1Biological Computation Research Department, Bell Laboratories, Lucent Technologies, Murray Hill, New Jersey 07974; and 2The Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria 3078, Australia
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ABSTRACT |
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Gelperin, A., J. Flores, F. Raccuia-Behling, and I.R.C. Cooke. Nitric Oxide and Carbon Monoxide Modulate Oscillations of Olfactory Interneurons in a Terrestrial Mollusk. J. Neurophysiol. 83: 116-127, 2000. Spontaneous or odor-induced oscillations in local field potential are a general feature of olfactory processing centers in a large number of vertebrate and invertebrate species. The ubiquity of such oscillations in the olfactory bulb of vertebrates and analogous structures in arthropods and mollusks suggests that oscillations are fundamental to the computations performed during processing of odor stimuli. Diffusible intercellular messengers such as nitric oxide (NO) and carbon monoxide (CO) also are associated with central olfactory structures in a wide array of species. We use the procerebral (PC) lobe of the terrestrial mollusk Limax maximus to demonstrate a role for NO and CO in the oscillatory dynamics of the PC lobe: synthesizing enzymes for NO and CO are associated with the PC lobes of Limax, application of NO to the Limax PC lobe increases the local field potential oscillation frequency, whereas block of NO synthesis slows or stops the oscillation, the bursting cells of the PC lobe that drive the field potential oscillation are driven to higher burst frequency by application of NO, the nonbursting cells of the PC lobe receive trains of inhibitory postsynaptic potentials, presumably from bursting cells, due to application of NO, and application of CO to the PC lobe by photolysis of caged CO results in an increase in oscillation frequency proportional to CO dosage.
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INTRODUCTION |
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Nitric oxide (NO) acts as a neurotransmitter at a
number of sites in the central and peripheral nervous systems
(Garthwaite and Boulton 1995; Moncada and Higgs
1993
; Schuman and Madison 1994
; Vincent
1994
; Zhang and Snyder 1995
). NO is a highly
reactive free radical synthesized from arginine by nitric oxide
synthase (NOS), a calcium/calmodulin activated enzyme (Bredt and
Snyder 1990
; Knowles et al. 1989
; Stuehr
and Griffith 1992
). NO is mobile and membrane permeant so it
acts within a sphere centered at its site of synthesis with radius
determined by its diffusion constant and average life time
(Gally et al. 1990
; Wood and Garthwaite 1994
). NO can influence ion channels directly (Broillet
and Firestein 1997
; Lynch 1998
; Summers
et al. 1999
) or can activate soluble guanylate cyclase and
elevate cGMP levels, thereby altering the gating kinetics of
cGMP-sensitive ion channels (Ahmad et al. 1994
; Chen and Schofield 1995
; Firestein 1996
;
Nawy and Jahr 1990
; Shiells and Falk
1992
; Yau 1994
).
Presumptive NO synthesizing neurons are particularly abundant in the
olfactory systems of mammals (Bredt et al. 1991;
Kishimoto et al. 1993
; Spessert and Layes
1994
; Vincent and Kimura 1992
; Zhao et
al. 1994
), mollusks (Cooke et al. 1994
;
Sánchez-Alvarez et al. 1994
), and insects
(Elphick et al. 1995
; Müller 1994
; Müller and Bicker 1994
; Müller and
Hildebrandt 1995
; Nighorn et al. 1998
;
Stengl and Zintl 1996
). Suggested roles for NO in olfaction include peripheral effects on receptor adaptation and central
effects that may contribute to independent activation of adjacent
glomeruli (Breer and Shepherd 1993
). NO also may
contribute to the specificity of central synaptic connections made by
axons of receptor cells in the olfactory bulb (Gally et al.
1990
; Hess et al. 1993
; Peunova and
Enlkolopov 1995
; Wu et al. 1994
).
Carbon monoxide (CO) is another highly mobile molecule implicated in
neuronal communication (Glaum and Miller 1993;
Ingi and Ronnett 1995
; Leinders-Zufall et al.
1995
; Marks et al. 1991
; Schmidt
1992
; Stevens and Wang 1993
; Verma et al.
1993
; Zakhary et al. 1997
; Zhuo et al.
1993
), reviews in (Dawson and Snyder 1994
;
Hawkins et al. 1994
; Maines 1993
). CO is
produced in brain by the enzyme heme oxygenase 2 (HO-2), which
oxidatively cleaves the heme ring to biliverdin and CO (Maines
1988
). HO-2 is a constitutive isoform of HO highly abundant in
brain (Sun et al. 1990
). CO, like NO, can activate
soluble guanylyl cyclase by binding to the iron in its heme moiety,
leading to increased levels of cGMP (Brune and Ullrish
1987
; Furchgott and Jothianandan 1991
).
CO has a particularly clear association with the olfactory system.
Olfactory receptor neurons and parts of the olfactory bulb show the
highest concentration of HO-2 in the mammalian nervous system
(Ewing et al. 1993; Verma et al. 1993
).
Studies on olfactory sensory neurons provide evidence for a role of CO
in receptor function (Ingi and Ronnett 1995
;
Leinders-Zufall et al. 1995
). Adult olfactory receptors
lack NO synthase (Bredt and Snyder 1994
; Kulkarni
et al. 1994
; Roskams et al. 1994
) and have a
cyclic nucleotide gated ion channel with a higher affinity for cGMP
than for adenosine 3',5'-cyclic monophosphate (cAMP) (Frings et
al. 1992
; Zufall et al. 1991
). CO production and
consequent cGMP production in olfactory receptor neurons have been
measured directly (Ingi et al. 1996a
,b
;
Ingi and Ronnett 1995
) as has cGMP gating of ion channels in olfactory receptor cells stimulated by exogenous CO (Leinders-Zufall et al. 1995
). The actions of CO on
central olfactory circuits are unknown.
Central olfactory circuits characteristically display oscillatory local
field potential responses to odor stimulation (Gelperin 1999; Gray 1994
; Tank et al.
1994
), a mode of activity potentially modulated by NO
(Ding et al. 1994
; Gelperin 1994
;
Pape and Mager 1992
) and perhaps by CO. The present
experiments explore the affects of NO and CO on the oscillatory
activity of olfactory interneurons in the procerebral (PC) lobe of the
terrestrial mollusk Limax maximus, a structure
implicated in olfactory learning (Gelperin 1999
;
Kimura et al. 1998
) that may be NO dependent
(Teyke 1996
). Results indicate that the
Limax cerebral ganglion and PC lobe contain cells and
fibers generating NO and CO and that endogenous NO and CO levels may
set the frequency of oscillation and perhaps wave propagation
(Ermentrout et al. 1998
; Kleinfeld et al.
1994
) in the PC lobe.
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METHODS |
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Anatomy
Slugs (L. maximus) weighing 0.5-2 g were collected locally near Melbourne, Australia, and chilled in crushed ice for 1 h before dissection. The entire CNS, together with the major anterior nerve roots and the attached tentacles and lips, were removed and fixed for 1 h in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.6). After fixation the tissues were washed in several changes of PBS, infiltrated with 30% sucrose, frozen, and sectioned on a cryostat at a thickness of 8 µm. Serial sections were mounted on gelatin-coated slides, air-dried, and then processed for the visualization of NADPH diaphorase activity or immunoreactivity to heme oygenase-1 (HO-1) and heme oxygenase-2 (HO-2).
The method of Scherer-Singler et al. (1983) was used to
visualize NADPH diaphorase activity in five preparations. Control preparations were stained with the NADPH diaphorase procedure with
either NADPH or Nitro Blue tetrazolium omitted (2 preparations each).
No staining occurred in control preparations.
Polyclonal antisera to native rat HO-1 and HO-2, purchased from StressGen Biotechnologies (Victoria, Canada), were used to examine the distribution of immunoreactivity to HO-1 (5 preparations) or HO-2 (6 preparations) in the nervous system. Sections were washed in PBS and incubated with the primary antibody (diluted 1:200 in PBS) for 24 h at room temperature. The sections then were washed in several changes of PBS and processed with an avidin-biotin immunoperoxidase method using a Sigma Extravidin kit (Sigma) and diaminobenzidine tetrachloride (Sigma) as the chromogen. The primary antiserum was omitted from alternate serial sections in two preparations for control purposes.
Electrophysiology
Specimens of L. maximus were obtained from a laboratory colony maintained on a 14:10 light-dark cycle at 16°C with ad libitum access to Purina lab chow supplemented with vitamins, sea sand, and the fungicide Tegosept. Slugs were anesthetized with cold, and the brain and buccal mass quickly isolated into saline previously chilled to 4°C. The PC lobe was isolated from the cerebral ganglion by fine dissection.
Recordings were made from both intact desheathed PC lobes and slice preparations made from desheathed lobes. Slices of PC lobe for electrophysiological recording were made by embedding the freshly dissected desheathed lobe in 2% agarose in saline at 39°C. The PC was sectioned on a tissue slicer (Vibratome) at room temperature with 5 mg/ml bovine serum albumin added to the saline. Sections can be cut as thin as 50 µm; however, reliable oscillations are obtained from sections >125-µm thick. By orienting the tissue in the agar and trimming the agar block before cutting, PC lobe sections can be obtained normal or parallel to the apical-basal axis of wave propagation. Sections are collected from the saline-filled well of the Vibratome, transferred to a silicone elastomer (Sylgard)-lined recording chamber and fixed to the Sylgard substrate by pins through the agar surrounding the tissue section. Signals were digitized at 1 kHz and stored on a computer (Mac IIfx). Custom software controlled data acquisition and display. Commercial software packages were used for data analysis (Igor Pro) and statistical analysis (StatView II).
NO was applied to PC lobes and slices using brief flashes of near UV
(360 ± 10 nm) light delivered to preparations previously bathed in the caged NO compound dipotassium
pentachloronitrosylruthenate (NPR) (Alfa, No.10534) (Bettache et
al. 1993; Komozin et al. 1983
; Makings
and Tsien 1994
). Flashes delivered in the absence of caged NO
had no effect. To apply uncaging flashes at a fixed latency relative to
the peak of the local field potential (LFP) oscillation, the amplified
and filtered oscillation signal was fed to a window discriminator the
output of which triggered the uncaging flash at a preselected latency.
The window discriminator output was enabled at 30-s intervals to allow
recovery from each uncaging event before the next uncaging event.
Oxymyoglobin was made essentially according to the methods of
Lev-Ram et al. 1995
.
CO was applied to slice preparations of the PC lobe incubated in the
caged CO compound bicyclo[2.2.1]hepta-2.5-diene-7-one. cyclic
1-(4,5-dimethoxy-2-nitrophenyl)-2-acetoxymethyloxycarbonyl-1.2-ethanediyl acetal (NV-CO/AM) (Kao and Keitz 1997), a
membrane-permeant form that releases CO on exposure to
ultraviolet light. After flash photolysis, release of CO from NV-CO is
complete on the millisecond time scale (J.P.Y. Kao, unpublished data).
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RESULTS |
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NADPH diaphorase activity
NADPH diaphorase activity was found to be distributed extensively throughout the nervous system of Limax. In each of the five preparations examined, NADPH diaphorase-positive nerve cell bodies were found in all of the central ganglia and NADPH diaphorase-positive fibers were present in large numbers in the neuropil regions of all of the central ganglia, in all central connectives and in many peripheral nerve roots.
Especially strong NADPH diaphorase activity occurred within the
procerebral lobes of the cerebral ganglia. The internal mass neuropil
of the procerebral lobes exhibited by far the most intense NADPH
diaphorase activity of any region of the nervous system. The terminal
mass neuropil, which contains neurites of PC neurons and afferents from
olfactory receptors located in the major and minor noses, also
exhibited strong NADPH diaphorase activity but this was always less
intense than that in the internal mass (Fig. 1A). Tracts of
NADPH-diaphorase-positive fibers extended between the terminal mass
neuropil and the cell body layer of the procerebral lobe (Fig.
1A) and intense punctate staining was observed at the periphery of many cell bodies (Fig. 1, A and B).
This is in accord with previous NADPH diaphorase staining of pieces of
cell body layer from the PC, which also showed isolated reactive cells
(Gelperin 1994). No staining occurred in preparations
for which either NADPH or Nitro Blue tetrazolium was omitted from the
staining protocol.
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HO immunoreactivity
The antiserum to HO-1 did not stain any structures in the nervous system in any of the five preparations we examined. By contrast, the antiserum to HO-2 always stained specific regions of neuropil in each of the six preparations examined and, in two of these preparations, also clearly stained discrete groups of nerve cell bodies. Omission of the primary antiserum to HO-2 from the staining procedure abolished all staining.
Within the procerebral lobe, punctate staining was observed consistently in a discrete region of the internal mass neuropil at the base of the procerebral lobe (Fig. 2A). Punctate staining of specific regions of neuropil occurred in the proximal regions of the tentacular ganglia and in all central ganglia. Staining was especially prominent in the suboesophageal ganglia (Fig. 2B). Numerous stained fibers exhibiting immunoreactivity to HO-2 also were observed in the interganglionic connectives and in the tentacular nerves.
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We did not observe any immunoreactivity to HO-2 in cell bodies in the procerebral lobes, elsewhere in the cerebral ganglia, or in the tentacular ganglia in any of the preparations we examined. By contrast, strong staining of individual cell bodies and clusters of cell bodies was observed in all of the suboesophageal ganglia in two preparations (Fig. 2, B and C) and very weak staining of cell bodies was observed in the same regions in these ganglia in another two preparations.
Slice anatomy
The cell and neuropil regions of the PC are seen clearly in
a stained 200-µm-thick longitudinal section of an agar-embedded PC
lobe cut along the apical-basal axis (Fig.
3). The neuropil layer adjacent to the
cell body layer is the terminal mass, whereas the neuropil layer
furthest from the cell body layer is the internal mass (Zaitseva
1991; Zs.-Nagy and Sakharov 1970
). The cell body layer is thickest at the apex and thins progressively toward the base
of the PC. Scattered cells are evident in the neuropil regions. The
relative volumes of somata, terminal mass and internal mass vary at
different levels along the apical-basal axis of the PC lobe.
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Slice physiology
The oscillating local field potential (Gelperin and
Tank 1990) can be recorded from PC slices cut either parallel
or normal to the apical-basal axis of the PC (Ermentrout et al.
1998
). Figure 4A shows
two-site recordings from a 200-µm-thick longitudinal slice cut along
the apical-basal axis of the PC lobe, like the slice shown in Fig. 3.
The delay of 548 ms in the LFP peak from apical to basal recording
sites separated by 600 µm reflects the wave-like propagation of
activity that originates at the apical pole of the PC and propagates at
1.1 mm/s at room temperature to the base of the PC (Delaney et
al. 1994
; Kleinfeld et al. 1994
). The
longitudinal slices show occasional events in which wave propagation is
replaced by simultaneous activation for one or two cycles after which
wave propagation resumes (cf Fig. 5B in Ermentrout et
al. 1998
). Intermediate wave-propagation states are not
observed in these dual site slice recordings, i.e., the apical-basal
delay is either 548 ms or 0 with apical and basal recording sites
separated by 600 µm.
|
NO effects
The caged NO compound NPR has no effect on the oscillating LFP
when applied in the dark (data not shown). In response to an uncaging
flash of 360 ± 10 nm covering the entire PC slice, the frequency
of the LFP oscillation is increased. If the 50 ms uncaging flash is
localized to a 35-µm-diam spot centered on the apical electrode,
apical oscillation frequency is increased but basal frequency is not
(Fig. 5, A and B).
If the uncaging flash is localized to the region of the PC midway
between the apical and basal recording sites, oscillation frequency is
increased at both apical and basal sites (Fig. 5C) and the
apical-basal latency decreases (Fig. 5D). If the uncaging
flash is localized to the basal recording site, the basal oscillation
frequency is increased while the apical oscillation frequency remains
unchanged (Fig. 5, E and F). The fact that local
NO uncaging in the PC midregion speeds up wave propagation with no
local nonpropagating activity suggests that the bursting cells in the
PC midregion mediating this effect are different from bursting cells at
the apex or base. The results in Fig. 5 are typical of four experiments
(30 trials/experiment). The amplitude and duration of LFP oscillation
frequency increase are directly proportional to the intensity and
duration of the uncaging flash. The uncaging flashes have no effect in
the absence of NPR or in the presence of NPR which has been subjected
to intense and prolonged illumination to fully photolyse the compound
before its application to the preparation (data not shown). This is
similar to previous observations made with the intact PC lobe
(Gelperin 1994).
|
Application of 10 mM
N-nitro-L-arginine methyl ester
(L-NAME) slows the PC oscillation, an effect which is
reversed by addition of 5 mM L-arginine in the continued
presence of L-NAME (Fig. 6).
A higher dose of L-NAME (20 mM) stops the PC oscillation in
a fully reversible manner (data not shown).
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The PC lobe contains two types of local interneurons, bursting (B) and
nonbursting (NB) cells (Kleinfeld et al. 1994;
Watanabe et al. 1999
). The responses of B and NB cells
to NO were studied in intact desheathed PC lobes by making both
loose-patch extracellular and nystatin perforated-patch intracellular
recordings while applying brief uncaging flashes to preparations
previously bathed in NPR. Figure
7A shows a simultaneous
recording of the LFP at the base of the PC lobe and a loose-patch
extracellular recording from a B cell located more apically. NO
application increases burst frequency and decreases the number of
spikes per burst for the 5-s period after the 60-ms uncaging flash. The
definitive identification of a B cell depends on its production of a
double burst when a double event is recorded in the LFP, as shown in
Fig. 7B. Two double events are seen in the LFP before NO
stimulation in Fig. 7B, each accompanied by a double burst
in the B cell (Kleinfeld et al. 1994
). A series of
100-ms uncaging flashes delivered to a preparation previously exposed
for 1 h in 50 µM NPR produced an increase in oscillation
frequency from 0.4 to 1.3 Hz for the 10-s period after the NO stimulus
(10 flashes per preparation, 3 preparations, P < 0.01). All 10 confirmed B cells we have recorded responding to NO
showed an increased frequency of bursting.
|
The response of NB cells to an NO pulse is a decrement in activity,
caused by a flurry of IPSPs occurring one-for-one with peaks in the LFP
(Fig. 8A). Before NO
stimulation the NB cell receives a 5- to 7-mV inhibitory postsynaptic
potential (IPSP) coincident with the peak in the LFP. NB spikes are
produced during the intervals between IPSPs. The flurry of IPSPs
triggered by the NO pulse briefly clamps the membrane potential of the
NB cell at the reversal potential for chloride, the ion mediating the IPSP (Gelperin et al. 1993; Watanabe et al.
1999
). The IPSPs recorded from NB cells, both occurring
spontaneously and during NO stimulation, are presumed to result from
activity in one or more B cells presynaptic to each NB cell. The NO
uncaging flash triggers a period of high-frequency bursting in B cells
(cf Fig. 7) that results in a barrage of high-frequency IPSPs onto NB
cells. Further evidence for the NO dependence of the increase in IPSP
frequency in NB cells in response to an NO uncaging flash is the
significant reduction in IPSP frequency and LFP oscillation frequency
increase observed after an NO pulse delivered with the PC lobe bathed
in 230 µM oxymyoglobin, an NO scavenger (Fig. 8B). The
effect of myoglobin in blocking NO-mediated synaptic input to NB cells
is not seen when metmyoglobin is used (data not shown).
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CO effects
CO also plays a role in mediating olfactory oscillations in
Limax. If CO is applied to the isolated PC lobe by
photolytic release from NV-CO/AM (Kao et al. 1995), the
frequency of the LFP oscillation is transiently increased (Fig.
9, A-C). As shown in Fig. 9,
increasing the duration of the uncaging flash increased the magnitude
of the frequency increase. The averaged results (± SD) of a series of
uncaging flashes, 10 trials per flash duration, corresponding to the
flash durations represented in Fig. 9, are shown in Fig.
10, A-C. A summary of
responses to flash durations from 10 to 300 ms is shown in Fig.
11, where for each of the 10 trials at
a given uncaging flash duration, the three peak LFP oscillation
frequency responses from each trial were averaged. The responses to all
flash durations are significantly different from control prestimulation
values (for 10-ms flash, t =
3.625, df =26,
P = 0.0012; for 300-ms flash, t =
18.846, df =29, P < 0.001; intermediate flash
durations all significant at P < 0.001). This result
was obtained in four experiments. It will be interesting to see if the
effects of CO on LFP oscillation frequency result from excitation of
burster cells in the PC, as seen for NO.
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DISCUSSION |
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The distribution and intensity of NADPH diaphorase activity in the
Limax procerebral lobe and tentacular ganglion are similar to those described previously for Helix (Cooke et al.
1994). The extensive distribution of NADPH diaphorase activity
in olfactory systems of mollusks (Dyakonova 1997
;
Moroz and Gillette 1995
; Sánchez-Alvarez et
al. 1994
), arthropods (Elphick et al. 1995
; Johansson and Mello 1998
; Nighorn et al.
1998
; Scholz et al. 1998
), and mammals
(Croul-Ottmann and Brunjes 1988
; Egberongbe et
al. 1994
; Schmidt et al. 1992
) suggests that a
diffusible intercellular messenger may play a critical role in the
pattern recognition and learning functions of the olfactory system
(Kendrick et al. 1997
). In mouse olfactory bulb, the
local inhibitory interneurons, the GABAergic granule and periglomerular
cells, have been shown to contain nitric oxide synthase by a variety of
techniques (Kishimoto et al. 1993
).
Indirect evidence suggests that NO signaling in the Limax
CNS is necessary for odor learning or odor discrimination. The LFP oscillation is dependent on NO signaling and is blocked by
L-NAME application to the PC lobe (Gelperin
1994). Injection of L-NAME into Helix
disables odor learning but not odor orientation (Teyke 1996
), a result also found in the honeybee (Müller
1996
) and mouse (Okere et al. 1996
). It is
possible that L-NAME injection disabled odor learning
because it altered the dynamics of activity in the PC lobe. This can be
tested by making L-NAME injections in a Limax
previously implanted with a pair of fine-wire recording electrodes in
its PC lobe (Gelperin et al. 1996
). Block of olfactory oscillations in the Limax PC lobe with L-NAME
application using an in vitro nose-brain preparation degraded odor
discrimination (Teyke and Gelperin 1999
), similar to
results in honeybee after pharmacological block of odor-triggered
oscillations in the antennal lobe (Stopfer et al. 1997
).
Within the PC lobe of Limax, NO is a candidate for the agent
mediating coupling between the local inhibitory interneurons, the B
cells. B cells must be coupled as they drive the activity wave and
there is only one activity wave in the PC lobe at a time (Ermentrout et al. 1998). Activity in apical B cells
initiates the wave, which then propagates from apex to base
(Kleinfeld et al. 1994
). NO production by apical B cells
triggered by calcium influx during their bursting could lead to
excitation of more basal B cells and hence mediate wave propagation.
Application of a concentrated solution of hemeproteins (10-20 mM)
slows or eliminates the LFP oscillation (Gelperin 1994
),
consistent with the possibility that spike-triggered NO production by B
cells might subserve excitatory coupling between them. Evidence for spike-triggered NO production by molluskan central neurons has been
obtained at several sites in the CNS of Aplysia
(Jacklet 1995
; Meulemans et al. 1995
;
Sawada et al. 1995
, review in Jacklet 1997
, see also Moroz et al. 1999
).
The changes in waveform of the LFP signal caused by a pulse of NO help
clarify the sources of transmembrane current contributing to the LFP
waveform. The LFP waveform shows a sharp transition during and
immediately after the NO pulse, seen most clearly in Fig.
8A. The flurry of NO-triggered IPSPs onto NB cells keeps their membrane potential clamped at ECl so the
normal source of transmembrane current producing the dominant part of
the LFP event is removed. Simultaneously, the population of B cells is
strongly and synchronously activated so that extracellular currents
arising from action potentials in B cells are more likely to summate
and make a larger contribution to the LFP event. The resultant of these
two processes, suppression of IPSP amplitude in NB cells and
enhancement of spiking synchrony in B cells, leads to a change in sign
of the LFP event. As B cell bursting wanes and NB cell IPSP amplitude
returns to normal, the brief upward deflection of the LFP due to B cell
spikes again becomes dominated by the large downward deflection due to
synchronous large-amplitude IPSPs in NB cells. As deduced from previous
optical and electrical recordings of PC lobe activity waves, in the
unstimulated lobe the B cell spikes contribute to a small upward
deflection in the LFP signal just before the large-amplitude downward
deflection in the LFP signal (Kleinfeld et al. 1994).
NO has been found to control oscillatory activity at several sites in
the mammalian CNS. NO release onto thalamocortical neurons leads to
reduced oscillatory activity, mediated by a cGMP-dependent change in a
hyperpolarization-activated cation conductance (Pape and Mager
1992). In hamster suprachiasmatic nucleus (SCN), NO production
provides part of the link between retinal light signals and resetting
of the SCN clock (Ding et al. 1994
). The control of the
sleep-wake cycle by cholinergic NOS-containing mesopontine cells in the
pedunculopontine tegmental nuclei is dependent on endogenous NO
production (Datta et al. 1997
). NO also potentiates responses of ventrobasal thalamic units to sensory inputs (Shaw and Salt 1997
).
In this study, we have demonstrated the presence of immunoreactivity to
HO-2, the enzyme responsible for the generation of CO by neurons, in
specific regions of the Limax nervous system that are
involved in olfactory processing. We also have shown that exogenously
applied CO can modulate the physioloigcal function of one of these
regionsthe procerebrum. Together, these data support the hypothesis
that CO plays a role in the normal processing of olfactory information
by Limax.
The immunoreactivity to HO-2 demonstrated here in the Limax
CNS is the first such description for any invertebrate. We also have
observed discrete regions of strong punctate staining for HO-2 in the
neuropil of the procerebral lobes and tentacular ganglia of Helix
aspersa, another pulmonate mollusk, in positions corresponding exactly to those that we have described here for Limax
(unpublished observations). The specific staining pattern in the
nervous system we observed for HO-2 but not HO-1 in both
Limax and Helix is consistent with previous
studies in vertebrates showing specific HO-2 immunoreactivity but
little HO-1 immunoreactivity (Ewing and Maines 1992;
Vincent et al. 1994
). In mammals the olfactory
epithelium and the mitral and granule cell layers of the olfactory bulb
show the most intense staining for HO-2 mRNA (Verma et al.
1993
).
The occurrence of immunoreactivity to HO-2 in discrete regions of
neuropil in the procerebral lobes and tentacular ganglia of
Limax is consistent with a potential role for CO in the
processing of olfactory information. The location of the cell bodies of
the neurons, the terminals of which exhibit NADPH diaphorase activity in the procerebral lobe neuropil and immunoreactivity to HO-2 evident
in the proximal region of the internal mass neuropil, is not clear.
Most of the neurons projecting to the procerebral neuropil are
intrinsic to the procerebrum (Ratté and Chase 1997, 1999
). Projections to the procerebral neuropil also have been shown to originate from the superior and inferior tentacular ganglia (Kawahara et al. 1997
), the pedal ganglia (Chase
and Tolloczko 1989
), and the buccal ganglia (Gelperin
and Flores 1997
).
The evidence that CO functions as a neuromodulator is particularly
clear in the olfactory system. We present here both immunocytochemical and physiological evidence for the role of CO in the olfactory analyzer
of the Limax PC lobe. Isolated salamander olfactory receptor neurons have cyclic nucleotide gated channels activated by CO (Zufall et al. 1995) and display a form of odor response
adaptation lasting several minutes that is dependent on CO stimulation
of cGMP formation (Zufall and Leinders-Zufall 1997
). The
cyclic nucleotide-gated channel of olfactory receptors can be activated
directly by NO (Broillet and Firestein 1996
, 1997
) so it
may be that CO also has direct as well as indirect effects on the
receptor neuron ion channels. CO production by cultured rat olfactory
receptor neurons has been measured directly (Ingi and Ronnett
1995
). Evidence from cultured olfactory receptors indicates
that CO activation of soluble guanylyl cyclase may regulate the
efficacy of the NO-cGMP-signaling system (Ingi et al.
1996a
,b
).
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ACKNOWLEDGMENTS |
---|
We thank W. Denk for suggesting the use of caged NO and J.P.Y. Kao for a generous gift of caged CO.
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FOOTNOTES |
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Address for reprint requests: A. Gelperin, Rm. 1C464, Bell Laboratories, 600 Mountain Ave., Murray Hill, NJ 07974.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 17 May 1999; accepted in final form 16 September 1999.
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