Coherent Oscillations in Membrane Potential Synchronize Impulse Bursts in Central Olfactory Neurons of the Crayfish

DeForest Mellon Jr. and Christopher J. Wheeler

Department of Biology and Center for Biological Timing, University of Virginia, Charlottesville Virginia 22903


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mellon Jr., DeForest and Christopher J. Wheeler. Coherent oscillations in membrane potential synchronize impulse bursts in central olfactory neurons of the crayfish. Lateral protocerebral interneurons (LPIs) in the central olfactory pathway of the freshwater crayfish Procambarus clarkii reside within the lateral protocerebrum and receive direct input from projection neurons of the olfactory midbrain. The LPIs exhibit periodic (0.5 Hz) changes in membrane potential that are imposed on them synaptically. Acute surgical experiments indicate that the synaptic activity originates from a group of oscillatory neurons lying within the lateral protocerebrum. Simultaneous intracellular recordings from many LPI pairs indicate that this periodic synaptic input is synchronous and coherent among the population of ~200 LPIs on each side of the brain. In many LPIs, specific odors applied to antennules in isolated head preparations generate long-lasting excitatory postsynaptic potentials and impulse bursts. The impulse bursts are generated only near the peaks of the ongoing depolarizations, ~1 s after stimulus application, and so the periodic baseline activity is instrumental in timing burst generation. Simultaneous recordings from pairs of LPIs show that, when impulse bursts occur in both cells after an odorant stimulus, they are synchronized by the common periodic depolarizations. We conclude that the common, periodic activity in LPIs can synchronize impulse bursts in subsets of these neurons, possibly generating powerful long-lasting postsynaptic effects in downstream target neurons.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fundamental questions concerning common brain mechanisms in olfactory processing remain unanswered. One of the most perplexing aspects of this ancient sensory system is oscillatory electrical activity observed in neurons of the central olfactory pathways of all major animal groups. These oscillations, which have been observed at various cellular levels in the olfactory system for one-half a century, were originally recorded in the hedgehog olfactory bulb (Adrian 1942) and were identified in other mammals (Freeman 1975), fish (Satou 1990), mollusks (Delaney et al. 1994; Gelperin and Tank 1990), and arthropods (Laurent et al. 1996; Mellon et al. 1992b). Nonetheless, the functional significance of oscillatory activity with regard to neural processing of olfactory information in any animal remains elusive.

Among invertebrates, conspicuous oscillations in neuronal membrane potential have been discovered in the olfactory forebrain of Limax, a pulmonate mollusk (Gelperin and Tank 1990), Schistocerca, an orthopteran insect (Laurent et al. 1996), and in Procambarus, a freshwater crayfish (Mellon and Alones 1997; Mellon et al. 1992b). Although the physiological significance of these oscillations is no more apparent in invertebrates than in vertebrate systems, their presence across phyla is of considerable interest, as is the similarity in depolarizing-hyperpolarizing sequences seen in central olfactory neurons of vertebrates and invertebrates after electrical stimulation of primary olfactory afferents (e.g., Hamilton and Kauer 1988; Meredith and Moulton 1978; Mori et al. 1981; Wachowiak and Ache 1994). These similarities in central olfactory neural activity may exist in all metazoans, and therefore it is entirely possible that insights obtained about the functional organization of invertebrate olfactory pathways can be generalized to vertebrate systems.

In the freshwater crayfish P. clarkii electrophysiological evidence has been obtained for important timing relationships among neurons comprising a critical link in the central olfactory pathway---the lateral protocerebral interneurons (LPIs)---and may have a crucial role in both the initiation of olfaction-mediated behavior and by analogy with the insect mushroom bodies in the establishment of olfactory memory. We find that oscillations in the LPI population are coherent, apparently in consequence of the action of local interneurons for which the LPIs are common postsynaptic targets. Furthermore, odor-evoked impulse bursts occur only at the peak of the ongoing periodic depolarizations. The oscillatory background activity may thus act as an entrainment signal to synchronize bursts in different members of the LPI population. Our electrophysiological evidence indicating important timing relationships among these neurons is discussed in light of the physiological role of impulse bursts and oscillatory activity in other neural systems.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Freshwater crayfish P. clarkii obtained from a dealer in Louisiana were used. The crayfish were housed in communal tanks of filtered, circulating fresh water and were kept at a temperature of 18°C and a light/dark cycle of 13 h/11 h. They were fed the green plant Elodea.

The dissection procedures devised to maintain vascular circulation in the brain and the exposure of the lateral forebrain were described elsewhere (Mellon and Alones 1997) but will be detailed here. Animals were quickly decapitated, and the isolated heads were fastened within a recording chamber with the antennules projecting into an olfactometer, as described subsequently. The median artery, which delivers blood to the brain and the neural centers in the eyecups, and one of the lateral cephalic arteries, which deliver blood to the antennae, the antennules, and the eyecup neural centers, were cannulated and flushed with chilled (15°C), oxygenated crayfish saline (Van Harreveld 1936). The organs of olfaction, antennules, were drawn into one arm of an odorant delivery device (olfactometer) consisting of a plastic T-tube. Vaseline was used to seal the base of the antennules within the T-tube arm to prevent saline in the recording chamber from contacting the odorant receptors on the distal one-half of the antennules. Dechlorinated fresh water entered the stem of the T-tube by means of small-diameter silastic tubing and washed over the antennules continuously except during passage of a pulse of stimulating solution. A second length of silastic tubing also was fitted into the stem of the T-tube, and odorant pulses from different reservoirs were delivered via a manifold on demand through this conduit. Both stimulant and wash water, after washing over the distal one-half of the antennules, were exhausted through the opposite arm of the T-tube into a sump that was emptied by suction.

Both the flow of wash water and the stimulus release were controlled by electrically switched valves that were driven by a digital pulse generator. The wash water flow through the olfactometer was terminated 10 ms before the onset of the stimulus pulse and remained off for the duration of the stimulus pulse. Stimulus pulses lasted 100 or 500 ms or 1 s. Because of stimulus tailing, stimulus duration was only approximate (Mellon and Alones 1997). Two minutes was allowed to elapse between successive presentations of the same odorant stimulus or of different odorants to assure that the olfactory receptor cells disadapted to the previous stimulus exposure. A pair of silver wires within the olfactometer provided electrical stimulation to the antennules as required.

Exposure of the right-hand hemi-ellipsoid body (HEB) in the lateral protocerebrum was accomplished by cementing one end of a semi-rigid support to the right compound eye and securing the other end to one wall of the recording chamber. Dissection of the dorsal exoskeleton over the base of the eyecup was performed with microrongeurs. Removal of the overlying muscles then exposed the proximal neural axis, consisting of the distal lateral protocerebral tract, the terminal medulla, and the HEB (see Fig. 1). The sheath surrounding the dorsal aspect of the HEB was carefully removed, and a gentle stream of saline was used to wash away blood and loose glial tissue. Pairs of micropipette electrodes filled with 2 M potassium acetate and having resistances of 100-150 MOmega were used to record simultaneously from the dendrites of different LPIs within the hemi-ellipsoid neuropil. Electrode holders were mounted on Leitz micromanipulators. Once established, recordings were usually stable for >= 1 h, permitting various testing procedures.



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Fig. 1. Diagram of the crayfish brain viewed from above and slightly anterior to the left side. A pair of prominent axon tracts connects the medial forebrain to the areas of lateral forebrain on either side. The olfactory-globular tract (OGT), containing axons of the olfactory projection neurons, is indicated by - - - , its position within the lateral protocerebral tract indicated by the cutaway view. Cell cluster 10, containing the somata of the olfactory projection neurons, is indicated on both sides. The nerve roots labeled 1 and 2 are motor nerves to the muscles of the eyestalks. *: approximate location of lateral protocerebral interneuron (LPI) recording sites within the hemi-ellipsoid body (HEB). Sites of focal electrical stimulation of the olfactory projection neurons are indicated by an X. The 3 optic ganglia, distal to the medulla terminalis (MT), are not included in the diagram. OSC, possible source of LPI oscillations in the MT; CC, circum-esophageal connectives; LPT, lateral protocerebral tract.

The membrane potential of LPIs near the recording site could be manipulated by passing depolarizing or hyperpolarizing current through the recording electrode. In either case, current levels varied between 1 and 10 nA. Neither the precise magnitude of the current steps nor the resulting voltage changes were measured because these procedures were used primarily to 1) accentuate the amplitude of the depolarizing oscillations and 2) determine in a qualitative sense whether injected currents that modified the impulse frequency of one impaled neuron affected the frequency of oscillations in another simultaneously recorded LPI.

Recorded signals were led to unity-gain electrometer input stages and then to an operational amplifier manifold where the signals were amplified 10-fold at wideband. Amplified signals were then led to the DC input of an analog storage oscilloscope (band-pass: DC-10 kHz) and a multichannel VHS tape recorder for viewing and storage, respectively. Some segments of recorded data were later digitized by Axotape software (Axon Instruments) and stored in a computer file (DC-10 kHz band-pass) or, as in most cases, photographed directly from the screen of the oscilloscope. In one instance (records in Fig. 3, E and F), before photography signals stored on VHS tape were amplified by an AC amplifier having a filter band-pass of 1-3,000 Hz. Digitized impulse burst files were analyzed statistically by hand measurement from the screen of the storage oscilloscope.

Direct electrical stimulation of the olfactory projection neurons was accomplished with a glass suction electrode filled with crayfish saline and having a tip diameter of ~100 µm. The tip of the electrode was gently pressed against the surface of the anterior part of cell cluster 10 or against the lateral protocerebral tract at its junction with the brain (see Fig. 1). Stimulus pulses 0.5 ms in duration were used to evoke impulse volleys of varying strength in the projection neuron pathway.

Anatomy of the olfactory pathway

As in other decapod crustaceans, olfaction is mediated by blunt sensilla on the lateral filaments of the paired antennules. In crayfishes, each of these sensilla, which are referred to as aesthetascs, houses the distal dendrites of ~250 bipolar sensory neurons, the cell bodies of which reside in a sensory ganglion near the base of each aesthetasc. The distal dendrites are believed to be the site of olfactory transduction (Hatt and Ache 1994). Axons of the olfactory receptor cells run within the antennular nerve to the ipsilateral midbrain, where they terminate within glomeruli in the olfactory lobe. Olfactory information is processed within the olfactory lobe through local midbrain interneurons, some of which were electrophysiologically characterized (Mellon and Alones 1995; Schmidt and Ache 1997).

Figure 1 presents a diagram showing the dorsal surface of the crayfish brain, seen from the left side in a slightly anterior view. Olfactory information that was processed within the olfactory lobe ascends to the lateral forebrain via olfactory projection neurons. These cells have extensive dendritic arborizations within the olfactory lobe, and their axons run within the olfactory globular tract, a distinct fascicular pathway within the lateral protocerebral tract, to the lateral protocerebrum. The projection neuron axons from each side terminate in the HEBs, a localized region of the medulla terminalis (MT), which together constitute the lateral protocerebrum. The cell bodies of the olfactory projection neurons reside within a latereral cluster on each side of the brain, cell cluster 10.

The diagram in Fig. 1 includes the prominent lateral protocerebral tracts (LPT) and the lateral forebrain regions found within the eyecups on each side, which include a terminal ganglion (MT) and the HEB. Located distal to the MT are three visual ganglia, the retina, and the cornea of each compound eye, none of which is shown in the diagram. The HEBs on each side are positioned prominently on the medial aspect of the neural axis within the eyecup.

Figure 2 is a longitudinal section of the neural axis of the compound eye. The HEB, stained here with the ethyl gallate/osmium technique (Leise and Mulloney 1986), is a bilobed, roughly hemispherical, medial projection of the MT. Microscopically, it consists of a cortical region, in which terminals of ~100,000 projection neuron axons in the olfactory-globular tract (OGT) make synaptic contact with fine distal terminations of dendritic arborizations from ~200 local interneurons (LPIs) and a medullary region comprising extensive dendritic branches of the LPIs. LPI somata reside in clusters adjacent to the MT, out of the plane of section in the micrograph. The major dendrites of the LPIs exit together in a central tract at the base of the HEB and join the axons and neuritic segments in the center of the MT. The axons terminate in neuropil near the ventral surface of the MT (Mellon et al. 1992a).



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Fig. 2. Sagittal section of the neural axis in the eyestalk stained with osmium-ethyl gallate. The OGT, containing axons of the olfactory projection neurons, is indicated by *, and the HEB is indicated by arrowheads. The projection neuron axons diverge around the periphery of the HEB and then terminate individually within microglomeruli, just beneath the surface. Within each microglomerulus, a projection neuron axon terminal expands into a rosette-like structure to make ~165 synaptic connections with various LPIs. The plane of section passes through only a lateral portion of the MT, located below the base of the HEB. The labeled optic ganglia are the lamina ganglionaris (L), the medulla externa (ME), the medulla interna (MI), and the MT. Scale marker: 1 mm.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sharp electrodes advanced into the HEB medulla penetrate large dendritic processes of LPIs. Resting potentials of 60-75 mV and overshooting action potentials are encountered at penetration of an LPI. As the recording situation stabilizes over several minutes, ongoing periodic depolarizations of 10-15 mV in amplitude are characteristically observed in the LPIs, as previously reported (Mellon and Alones 1997). Concomitant with recovery from the effects of initial penetration, many cells also exhibit occasional spontaneous impulse bursts, each lasting ~100 ms.

Synchronous oscillatory activity in the LPIs

Figure 3 presents typical simultaneous intracellular recordings from three pairs (A-D, E, and F) of LPIs in the same HEB under conditions stated in the figure legends. Subthreshold depolarizing excursions of the membrane potential in each cell pair have the same frequency and are in approximate phase. Figure 3, C and D, shows activity recorded during a period when both neurons in the paired recording were hyperpolarized by injected current to accentuate the amplitude of the depolarizing excursions. Although the successive individual waveforms comprising the depolarizing oscillations are not entirely alike, they are mostly similar in detail with respect to corresponding events in each cell of the pair. Occasionally, although not typically, some components present in the complex waveforms seen in one cell are missing at the corresponding time point in the second cell, and these components are indicated by asterisks. Similar properties are seen in the paired records of Fig. 3, E and F, from cell pairs in two additional preparations. The records from both of these latter cell pairs represent synchronous activity that was amplified and filtered to accentuate the subcomponents. These results clearly indicate a temporal correlation among respective waveform trains in the LPI pairs.



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Fig. 3. Multiple excerpts of simultaneous recordings from 3 different pairs of LPIs (A-D: 061797, A and B; E: 062696, A and B; F: 071797, A and B). A and B indicate that the underlying slow depolarizations are synchronous in both cells, although the impulses generated by them are not necessarily synchronous. In C and D both neurons were hyperpolarized to emphasize the depolarizing excursions of the membrane. An * in each trace indicates a synaptic component in that cell that appears to have no counterpart in the other cell. In each frame, voltage calibration 1 applies to the topmost trace, and calibration 2 applies to the bottom trace. Voltage calibrations: 20 mV. E and F illustrate records from 2 different pairs of LPIs, identified previously, amplified and filtered (band-pass: 1 Hz to 3 kHz) by an AC amplifier to maximize identification of synaptic components. Nearly every subcomponent in 1 cell is synchronous with a component in its pair. Voltage calibrations: 10 mV.

A possible explanation for the similarity of activity in neighboring LPI neurons could be electrical or chemical synaptic coupling between cells. We tested for coupling by passing both hyperpolarizing and depolarizing current pulses sequentially into each electrode while we recorded from cell pairs. The records of Fig. 4, which are typical, show three separate instances of depolarizing current injections into one pair of neurons. In recordings obtained from ~50 LPI pairs, we never observed any type of functional coupling between the cells. If functional coupling among the LPIs exists but is weak it is possible that subtle changes in coupling among the LPIs might have occurred but went unobserved. Such coupling could potentially be of importance among subgroups of cells that might have similar stimulus preferences, because their individual responses to an odorant might be strengthened through such a pathway via an enhancement of the background depolarizations. This possibility, although reasonable, was not examined further because the depolarizing responses of the LPIs to odorants presented to the antennules was far weaker than the apparently ineffective experimentally applied current, as judged by the increase in impulse frequency caused in the LPIs, respectively, by the two types of stimulation.



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Fig. 4. Simultaneous recording from 3 different cell pairs (061297a and b; 071797) in which depolarizing current pulses were passed into 1 cell of the pair in each case. In neither these trials nor any others, in which both hyperpolarizing and depolarizing current was passed into each cell of the pair, were any forms of functional coupling among the cells observed. Calibrations: 50 mV and 5 s.

Origins of oscillatory input

The olfactory midbrain projection neurons were previously physiologically and anatomically identified as excitatory inputs to the LPIs (Mellon et al. 1992a,b). We confirmed this by recording from LPIs while electrically stimulating sets of projection neurons via a suction electrode placed on cell cluster 10 (the location of the somata of olfactory projection neurons), prominently located on the side of the brain, or on the lateral protocerebral tract, within which run the axons of the projection neurons. Electrical shocks to either site ought to generate impulse volleys in the projection neuron axons and thus synaptic potentials in the LPIs, although at different latencies. Figure 5 shows that brief electrical shocks to projection neuron somata from either side are effective in generating prolonged excitatory postsynaptic potentials (EPSPs) in the LPIs. The EPSPs evoked by even these single brief stimuli lasted 0.5-1.0 s and therefore would be expected to encroach on the depolarizing phase of the periodic activity observed in the LPIs. This point is important and will be touched on in the DISCUSSION. Electrical stimulation of the LPT had no apparent effect on the period of the oscillations observed in the LPIs (data not shown). However, because of the variable period of the oscillations, small perturbations in period length would not easily be detected.



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Fig. 5. Responses of an LPI (051997) to single electrical stimuli of increasing strength (1-5, in A and B; 1 to 2 in C) delivered, as indicated by the arrows in the diagram, to ipsilateral (B) or contralateral (C) somata of olfactory projection neurons in cell cluster 10 on each side or (A) to the ipsilateral lateral protocerebral tract, containing projection neuron axons. Recording site in the HEB indicated by an asterisk.

LPI oscillations previously were attributed to activity in the axons of the olfactory projection neurons (Mellon and Alones 1997). However, we performed acute surgical experiments with six different preparations in which the entire lateral protocerebral tract including the OGT was severed. The results of this treatment demonstrate that our initial assumption was in error, because the oscillations continued in LPIs after ipsilateral LPT transection. Simultaneous recordings from a pair of LPIs after transection of the ipsilateral LPT indicate that the periodic activity persists in a robust fashion after this surgery, and the underlying depolarizations, emphasized by hyperpolarizing both neurons, are clearly synchronous (Fig. 6, A and B). Furthermore, cross-correlation analysis, shown in Fig. 6C, indicates there is a high degree of temporal correlation (r = 0.73; P < 0.001) between the activity peaks in the LPI pair.



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Fig. 6. Simultaneous records obtained from a pair of LPIs (111197A and B) after transection of the ipsilateral LPT. A: activity at normal levels of membrane potential. In B, both cells were hyperpolarized to eliminate impulse activity and increase the amplitude of the synaptic potentials. Calibrations (C): auto- and cross-correlograms of activity in the 2 cells. R = 0.73.

It was of interest to consider the anatomic site of the periodic synaptic input to the LPIs. Projection neuron synapses with LPIs are confined to a microglomerular layer close to the periphery of the HEB (Mellon et al. 1992a), suggesting that only the most terminal branches of the LPI dendrites take part in these connections. By hyperpolarizing an LPI to a fixed level and then comparing the extent of the resulting amplitude change in EPSPs evoked by projection neuron impulse volleys with that seen in the periodic EPSPs, some measure can be obtained for the relative electrotonic distances of the two synaptic sites from the recording/current passing electrode. Ideally, such measurements should be made on individual miniature EPSPs or unitary EPSPs evoked by a single presynaptic axon rather than multi-axonal volleys. Moreover, there is now ample evidence in some systems that dendritic membranes are not necessarily passive elements, and measurements can be skewed by changes in their membrane conductance properties as membrane potential levels are varied (Haag and Borst 1996; Laurent 1993). However, because it is the relative change in two different sources of excitatory synaptic input that would be measured in such experiments, any differences could, to a first approximation, be attributed to the relative extent of degradation of applied hyperpolarization encountered by the two classes of EPSPs. Accordingly, we examined this question by quantitative comparisons in two LPIs. The maximum amplitudes of periodic EPSPs and EPSPs evoked by single brief electrical shocks delivered to the projection neuron somata via cell cluster 10 were compared at the normal membrane potential and at one or two different levels of membrane hyperpolarization, obtained by passing DC current via the recording electrode. As indicated in Fig. 7, changes observed in the mean amplitude of evoked EPSPs were essentially the same (~30% increase) as the mean changes observed in the periodic EPSPs. Assuming that active membrane processes did not interfere with the electrotonic spread of one class of synaptic potential compared with the other and that both sources of synaptic potential have the same reversal potential, we conclude that the region of periodic synaptic input is electrotonically equidistant from the recording site compared with the most distal dendritic branches, where the evoked potentials are known to originate. One possible explanation for these observations would be the siting of both kinds of synaptic input at the peripheral microglomeruli.



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Fig. 7. Relative amplitude changes in periodic and evoked excitatory postsynaptic potentials (EPSPs) in an LPI (040798) during membrane hyperpolarization. A and B: excerpts of spontaneous EPSPs at normal membrane potential and (C and D) at a hyperpolarized level of membrane potential. E and F: sample-evoked EPSPs at normal and (G and H) the same hyperpolarized membrane potential level. Arrows indicate potential levels where amplitude measurements were made from spike-generating EPSPs. Calibrations: 25 mV; A-D, 8 s; E-H, 1 s.

Synchronization of impulse burst responses to oscillations

Impulse bursts are a characteristic, long latency feature of the response to odors in ~35% of the LPIs that we examined (Mellon and Alones 1997). As illustrated by the record inset in Fig. 8A, the bursts consist of brief (100 ms) trains of 4-10 spikes recurring at frequencies of <= 110 Hz. They occur not only spontaneously but also in response to exposure of the antennules to acceptable odorant stimuli (Mellon and Alones 1997). We measured the intraburst spike frequency in a number of cells to determine whether the frequency structure was constant or variable. Figure 8, A and B, illustrates normalized data from four different preparations. The data show that for each LPI the interspike interval profile within each burst was consistent, as adjudged by the relatively small SDs to the mean values. Furthermore, comparisons of burst envelopes from the four different preparations are very similar. These data are suggestive that the impulse bursts may be generated by membrane events that have a fixed waveform, such as plateau potentials.



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Fig. 8. A, inset: impulse burst structure in an LPI (123094) showing a roughly parabolic frequency envelope. A and B: intraburst interspike interval profiles from different LPIs. A shows data from 4 LPIs (123094, 010497, 072694, and 013198A) in which bursts containing 6 impulses occurred. B presents data from 3 of the same LPIs (010497, 013198A, and 123094) for bursts in which 7 impulses occurred. The histograms indicate that, when normalized for burst length, the interspike interval profiles within and across preparations are similar. Error bars indicate ± SD.

Records in Fig. 9 illustrate the impulse bursts of an LPI in response to several different odorants, including three individual amino acids, a mixture containing four amino acids, a solution made from the commercial tropical fish food tetramin, and D-glucose. Here the impulse bursts occur after the prolonged EPSPs generated in response to most odors by projection neuron input. At least one other amino acid odorant we tried did not evoke a burst response in this cell; furthermore, burst latencies varied somewhat depending on the odorant being tested (e.g., compare the latency of the burst response to the amino acid mixture in Fig. 9D with the latency of the burst to tetramin in Fig. 9E). It is not clear, however, whether this variation depended in a systematic manner on the tested odorant; clearly, the relative timing of stimulus application within the oscillatory period could be a factor in generating latency variations. However, because of the extensive series of odorant applications that such an examination would entail and the uncertainty caused by the natural variations in the oscillatory period, this potentially interesting aspect of the burst responses was not examined in the current series of experiments. Examination of timing of the bursts indicates that they do not arise directly from the peak of the projection neuron EPSPs; rather they occur in synchrony with of one of the baseline oscillations after an EPSP. Additional data showing synchronization of the burst activity with the baseline oscillations are shown in Fig. 10. The recordings are from an LPI that exhibited robust impulse bursts in response to an antennular application of 0.02% tetramin solution, a standard broad-spectrum odorant that we routinely use as a stimulus for the crayfish olfactory system (e.g., Mellon and Alones 1997). In this cell, the impulse burst responses to tetramin were clearly superimposed on the depolarizing phase of the periodic activity, the frequency of which increased somewhat for several seconds after the stimulus. During injection of hyperpolarizing current through the recording electrode, the amplitude of the baseline oscillations increased, emphasizing the synchronization of the burst responses with depolarization. Because the question of timing of the burst responses with respect to the depolarizations is an important one, we also studied a number of LPIs in paired simultaneous recordings, when one or both cells of the pair responded to odors with typical impulse bursts. As indicated in Fig. 11, the impulse bursts in one cell always were associated with the background depolarizations observed in the other LPI of the pair. Additional data emphasizing this point are shown in the frequency histograms of Fig. 12. Spontaneous burst data from LPIs 013198A and B were used to determine when the initial spike of each burst in LPI 013198B occurred with respect to the background depolarization period. Approximately 365 EPSP intervals in 013198A were measured as the distance between peak amplitudes. The timing of 43 spontaneously occurring bursts in 013198B was measured with respect to the nearest previous depolarization peaks. The data in Fig. 12 indicate that the intervals between the bursts and the nearest previous peak EPSP have values that are primarily between 50 and 325 ms. Because most of the EPSPs persisted for >= 300 ms, the bursts and the EPSP occurrences are clearly clustered together.



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Fig. 9. Response patterns (top traces) of an LPI (123094) to 1-s applications (indicated by excursions on bottom traces) of (A) 10-4 M glycine, (B) 10-4 M taurine, (C) 10-4 M L-alanine, (D) a mixture of equal parts of glycine, taurine, L-alanine, and L-glutamate, all at 10-4 M concentration, (E) 0.02% tetramin, and (F) 10-3 M D-glucose to the antennules of an isolated head preparation. Impulse bursts (*) were a long latency response feature in each case but were generated at peaks of the periodic depolarizations, not by the EPSPs imposed on the LPIs by the projection neurons. In A and D-F they occurred at single or double depolarizing interval peaks predicted by the last spontaneous impulse before the stimulus. Calibrations: 50 mV and 5 s.



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Fig. 10. Activity in an LPI (030198) in response to 1-s pulses of standard tetramin. A: spontaneous oscillatory activity, before odor stimulation of the preparation. B-D: successive responses to tetramin stimulation (indicated by bottom traces). A short-latency EPSP response to each odor presentation is followed by 3 separate impulse bursts, each appearing to arise from a peak of the baseline depolarization. E: control in which the wash water was terminated for 1 s but no odorant was delivered. In F, a pulse of tetramin was delivered while the LPI was being hyperpolarized by injected current. The delayed impulse burst arises from a baseline depolarization. Calibrations: 50 mV and 5 s.



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Fig. 11. Six excerpts of simultaneous intracellular records from a pair of LPIs (013198, A and B) attesting to the synchrony of spontaneous impulse bursts in 1 cell with peaks of the periodic background depolarization in the other. Spontaneous bursts in any cell usually occurred only once or twice each minute. These records support the contention that bursts are generated only at the maximum excursions of membrane depolarizations.



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Fig. 12. Comparisons of 365 intervals among successive background depolarization peaks (open bars) and 43 intervals between the beginning of a spontaneous burst and the nearest previous depolarization peak (solid bars) in cell 013198B. With two exceptions, the bursts begin within 325 ms of the peaks, indicating that they are specifically clustered at times during the depolarizations.

We next obtained recordings from an additional LPI pair that exhibited bursting activity and was also driven by tetramin applied to the antennules. Figure 13 indicates responses of these two cells to both chemical and electrical stimulation of the antennules. Burst responses in both cells occurred closely in time after respective antennular stimulations, confirming that generation of burst responses in phase with the same depolarizing peak is observed in some LPI pairs.



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Fig. 13. Paired intracellular records from two LPIs (051398, A and B) that both responded to (A) an amino acid mixture and (B) electrical stimulation of the antennules. The records indicate that both cells generated impulse bursts in response to the same periodic baseline depolarization.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The work presented here suggests that the oscillatory electrical activity in the hemi-ellipsoid LPIs of the freshwater crayfish P. clarkii is imposed synchronously on the entire population of hemi-ellipsoid LPIs. This conclusion emerges both from visual inspection of >50 paired recordings from these forebrain neurons and from cross-correlation analysis of subthreshold depolarizations in one pair of LPIs that were surgically isolated from the olfactory midbrain.

LPIs possess highly branched dendritic arbors that fill the HEB. Their primary dendrites have the largest diameter of any neural structures within the HEB and therefore are the ones most likely to be successfully penetrated by sharp electrodes. These major branches of the dendrites are also electrically excitable, as indicated by the presence of overshooting action potentials. In this respect, the LPIs are similar to Kenyon cells of the insect mushroom body, with which they share certain gross anatomic features. Kenyon cell secondary dendrites are also characterized by having voltage-gated depolarizing conductances that are believed to amplify synchronized input signals (Laurent 1996). Possible branch spikes were previously noted in crayfish LPIs (Mellon and Alones 1997; Mellon et al. 1992b) and may have a similar functional significance.

Two sources of excitatory synaptic input contribute to activity in the LPIs, the olfactory projection neuron axons from the midbrain and the oscillatory cells from other, unidentified eyecup neural centers. The olfactory projection neuron synaptic input zone was determined previously by electron microscopy to be microglomeruli within a peripherally placed shell of the HEB (Mellon et al. 1992a). Within each microglomerulus a projection neuron axon terminal forms an expanded rosette ending, and each rosette may make as many as 160 synaptic connections with LPI dendritic twigs (Mellon et al. 1992a). Because ~100,000 projection neuron axons make synaptic contact with ~200 LPIs, considerable synaptic convergence occurs in this region of the HEB.

The data presented here indicate that local oscillatory neurons drive all of the LPIs. Although our recording methods allowed for simultaneous observations of activity from only two LPIs at one time, the consistency of observations of phase-locked periodic activity in every paired recording from these hemi-ellipsoid cells provides a strong implication that the entire group of LPIs on each side of the brain undergoes oscillatory activity in a coherent, unified, population response to imposed synaptic input. The input zone within which oscillatory neurons make synaptic connections with the LPIs is currently not anatomically defined. However, because the same levels of injected hyperpolarizing currents increase the amplitude of the oscillatory potentials by roughly the same extent as the EPSPs generated by olfactory projection neuron volleys and assuming that both synaptic sources have the same reversal potential, we conclude that the periodic input synapses are at the same electrotonic distance from the recording site as those of the olfactory projection neurons. Additional details about the cellular location of these synapses must wait until the oscillatory cells are identified anatomically and their processes are stained.

Two distinct sources of voltage-gated electrogenesis apparently exist in the LPIs. Individual impulses, or impulse pairs, commonly arise from the baseline oscillations and in most cells studied are always present. On the other hand, impulse bursts occur only sporadically or in response to stimulation of the antennular afferents. The apparent threshold for impulse bursts is higher than that for individual spikes, suggesting that, although both forms of activity invade the recording site, their respective loci of origin in these morphologically complex neurons may be different from each other. This separation may have a bearing on the output signal transmitted by these neurons. The data also indicate that spontaneous impulses bursts are loosely synchronized with the periodic background depolarizations. This conclusion follows both from visual observations of recorded bursts as well as from phase histograms of burst occurrence with respect to the mean period of the background depolarization in six different preparations. As Fig. 12 indicates, spontaneous bursts occurred predominantly close in time with the depolarization peaks.

Impulse bursts in response to odorant input are also loosely synchronized with the depolarizing peaks of the LPI baseline oscillations. The bursts may in fact be triggered by a summation of the prolonged EPSPs generated by projection neuron input and the peak depolarizations generated by the unidentified oscillatory neurons. This supposition, however, requires experimental support, which we now lack. When multiple bursts are present in the response these occur at successive peaks of the background oscillations. Each burst appears thus to have a high-threshold for activation but once triggered generates a characteristic envelope of impulse firing. Many different crustacean central neurons characteristically fire in highly consistent impulse bursts, and they are often found within restricted networks that generate stereotyped behaviors or that ensure maximum effective periodic activation of the muscle groups responsible for cyclical contraction patterns (Mellon 1997). The most extensively examined systems exhibiting bursting are the crab heart ganglion (Russell and Hartline 1983; Tazaki and Cooke 1983) and the lobster stomatogastric ganglion (Moulins and Nagy 1985). In all of these neurons, the impulses within the burst responses are consistent in their frequency and duration, indicating that the underlying waveform (sometimes referred to as plateau potentials) has a constant time course and amplitude profile. Voltage- and current-clamp studies of these potentials have shown that they are generated by voltage- and time-dependent membrane properties. The consistency of the burst envelope in LPIs is noticeable and may indicate that plateau potentials also constitute the membrane generator for these stereotyped impulse trains.

Because there is no evidence to suggest that impulse burst activity in the LPIs generates cyclical or stereotyped behaviors, what is the functional significance of the oscillations and impulse bursts within these neurons? Because bursts are generated during the common depolarizing oscillatory waves after odor stimulation, we suggest that the background oscillations constitute a mechanism that synchronizes impulse burst responses to odors in different subsets of LPIs. This apparently occurs because bursts are generated when odor stimulation provides a trigger, after which they appear superimposed on one or more of the oscillatory changes in membrane potential that are present at the same time in all LPIs. If this is the case the effects of high-frequency bursts in several LPI axons activated in parallel could have a powerful modulatory influence on their common postsynaptic targets. Impulse bursts are considerably more effective than single spikes for information transfer at some central synapses (Kim and McCormick 1998; Lisman 1997). As exemplifed by some types of neuromuscular junctions in crustaceans, such synapses in effect act as high-pass filters of electrical activity so that single impulses or even trains of impulses at low frequency may have little effect as agents of synaptic transfer. The same synapses, on the other hand, can exhibit robust trans-synaptic activation in response to a brief, high-frequency impulse train, such as a burst (Mellon 1997). If this is the case at the synaptic junctions between the LPIs and their target neurons within the MT, it may be speculated that the single impulses generated in LPIs by the 0.5-Hz oscillatory activity do not act as carriers of information, that is, they may constitute noise. Single impulses may have some functional significance, however, in priming or enhancing the ability of the synaptic terminals to respond maximally to impulse bursts.

Synchronization of multiple presynaptic pathways combined with the filtering could generate highly effective postsynaptic voltage changes. Moreover, as is well known, synchronization of bursts in relevant parallel presynaptic pathways can promote long-term potentiation (LTP) at synapses in the mammalian hippocampus (Bliss and Collinridge 1993). Among other pathways within the hippocampus, the excitatory synapses made by CA3 pyramidal cells onto CA1 cells via the Schaeffer collateral pathways exhibit dramatic LTP when numbers of the presynaptic axons are stimulated in concert. CA1 pyramidal neurons in vivo exhibit 4- to 7-Hz theta oscillatory activity, and they can be induced to oscillate in slice preparations by the application of carbachol. If a single burst of presynaptic action potentials (e.g., 4 spikes at 100 Hz) is evoked in the Schaeffer collaterals coincident with the depolarizing peak of the theta oscillations, LTP that lasts for hours is established at the synapses with CA1 neurons. If, however, the burst coincides with the hyperpolarizing trough of the theta rhythm, no change occurs in the normal synaptic strength (Huerta and Lisman 1995). This type of synaptic memory trace thus requires the temporal superposition of pre- and postsynaptic potential changes at specified synapses on single neurons. Because the theta oscillations appear to be synchronous in large numbers of hippocampal cells, a single burst evoked in several presynaptic pathways could, if timed correctly to the theta rhythm, effect LPT in a large population of CA3-CA1 synaptic contacts. More recently, Thomas et al. (1998) have shown that high-frequency bursts of action potentials backpropagating within CA1 pyramidal cells will induce LTP when such bursts occur during theta frequency synaptic stimulation of CA1 neurons. The hippocampal model therefore has obvious relevance to the olfactory system, as well as to other brain regions, where the functional significance of ongoing oscillatory activity has been pondered over so many years. Future work will disclose whether the odor-evoked synchronous impulse bursting within subsets of LPIs is instrumental in modifying synaptic transfer at their target neurons.


    ACKNOWLEDGMENTS

The authors are grateful to K. Dame for insightful editorial changes in the manuscript.

This research was supported in part by research grants from the National Science Foundation (IBN 93-19406) and the National Institute of Deafness and Other Communications Disorders (RO1 DC-02376).

Present address of C. J. Wheeler: 311 Eddy St., Apt. 4, Ithaca, NY 14850.


    FOOTNOTES

Address for reprint requests: D. Mellon, Jr., Dept. of Biology, Gilmer Hall, University of Virginia, Charlottesville, VA 22903.

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 11 June 1998; accepted in final form 13 November 1998.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society