The Journal of Neurophysiology Vol. 80 No. 2 August 1998, pp. 1011-1015
Copyright ©1998 by the American Physiological Society

RAPID COMMUNICATION

Presynaptic Afferent Inhibition of Lobster Olfactory Receptor Cells: Reduced Action-Potential Propagation Into Axon Terminals

Matt Wachowiak^{1, 3} and Lawrence B. Cohen^{1, 2}

¹ Marine Biological Laboratory, Woods Hole, Massachusetts 02543; ² Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; and ³ The Whitney Laboratory, University of Florida, St. Augustine, Florida 32086

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Wachowiak, Matt and Lawrence B. Cohen. Presynaptic afferent inhibition of lobster olfactory receptor cells: reduced action-potential propagation into axon terminals. J. Neurophysiol. 80: 1011-1015, 1998. Action-potential propagation into the axon terminals of olfactory receptor cells was measured with the use of voltage-sensitive dye imaging in the isolated spiny lobster brain. Conditioning shocks to the olfactory nerve, known to cause long-lasting suppression of olfactory lobe neurons, allowed the selective imaging of activity in receptor cell axon terminals. In normal saline the optical signal from axon terminals evoked by a test stimulus was brief (40 ms) and small in amplitude. In the presence of low-Ca²⁺/high-Mg²⁺ saline designed to reduce synaptic transmission, the test response was unchanged in time course but increased significantly in amplitude (57 ± 16%, means ± SE). This increase suggests that propagation into receptor cell axon terminals is normally suppressed after a conditioning shock; this suppression is presumably synaptically mediated. Thus our results show that presynaptic inhibition occurs at the first synapse in the olfactory pathway and that the inhibition is mediated, at least in part, via suppression of action-potential propagation into the presynaptic terminal.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Presynaptic inhibition of transmitter release from primary afferent fibers is a well-established strategy for regulating sensory input to the CNS, occurring in vertebrates and invertebrates (Rudomin 1990; Watson 1992). Presynaptic inhibition enables regulation of input from specific sets of afferent fibers and can sharpen receptive fields, prevent habituation, and maintain the sensitivity of inputs across a wide dynamic range (Burrows and Matheson 1994; Watson 1992;). One mechanism for presynaptic inhibition is a reduction in size or penetration of the action potential into the nerve terminal (e.g., Wall 1994; Zhang and Jackson 1993). Another is an effect on subsequent events that control transmitter release (e.g., calcium influx into the terminal) (Wu and Saggau 1997).

There are suggestions that presynaptic afferent inhibition may play a role in olfaction. Presumptive inhibitory interneurons contact receptor axons in the cockroach (Distler and Boeckh 1997), and inhibitory D2 dopamine receptors are found in the olfactory nerve layer of the rat olfactory bulb (Nickell et al. 1991). Physiological data from several studies suggest presynaptic inhibition. In the spiny lobster, Panulirus argus, olfactory receptor cells are hyperpolarized by histamine, a known inhibitory transmitter in olfactory glomeruli (Bayer et al. 1989; McClintock and Ache 1989). In vertebrates, mitral cells and periglomerular interneurons show strong suppression after a conditioning nerve shock, which cannot be attributed to activation of other known interneurons (Aroniadou-Anderjeska et al. 1997; Freeman 1974; Mori et al. 1984). However, no direct physiological evidence exists, largely because electrode measurements of transmembrane potential in pre- or postsynaptic sites are prohibited because of the small diameter of the neuronal processes.

We used voltage-sensitive dyes to directly measure action potentials in the axon terminals of olfactory receptor neurons in the spiny lobster and also conditioning olfactory nerve shocks and low-Ca²⁺/high-Mg²⁺ block to suppress activation of postsynaptic neurons. Our results suggest that olfactory lobe interneurons mediate presynaptic inhibition of olfactory receptor cells, either by reducing the height of action potentials or by blocking propagation into terminal branches.

	METHODS

Abstract Introduction Methods Results Discussion References

Recordings were obtained in an isolated brain preparation (Wachowiak and Ache 1994). The brain was perfused with oxygenated Panulirus saline containing (in mM) 460 NaCl, 13 KCl, 13 CaCl₂, 10 MgCl₂, 14 Na₂SO₄, 1.7 glucose, and 3 N-2-hydroxyethyl piperazine-N'-2-ethane sulfonic acid (HEPES), pH 7.4. Paired electrical shocks [250-800 ms interstimulus interval (ISI)] were delivered to the olfactory (antennular) nerve. For the low-Ca²⁺/high-Mg²⁺ substitution experiments, the saline was transiently replaced with saline containing 1 mM CaCl₂ and 22 mM MgCl₂.

Brains were stained with a voltage-sensitive dye, di-2-ANEPHQ (JPW-2081, 0.06-0.1 mg/ml; synthesized by J. P. Wuskell and L. M. Loew, University of Connecticut Health Center, Farmington, CT) or di-4-ANEPPS (Molecular Probes, Eugene, OR; 0.05 mg/ml) by introducing 1-2 ml dye into the perfusion line or by applying dye (di-2-ANEPHQ; 0.24 mg/ml) into 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. One brain was stained by bath application and perfusion.

To record optical signals, light from a tungsten halogen lamp was passed through a 520 ± 45 nm interference filter, reflected from a 590-nm, long-pass dichroic mirror and focused on the preparation with the use of a 25 mm, 0.95 f, closed circuit television lens (Kleinfeld and Delaney 1996). A 3.5-mm-diam field was imaged with a 0.4 numerical aperture onto an array of 464 photodiodes. Each photodiode received light from a 150 × 150 µm area of the object plane. The photocurrents from each diode were amplified separately, band-pass filtered (0.06-500 Hz), and digitized at 1,010 Hz (Wu and Cohen 1993) under the control of NeuroPlex software (OptImaging, LLC, Fairfield, CT) on an IBM PC computer. In the five preparations, the mean photocurrent from the diodes was about 10 nA. Afterward, acquisition signals were digitally low-pass filtered (150 Hz Gaussian) and analyzed with the use of NeuroPlex. Additional digital filtering and spatial averaging was performed as described in the figure and table legends. All results come from single trials. Intracellular recordings from single olfactory lobe interneurons were obtained in separate preparations as described previously (Wachowiak and Ache 1994).

	RESULTS

Abstract Introduction Methods Results Discussion References

Figure 1 shows optical and electrical responses to paired-pulse stimulation of the antennular nerve at three levels of the olfactory pathway. In the olfactory nerve (Fig. 1A) the optical signals evoked by the first (conditioning) and second (test) stimuli consist of identical spikelike waveforms reflecting action potentials in receptor cell axons. In the olfactory lobe (Fig. 1B), the optical signal evoked by the conditioning stimulus consists of a large, fast component and a smaller, slow component. The test response consists of a single, small-amplitude fast component, similar in duration to that in the olfactory nerve. Electrical recordings from olfactory lobe interneurons show no intracellular response to the test stimulus (Fig. 1B, top trace), indicating a total suppression of postsynaptic activity after the conditioning stimulus. This suppression was observed in intracellular recordings from all known types of olfactory lobe interneurons and lasts from 1 s up to 30 s (Wachowiak et al. 1996, 1997). Complete suppression is also seen in the accessory lobe, a second-order olfactory neuropile, where the test stimulus elicits no response in either the optical or intracellular recordings (Fig. 1C).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Schematic drawing of the lobster brain (bottom right) and electrical (intracellular) and optical signals recorded in response to paired electrical stimulation of the antennular (olfactory) nerve. (Clockwise from lower left) A: optical signal from the nerve. , time of the antennular nerve stimulus. Olfactory receptor cell axons entering the olfactory lobe are shown in the drawing; di-2-ANEPHQ, bath applied. B: optical signal (bottom: di-2-ANEPHQ, perfused) from the olfactory lobe and intracellular recording (top) from an olfactory lobe projection neuron. C: optical signal (bottom: di-4-ANEPPS, perfused) from the accessory lobe and intracellular recording (top) from an accessory lobe projection neuron. All optical traces were taken from single photodiodes with no additional filtering. Optical and intracellular records are from different preparations. A, B, and C are from different preparations, and different interstimulus intervals (ISIs) were used. In this and subsequent figures the vertical calibration bar represents (either or both) the intracellular potential measured with a microelectrode and/or the fractional fluorescence change, F/F, in the optical measurement.

We compared responses in the olfactory lobe to paired-pulse stimulation in normal and low-Ca²⁺/high-Mg²⁺ saline designed to reduce synaptic transmission. As controls, we monitored optical signals in both the antennular nerve and also in the accessory lobe, which receives polysynaptic but not direct input from olfactory afferents. Optical signals in the antennular nerve were unchanged in low-Ca²⁺/high-Mg²⁺ (Fig. 2A). The accessory lobe signal was eliminated in low-Ca²⁺/high-Mg²⁺ saline (Fig. 2B), indicating that polysynaptic transmission was effectively blocked. We also recorded intracellularly in separate preparations from olfactory lobe projection neurons, which are believed to receive monosynaptic afferent input (Wachowiak and Ache 1994). In three preparations, low-Ca²⁺/high-Mg²⁺ perfusion eliminated the spike burst normally evoked by electrical stimulation and the subsequent hyperpolarization (Fig. 2C). However, a slow-onset, prolonged depolarization persisted in low-Ca²⁺/high-Mg²⁺, suggesting that monosynaptic transmission was significantly reduced but not blocked completely. Nonetheless, low-Ca²⁺/high-Mg²⁺ saline clearly attenuated and altered the time course of any postsynaptic response, eliminating rapid-onset postsynaptic potentials.

View larger version (11K): [in this window] [in a new window]   FIG. 2. A: low-Ca2+/high-Mg2+ saline does not affect the optical signal measured in the antennular nerve. Traces are spatial average of 5 diodes and were digitally high-pass filtered at 1.3 Hz; di-2-ANEPHQ, bath applied. B: low-Ca2+/high-Mg2+ saline eliminates the accessory lobe signal. Spatial average of 4 diodes; di-4-ANEPPS, perfused. C: intracellular recording from a single olfactory lobe projection neuron. Low-Ca2+/high-Mg2+ saline eliminates the spiking and hyperpolarizing response of projection neurons. A slow-onset, prolonged depolarization persists in low-Ca2+/high-Mg2+. Optical signals were not recorded in this preparation.

Surprisingly, in the olfactory lobe, the optical signal elicited by the test stimulus increased in amplitude in low-Ca²⁺/high-Mg²⁺ saline, whereas the amplitude of the conditioning response decreased, so that both stimuli elicited nearly identical signals with short latency and rapid onset. Examples from two preparations are shown in Fig. 3. Results from each of the five preparations are summarized in Table 1. The mean increase in the amplitude of the test response was 57 ± 16% (SE). Although these measurements were from averages of several photodiodes receiving light from the olfactory lobe, measurements from individual photodiodes indicated that the increase in test response amplitude occurred in all regions of the lobe (data not shown). We also compared the time courses of the test responses in normal and in low-Ca²⁺/high-Mg²⁺ saline after scaling the signals to the same size; the time courses did not differ in the two salines (Fig. 3B).

View larger version (12K): [in this window] [in a new window]   FIG. 3. Effect of low-Ca2+/high-Mg2+ saline on the optical signal recorded from the olfactory lobe. A: test response amplitude is increased in low-Ca2+/high-Mg2+. The conditioning response amplitude is reduced and a slow depolarizing component is seen. Spatial average of 9 diodes, high-pass filter, 2.6 Hz; di-2-ANEPHQ, perfused. B: traces from a different preparation showing the increase in the test response amplitude (top right) and decrease in the conditioning response (left). Heavy traces, normal saline; light traces, low-Ca2+/high-Mg2+. Test responses also shown (bottom) with the vertical scales adjusted so that the peak amplitudes in normal and low-Ca2+/high-Mg2+ salines are equal, illustrating that the kinetics of the test response are unchanged. Spatial average of 7 diodes. High-pass filter, 4.2 Hz; di-2-ANEPHQ, perfused.

Low-Ca²⁺/high-Mg²⁺ also affected the slower components of the conditioning response in the olfactory lobe. In all preparations a prolonged, slow-onset depolarizing optical signal emerged in low-Ca²⁺/high-Mg²⁺ (Fig. 3A, middle trace). This signal was similar in waveform and time course to the electrical response of single olfactory lobe projection neurons in low-Ca²⁺/high-Mg²⁺ (Fig. 2C, bottom trace).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

We measured voltage-sensitive dye signals from the lobster olfactory lobe in response to paired-pulse stimulation of the antennular nerve, a protocol that allows us to selectively image action-potential propagation into primary olfactory receptor axon terminals. In >200 neurons tested in previous intracellular studies, all known types of olfactory lobe interneurons are completely unresponsive to the second of a stimulus pair at the ISIs used in this study (Wachowiak et al. 1996, 1997). In addition, in the present study we observed no optical response to the second (test) pulse in the accessory lobe (see Fig. 1), which receives second-order fibers but no primary afferent input (Wachowiak et al. 1996). We thus conclude that the optical signal in the olfactory lobe evoked by a test stimulus reflects activity only in the terminals of primary olfactory receptor axons.

We used low-Ca²⁺/high-Mg²⁺ saline to block postsynaptic activity initiated by the first (conditioning) pulse, indicated by the fact that low-Ca²⁺/high-Mg²⁺ significantly reduced the amplitude of the conditioning response. The persistence of a slow, prolonged component in the conditioning response that parallels the slow olfactory lobe projection neuron response in low-Ca²⁺/high-Mg²⁺ indicates that synaptic transmission was not blocked completely under these conditions. However, the intracellular recordings from projection neurons show that monosynaptic transmission was severely attenuated, whereas the lack of response in the optical signal from the accessory lobe shows that transmission across multiple synapses was eliminated (see Fig. 2).

Our finding that the test response is larger under conditions of reduced synaptic transmission (low-Ca²⁺/high Mg²⁺ saline) implies that in normal saline, action-potential propagation into the receptor cell axon terminal is inhibited by interneurons activated by the conditioning pulse. An alternative explanation is that the increase in the test response in low-Ca²⁺/high-Mg²⁺ saline is caused by a reduction of a Ca²⁺-activated potassium current in receptor cell axon terminals, which would prolong and possibly increase the amplitude of the optical signal. However the test response increased significantly in amplitude but not in duration, and thus we believe this explanation is unlikely. We conclude that the strong paired-pulse suppression of olfactory lobe neurons is at least in part a result of presynaptic inhibition of action-potential propagation into olfactory receptor axon terminals.

Synaptically mediated propagation block occurs in several systems (Debanne et al. 1997; Wall 1993; Zhang and Jackson 1993), and a modeling study indicates that at branch points propagation can be blocked by relatively small conductance increases (Segev 1990). In the lobster, glomeruli are columnar and arranged radially around the olfactory lobe, and receptor cell axons branch and penetrate through three glomerular layers (Schmidt and Ache 1992). Thus inhibition in the outermost layer could potentially block propagation into the deeper layers, which contain the processes of olfactory lobe output neurons.

In vertebrates indirect evidence exists for presynaptic inhibition of olfactory receptor cells (see INTRODUCTION), although no anatomic contacts onto receptor cell axons were found (Pinching and Powell 1971). Our results show that in the lobster, presynaptic inhibition of olfactory afferents can be substantial. The functional role of presynaptic afferent inhibition in other systems includes gain control of input across a wide dynamic range (Burrows and Matheson 1994) and refining the tuning of afferent input (Levine and Murphey 1980), functions that are also presumed critical to olfactory coding. Future work will focus on characterizing the synaptic pathways underlying presynaptic afferent inhibition in the lobster and the functional role of this phenomenon in olfactory processing, using pharmacological probes to the known inhibitory transmitters in the olfactory lobe.

	ACKNOWLEDGEMENTS

  We thank B. Ache, B. Ehrlich, Y.-W. Lam, B. Ross, and D. Zecevic for comments on the manuscript and L. Milstead for providing the lobster brain drawing.

  This work was supported by National Science Foundation (NSF) Grant IBN9604356, National Institute of Neurological Disorders and Stroke Grant NS-08437, an MBL Research Fellowship, and NSF Grant IBN 9515307.

	FOOTNOTES

  Address for reprint requests: M. Wachowiak, Dept. of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520.

  Received 12 January 1998; accepted in final form 30 March 1998.

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