Nitric Oxide and Carbon Monoxide Modulate Oscillations of Olfactory Interneurons in a Terrestrial Mollusk

A. Gelperin,1 J. Flores,1 F. Raccuia-Behling,1 and I.R.C. Cooke2

 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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INTRODUCTION
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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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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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Fig. 1. NADPH diaphorase activity in the procerebral lobe of Limax. A: low-power view showing the distribution of NADPH diaphorase activity in the procerebral lobe. Note the very dense reaction product in the internal mass neuropil (IM), the less intense but nevertheless strong activity in the terminal mass neuropil (TM) and the staining of prominent fiber tracts within the cell body layers (C). Scale bar: 50 µm. B: high-power view of the procerebral lobe cell body layer showing regions of intense punctate staining at the periphery of individual cell bodies. Scale bar: 10 µm.

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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Fig. 2. Heme oxygenase 2 (HO-2) immunoreactivity in the nervous system of Limax. A: HO-2 immunoreactivity in the procerebral lobe. A discrete region of punctate staining (arrowed) was present at the base of the internal mass neuropil (IM). TM, terminal mass neuropil. Scale bar: 100 µm. B: HO-2-immunoreactive cells (arrowed) and neuropil regions (*) in the visceral ganglion (VG) and parietal ganglion (PaG). Scale bar: 50 µm. C: cluster of HO-2-immunoreactive cells (arrowed) in the parietal ganglion. Scale bar: 25 µm.

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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Fig. 3. Photomicrograph of a longitudinal slice of the Limax procerebral (PC) lobe stained with toluidine blue. PC lobe was embedded in agarose and 200-µm-thick sections cut on a Vibratome. PC lobe was cut along the apical-basal axis of wave propagation. Recording electrodes are shown in typical sites to record the apical and basal oscillatory local field potential.

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.



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Fig. 4. Recordings of the local field potential (LFP) at apical and basal sites in a PC lobe slice like that shown in Fig. 3. Distance between apical and basal recording sites and the time delay of the LFP peak between recording sites correspond to a wave propagation velocity of 1 mm/s. Polarity of the basal LFP signal has been inverted for display.

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).



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Fig. 5. Nitric oxide (NO) increases the frequency of the LFP oscillation. A 200-µm-thick apical-basal slice like that shown in Fig. 3 was exposed to 50-µM caged NO (NPR) for 45 min and then rinsed 4 times with saline. A 70-ms uncaging flash 35 µm in diameter was triggered at constant latency from the negative peak of the basal LFP oscillation. A: with the uncaging spot centered on the apical recording electrode, the apical oscillation frequency increased while the basal frequency did not. B: average of 10 trials like that shown in A (mean ± SD) C: with the uncaging spot centered midway between the apical and basal recording sites, the oscillation frequency increased coherently at both apical and basal sites. D: average of 10 trials like that shown in C showing decreased apical-basal latency during frequency increase of LFP oscillation. E: with uncaging spot centered on the basal recording electrode, the basal oscillation frequency increased but the apical frequency did not. F: average of 10 trials like that shown in E.

Application of 10 mM Nomega -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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Fig. 6. NO synthesis inhibitor Nomega -nitro-L-arginine methyl ester (L-NAME) reduces the frequency of the LFP oscillation in the procerebral lobe. Addition of 5 mM L-arginine in the continued presence of 10 mM L-NAME reverses the inhibitory effect of L-NAME on the PC oscillation frequency. A whole lobe preparation was used. Mean values (± SD) of 10 cycle samples of the oscillation frequency are shown. This response was seen in 4 trials on 3 preparations.

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.



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Fig. 7. NO increases the burst frequency of bursting (B) cells. A: extracellular loose-patch recording from the soma of a bursting (B) cell shows periodic bursts of action potentials preceding each peak in the LFP. A pulse of NO produced by flash photolysis of NPR transiently increases burst rate and decreases spike number during each burst. Note that there is a peak in the LFP for each burst in the B cell. B: extracellular recording from a B cell process shows 2 LFP doublet events just prior to NO stimulation. B cell gives a double burst during the double events in the LFP. A pulse of NO increases burst frequency and LFP oscillation frequency.

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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Fig. 8. Responses of nonbursting (NB) cells to NO. A: intracellular recording from an NB cell obtained using the nystatin perforated-patch technique. NB cell receives a large inhibitory postsynaptic potential (IPSP) coincident with each peak in the LFP. NO pulse increases the frequency of LFP events, each of which is accompanied by an IPSP in the NB cell, clamping its membrane potential at ECl. Note the change in waveform of the LFP signal during the NO pulse and transition to the prepulse LFP waveform after the NO pulse. B: response of the NB cell to an NO pulse is greatly attenuated in the presence of oxymyoglobin, an NO scavenger.

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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Fig. 9. Application of carbon monoxide (CO) to the PC lobe increases the frequency of the LFP oscillation in a dose-dependent manner. Preparation was bathed in 44 µM caged CO in the form of NV-CO/AM. CO stimulation was initiated with an uncaging flash of 360 ± 10 nm light covering 75% of the PC lobe. A: LFP recording of typical response to 25-ms uncaging flash. B: LFP recording of typical response to 100-ms uncaging flash. C: LFP recording of typical response to 200-ms uncaging flash.



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Fig. 10. Averaged responses of the local field potential oscillation to 10 trials of uncaging flashes of varying duration. A: averaged responses to uncaging flash duration of 25 ms. B: averaged responses to uncaging flash duration of 100 ms. C: averaged responses to uncaging flash duration of 200 ms.



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Fig. 11. Dose-response curve relating peak increase in LFP oscillation and duration of CO uncaging flash. Each CO uncaging flash duration was repeated 10 times. Thirty measurements producing the 3 peak oscillation frequency responses for each flash duration were taken from plots like those shown in Fig. 9 and averaged. These average values are shown (± SD). Values at flash durations from 10 to 300 ms are significantly different from their control prestimulation values, as indicated in the text.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 regions---the 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).


    ACKNOWLEDGMENTS

We thank W. Denk for suggesting the use of caged NO and J.P.Y. Kao for a generous gift of caged CO.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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