared to be
unaffected, whereas GABA did reduce the size of the second, larger
peak, and this effect was reversed by picrotoxin. This result suggests
that GABA receptors are expressed selectively on the olfactory axons
and not on the larger-diameter afferents.
Voltage-sensitive dye recordings in turtle olfactory bulb
We used the same paired-pulse stimulation and
Ca2+-free perfusion protocol to record
action potential propagation into axon terminals of olfactory receptor
neurons in the isolated olfactory bulb of the box turtle T. carolina. In normal turtle Ringer's solution, a single
conditioning shock to the olfactory nerve elicited a brief,
large-amplitude spike that could be observed propagating caudally
across the bulb, followed by a smaller, slowly decaying depolarization
(Fig. 5A). The initial spike
presumably reflects action potentials in incoming receptor cell axons,
whereas the slow component reflects the activation of postsynaptic
neurons. The slow, postsynaptic component could not be detected in the rostral region and became progressively larger in more caudal regions
of the bulb. The test pulse, delivered at the same ISI used in the
lobster experiments, elicited an identical initial transient but a much
smaller-amplitude and shorter-duration slow component (Fig.
5A).
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Figure 5.
Voltage-sensitive dye signals in in
vitro turtle olfactory bulb. The action potential signals are
not suppressed by a conditioning pulse and are not changed after
bathing in Ca2+-free saline. A,
Responses to paired-pulse stimulation recorded from four locations,
indicated on the drawing of the olfactory bulb at left.
Rostrally, the evoked signal is dominated by the compound action
potential from olfactory receptor axons (trace 1). In
the middle bulb the signal consists of a fast receptor axon component
and a slow depolarizing component (traces 2 and
3). The slow component of the test pulse response is
strongly suppressed. In the caudal bulb, mainly the slow component is
seen, and it too shows strong suppression of the test response
(trace 4). B, Evoked responses in
Ca2+-free Ringer's solution. Same detectors and
same preparation as in A are shown. The slow components
in the middle and caudal regions are significantly reduced, whereas the
compound action potential in the rostral region is unaffected. The peak
amplitude of the fast component in traces 2 and
3 is slightly smaller in Ca2+-free
Ringer's solution, possibly because of the reduction of the underlying
slow component. Digital filter cutoffs are 1.4 and 200 Hz.
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In six preparations, replacement of the normal Ringer's solution with
Ca2+-free/high-Mg2+
Ringer's solution significantly reduced the slow, postsynaptic component but did not affect the initial fast component reflecting receptor axon action potentials (Fig. 5B). The amplitude of
the test pulse-evoked spike in Ca2+-free
Ringer's solution was 97 ± 3% (n = 6) of its
control value (p = 0.3), whereas the amplitude
of the slow component, measured 25 msec after the spike, was 50 ± 4% of control (p < 0.0001). Thus, action
potential propagation in turtle olfactory receptor axons does not show
paired-pulse suppression as observed in the lobster.
Superfusion of the bulb with 1 mM GABA or 1 mM
dopamine in normal Ringer's solution greatly reduced or eliminated the
slow component of the evoked response with little or no effect on the fast component (Fig.
6A). In one experiment,
the D2 receptor agonist quinpirole (100 µM) also reduced the slow component (data not shown). In Ca2+-free ringers (which
eliminated postsynaptic signals), GABA, dopamine, and quinpirole did
not affect the amplitude or time course of the fast receptor axon
component (Fig. 6B). The mean amplitude of the test
pulse response in Ca2+-free ringers was
103 ± 3% of control values when GABA was applied (n = 4; p = 0.34) and 102 ± 6%
for dopamine application (n = 3; p = 0.75). Thus, while exogenous GABA and dopamine inhibit postsynaptic events in the turtle olfactory bulb, neither transmitter affects action
potentials in the receptor axon terminals.
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Figure 6.
GABA and dopamine suppress postsynaptic activity
but do not affect receptor cell action potentials in turtle olfactory
bulb. A, Effect of GABA and dopamine in normal turtle
Ringer's solution. Dopamine (left traces) and GABA
(right traces), both 1 mM, significantly
reduce the slow, postsynaptic component of the evoked response. The
left and right traces were taken from the
same preparation but from different detectors. B, Effect
of GABA and dopamine (1 mM) on the test response in
Ca2+-free Ringer's solution. Control and drug
traces are superimposed. Dopamine eliminates the remaining slow
component of the evoked response but does not affect the compound
action potential amplitude. GABA has little effect on the slow
component and no effect on action potential amplitude. Same detectors
and preparation as in A are shown. Digital filter
cutoffs are 1.4-200 Hz.
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Calcium dye recordings in turtle olfactory bulb
To test whether presynaptic inhibition might be mediated by
processes subsequent to action potential invasion of the axon terminal,
we selectively labeled olfactory afferents by applying a
dextran-conjugated calcium indicator to the olfactory epithelium and
allowing anterograde transport to the terminals in the olfactory bulb
(Friedrich and Korsching, 1997) (see Materials and Methods). Figure
7A shows the optical signal
evoked by olfactory nerve shock using the calcium dye (Calcium Crimson,
10 kDa), superimposed on the optical signal from the same location on
the olfactory bulb after staining with the voltage-sensitive dye
di-4-ANEPPS. The calcium signal is slightly delayed, slower in rise
time, and much longer lasting. In contrast to other figures, both
traces are shown with no digital high-pass filtering. Superfusing the preparation with Ca2+-free Ringer's
solution eliminated the evoked calcium signal (data not shown).
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Figure 7.
Optical signals recorded with dextran-conjugated
calcium indicators in turtle olfactory afferent terminals.
A, The calcium- and voltage-sensitive dye signals
evoked by olfactory nerve shock. The Calcium Crimson
dextran signal was recorded first, then the olfactory bulb was
stained with di-4-ANEPPS and the voltage-sensitive dye signal was
recorded using the same wavelength filters, stimulus intensity, and
detectors. Traces were digitally low-pass-filtered at 200 Hz (no
high-pass filtering). B, Calcium signal recorded with
Calcium Green-1 dextran, demonstrating paired-pulse
suppression of evoked calcium influx (top trace).
Bottom trace shows repetitive stimulation of the
olfactory nerve (arrows) causing summation of calcium
levels in the nerve terminal, indicating that the paired-pulse
suppression did not result from saturation of the calcium dye. In these
and all subsequent traces, upward indicates an increase in fluorescence
intensity (and Ca2+ levels). Traces were digitally
low-pass-filtered at 30 Hz.
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With paired-pulse stimulation (300-400 msec ISI), the peak amplitude
of the test pulse showed substantial suppression (46 ± 3% of
conditioning pulse amplitude; n = 12; p < 0.0001) (Fig. 7B, top trace). Because the
calcium signal evoked by a single shock decayed very slowly, the
apparent suppression could have been caused by saturation of the dye
when the test pulse-evoked calcium influx summated with the residual
calcium levels evoked by the conditioning pulse (Fig. 7B,
top trace). We tested this possibility in each preparation
by delivering a series of three shocks at 50 msec intervals. As shown
in Figure 7B (bottom trace), each shock caused
progressively smaller increases in calcium, but because of the slow
decay, the total calcium signal summated to levels above that evoked by
the conditioning pulse, indicating that the suppression of the test
response was not a result of dye saturation. Because there was no
evidence for dye saturation in the evoked signals, in Figures
8 and 9 the
data were digitally high-pass-filtered using a numerical RC
filter with a time constant of 1.4 Hz.
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Figure 8.
Effect of the glutamate receptor antagonists AP5
and DNQX, the transmitter GABA, and the dopamine D2 agonist
quinpirole on evoked calcium influx. A, Reducing
glutamatergic transmission from receptor cells to olfactory bulb
interneurons reduces paired-pulse suppression by increasing the test
response. Superimposed traces are in normal turtle
Ringer's solution and a mixture of AP5 (100 µM) and DNQX
(10 µM). B, Superimposed
traces in normal turtle Ringer's solution and 1 mM
GABA. C, Superimposed traces in normal
Ringer's solution and 100 µM quinpirole.
A-C are from different preparations. In
B and C, the conditioning response shows
the greatest amount of suppression. Recovery is not shown. Digital
filter cutoffs are 1.4 and 30 Hz.
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Figure 9.
Effect of the GABAB agonist baclofen
and the antagonist saclofen on evoked calcium influx. A,
Baclofen (50 µM) preferentially suppresses the
conditioning response so that both the conditioning and test pulses are
of similar amplitude (top two traces). Recovery of the
paired-pulse suppression has a longer time course than recovery of the
stimulus-evoked calcium levels, so that after 10 min of baclofen
washout the test pulse is actually larger in amplitude than before
baclofen application (third trace). After prolonged
washout, paired-pulse suppression recovers fully (bottom
trace). B, Saclofen (200 µM)
reduces paired-pulse suppression of calcium influx, causing an increase
in the test response amplitude as well as a smaller decrease in the
conditioning response. Recovery is not shown. Digital filter cutoffs
are 1.4 and 30 Hz.
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The same protocol used in the voltage-sensitive dye recordings was used
to test for transmitter-mediated suppression of calcium influx into
turtle olfactory receptor cell axon terminals, except that all
experiments were performed in normal turtle Ringer's solution. A
mixture of the NMDA receptor antagonist AP5 (100 µM) and
the non-NMDA receptor antagonist DNQX (10 uM) was used to reduce glutamatergic transmission from receptor cells to olfactory bulb
neurons (Berkowicz et al., 1994). In separate preparations stained with
voltage-sensitive dye, AP5/DNQX caused a large, although not complete,
suppression of the slow, postsynaptic component of the evoked response
and a partial suppression of the fast component (n = 2;
data not shown). As shown in the example of Figure
8A, in preparations stained with calcium dye,
AP5/DNQX caused a significant and reversible increase in calcium influx
after the test pulse (134 ± 7%; n = 4;
p = 0.01) without affecting conditioning pulse-evoked influx (98 ± 2%; n = 4; p = 0.2). Thus, AP5/DNQX significantly reduced paired-pulse suppression,
indicating that calcium influx into olfactory receptor cell axon
terminals is suppressed by evoked postsynaptic activity in the
olfactory bulb.
In contrast to the voltage-sensitive dye results in the turtle (Fig.
6), GABA (1 mM; n = 5) significantly and
reversibly reduced the amplitude of both the conditioning
and test pulses to 57 ± 8% (p = 0.005)
and 66 ± 6% (p = 0.006), respectively, of
their initial values (Fig. 8B). The
D2 agonist quinpirole (100 µM; n = 5) also affected
calcium influx, reducing the conditioning and test responses to 77 ± 4% (p = 0.04) and 86 ± 5%
(p = 0.06) of their initial amplitudes (Fig.
8C). The effects of quinpirole were long lasting, with
responses showing only partial recovery as much as 30 min after
returning to normal saline.
Baclofen (50 µM; n = 3), a
GABAB agonist, strongly inhibited evoked calcium
influx (Fig. 9A), reducing the conditioning response amplitude to 27 ± 2% (p = 0.001) of its
original amplitude, and a reduction to 62 ± 9%
(p = 0.04) for the test response. Both baclofen
and quinpirole had significantly greater effects on the conditioning
versus the test responses (baclofen, p = 0.01;
quinpirole, p = 0.02; unpaired t test)
(Figs. 8C, 9A). Likewise, 10 µM baclofen, tested in one experiment, reduced
the conditioning response to 59% of the control value and did not
affect the test response. One result of this preferential effect of
baclofen was to reduce or nearly eliminate paired-pulse suppression of
calcium influx. Interestingly, in all baclofen experiments, the
strength of paired-pulse suppression remained attenuated for up
to 20 min after washout of baclofen, whereas the effect on the absolute
response amplitude recovered after 5-10 min of washout (Fig.
9A).
A likely explanation for the preferential effect of baclofen (and
quinpirole) on the conditioning response is that the paired-pulse suppression already reflects GABAB- (and
D2-)mediated inhibition of
Ca2+ influx evoked by the conditioning
pulse. We therefore measured the effect of the
GABAB antagonist saclofen on the test response amplitude. Saclofen (200 µM; n = 5)
significantly increased the test response to 120 ± 6%
(p = 0.03) of its control value (Fig. 9B), supporting the idea that paired-pulse suppression is at
least partially mediated by GABAB receptors
activated by GABA release from olfactory bulb interneurons. At the same
time, saclofen caused a slight decrease in the amplitude of the
conditioning response, to 88 ± 3% (p = 0.01) of its control value.
| |
DISCUSSION |
The results from the experiments on the lobster extend the
findings of our preliminary study (Wachowiak and Cohen, 1998) showing that antennular nerve shock causes a long-lasting suppression of action
potential propagation in olfactory receptor axon terminals. Because no
known olfactory lobe interneurons show any response to a test shock at
the ISIs used in this study [>200 neurons tested in earlier studies
(Wachowiak et al., 1995, 1997)], the test pulse-evoked voltage-sensitive dye signal is a selective measure of receptor cell
activation. In the present experiments, presynaptic inhibition was
revealed experimentally as an increase in amplitude of the test pulse
response after synaptic blockade with zero
Ca2+/high-Mg2+
saline. In contrast to our preliminary study, which used low (1 mM) Ca2+ to reduce (but not
eliminate) synaptic transmission, in the present experiments we saw no
evidence of postsynaptic activity in the olfactory or accessory lobes
in the presence of zero Ca2+ saline.
Nonetheless, the increase in test response amplitude was similar in the
two studies: 57 ± 16% in the initial study versus 49 ± 15% in the present study.
Our results in the lobster do not discriminate between whether
presynaptic inhibition is mediated by reduced action potential amplitude or by conduction failure in the terminal region. However, both phenomena are well documented in sensory afferent terminals of
crustaceans and insects (Boyan, 1988; Burrows and Laurent, 1993;
Clarac and Cattaert, 1996). Because the terminal branching regions of
sensory afferents in the arthropod CNS appear to be largely passive,
shunting conductances such as those mediated by ionotropic GABA and
histamine receptors can have a large effect on action potential
amplitude at the axon terminal and, subsequently, on transmitter
release (Clarac and Cattaert, 1996).
Dual regulation of olfactory input in the lobster
The voltage-sensitive dye signal in lobster olfactory afferent
terminals was suppressed by exogenous GABA or histamine, two known
inhibitory transmitters in the lobster olfactory lobe (Wachowiak and
Ache, 1997). The suppression caused by each transmitter was selectively
blocked by known antagonists for GABA and histamine receptors,
suggesting that GABA and histaminergic interneurons in the olfactory
lobe constitute dual, functionally independent inhibitory pathways
mediating presynaptic inhibition. Interestingly, GABA and histamine
also mediate presynaptic inhibition of sensory afferents in the
crayfish thoracic ganglion (el Manira and Clarac, 1994), as well as
postsynaptic inhibitory events in the lobster olfactory lobe (Wachowiak
and Ache, 1997).
Differences in the morphology of GABA and histaminergic interneurons
suggest that GABA- and histamine-mediated presynaptic afferent
inhibition may serve distinct functional roles in olfactory processing.
Type II olfactory lobe interneurons, which appear to be GABAergic, have
extensive lateral branches innervating many adjacent glomeruli
(Wachowiak et al., 1997). Thus, GABAergic interneurons appear well
suited to mediate widespread lateral inhibition of olfactory afferent
input. This inhibition could serve to sharpen the receptive fields of
neurons innervating a particular glomerulus. By contrast, the
individual neurites of type I interneurons, which appear to be
histaminergic, remain confined to a single glomerulus (Wachowiak et
al., 1997). This branching pattern leads us to speculate that
histamine-mediated presynaptic inhibition may regulate input to a
specific postsynaptic target and refine intraglomerular processing. In
addition, the histaminergic type I interneurons respond to olfactory
nerve shock with a spike burst and a prolonged depolarization lasting
from 1 to 10 sec (Wachowiak et al., 1997), suggesting that presynaptic
inhibition mediated by these neurons could underlie the prolonged
paired-pulse suppression of olfactory lobe projection neurons
(Wachowiak and Ache, 1994).
Distribution of GABA and histamine receptors on lobster
olfactory afferents
Receptors for GABA and histamine are not only located at the nerve
terminal; they are also expressed on the somata of olfactory receptor
neurons, where they activate inhibitory conductances (Bayer et al.,
1989; Doolin et al., 1998). The absence of endogenous histamine in the
periphery (B. W. Ache, personal communication) led to the
hypothesis that these receptors are also expressed on the receptor cell
axon terminal where they mediate presynaptic inhibition, a hypothesis
confirmed by our results. In addition, our finding of suppression of
the compound action potential in isolated antennular nerve implies that
olfactory receptor neurons express GABA and histamine receptors on
regions of axon that are up to 10 mm distant from their terminal
branches. Interestingly, this result is similar to studies in mammals
showing that GABAA receptors suppress compound
action potentials in axons of primary sensory afferents (Brown and
Marsh, 1978; Morris et al., 1983). High concentrations of GABA and
histamine were required to cause suppression in the nerve relative to
that seen in the olfactory lobe (300 µM vs 10-30
µM), and GABA and histamine seemed roughly equally
effective in the nerve, whereas histamine was more potent than GABA at
causing suppression in the olfactory lobe. Although these differences
could reflect lower receptor density in the nerve, they could also be
explained by differences in access of agonists to receptors in the two
preparations (bath application in the nerve vs perfusion in the brain).
The functional relevance of this ectopic expression of transmitter
receptors on the cell body and axon remains unknown.
Dual regulation of olfactory input in the turtle
The paired-pulse suppression of calcium influx in the turtle
experiments, and the reduction of this suppression by glutamate receptor antagonists that inhibit synaptic transmission from receptor cells to olfactory bulb interneurons, indicates that olfactory input
activates a feedback inhibitory pathway that suppresses subsequent
calcium influx into receptor cell terminals, presumably reducing
transmission at the primary olfactory synapse. Our experiments also
show that presynaptic afferent inhibition occurs without affecting
action potentials in the axon terminal. Baclofen and quinpirole,
selective agonists for GABAB and
D2 receptors, respectively, suppressed
shock-evoked calcium influx. In other regions of the CNS, including the
hippocampus, cerebellum, and striatum, presynaptic GABAB and D2 receptors
inhibit transmitter release by coupling to G-proteins that directly
inhibit voltage-sensitive Ca2+ channels
(Hsu et al., 1995; for review, see Wu and Saggau, 1997; Miller, 1998).
A similar mechanism may underlie GABA- and dopamine-mediated presynaptic inhibition of turtle olfactory afferents.
The reduction of paired-pulse suppression of calcium influx by saclofen
and during recovery from baclofen application suggests that this
suppression is at least partly mediated by stimulus-evoked release of
endogenous GABA, presumably from juxtaglomerular neurons excited by the
conditioning pulse. This finding agrees with a recent study showing
that GABAB antagonists reduce paired-pulse suppression of postsynaptic activity in the rat (Keller et
al., 1998). Both GABA and dopamine are found in neurons branching
heavily within glomeruli of turtles as well as mammals (Halasz et al., 1982; McLean and Shipley, 1992), and GABAB and
D2 receptors are present in the glomerular and
olfactory nerve layers (Bouthenet et al., 1987; Bowery et al., 1987;
Nickell et al., 1991). However, EM studies of the synaptic organization
of the mammalian olfactory glomerulus have failed to find classical
output synapses from juxtaglomerular interneurons onto receptor cell
axon terminals (Pinching and Powell, 1971; White, 1972). Whether
classical synapses onto afferent terminals are in fact present in the
turtle or whether inhibitory transmitters are released at nonsynaptic
sites to affect distant targets, as has been shown for dopamine in
several systems (for review, see Maley et al., 1990), are questions for
further study. A more complete understanding of the mechanisms
underlying presynaptic inhibition of turtle olfactory afferents is
clearly necessary; however, our results suggest that this phenomenon
plays a significant role in the paired-pulse suppression that is
characteristic of olfactory bulb neurons and may also be important in
modulating or directly shaping the responses of olfactory bulb neurons
to odors. Our results show that two different transmitter substances mediate presynaptic inhibition of olfactory afferents. However, because
no other transmitter substances were tested, possible contributions
from other transmitters are not ruled out.
Phylogenetic and functional considerations
The organization of olfactory pathways in vertebrates and
invertebrates shares a number of common features that suggest a common
functional strategy for processing olfactory input that is shared
across phylogenetically distant groups (Boeckh et al., 1990; Ache,
1991; Hildebrand and Shepherd 1997). The present study suggests that
presynaptic regulation of transmission from olfactory receptor cells to
interneurons may be another feature of this common functional strategy,
because in both lobster and turtle, GABAergic and aminergic transmitter
pathways appear to act as dual mediators of presynaptic inhibition of
olfactory afferents. In addition, physiological evidence supports the
existence of presynaptic inhibition in mammals (see above), and in
insects, ultrastructural studies show that GABAergic interneurons
synapse onto receptor cell terminals (Distler and Boeckh, 1997).
Nonetheless, phylogenetic differences are apparent in the cellular
mechanisms mediating presynaptic inhibition, because we found no
evidence for suppression of action potentials in olfactory receptor
terminals in the turtle and pronounced suppression in the lobster
(Table 1). We were not able to test
whether, in the lobster, transmitters also directly inhibit calcium
influx into the terminal as they do in the turtle.
Although our results suggest that presynaptic inhibition plays a
significant role in the paired-pulse suppression seen in both turtle
and lobster, its role in odor processing remains unclear. Presynaptic
inhibition mediated by inhibitory interneurons innervating several
adjacent glomeruli could directly shape and refine spatial patterns of
input across glomeruli. This "pre-glomerular" level of processing
would be in addition to glomerular and subglomerular processing steps
already characterized in the vertebrate olfactory bulb (for review, see
Nickell and Shipley, 1992). Alternatively, presynaptic inhibition could
have a modulatory role, regulating sensitivity to olfactory input by
controlling the effectiveness of transmission from primary afferents to
second-order neurons. Additional experiments are needed to distinguish
between this and other possible roles in olfactory coding. Nonetheless,
it is interesting to speculate that presynaptic inhibition of olfactory input to the CNS has evolved independently in vertebrates and invertebrates in response to common functional demands in processing odor information.
| |
FOOTNOTES |
Received April 14, 1999; revised Aug. 5, 1999; accepted Aug. 5, 1999.
This work was supported by National Institutes of Health Grant NS08437
and a Marine Biological Laboratory Summer Research Fellowship. We thank
Barry Ache, Charles Greer, Brian Salzberg, and Dejan Zecevic for useful
comments on this manuscript.
Correspondence should be addressed to Dr. Matt Wachowiak, Department of
Cellular and Molecular Physiology, Yale University School of Medicine,
333 Cedar Street, New Haven, CT 06510.
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ABSTRACT
INTRODUCTION
