Department of Biology and Center for Biological Timing, University of Virginia, Charlottesville Virginia 22903
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ABSTRACT |
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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.
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INTRODUCTION |
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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
pathwaythe 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.
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METHODS |
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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 M 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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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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RESULTS |
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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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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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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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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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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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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.
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FOOTNOTES |
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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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REFERENCES |
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