Department of Biological Computation, Bell Laboratories, Lucent Technologies, Murray Hill, New Jersey 07974
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ABSTRACT |
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Wang, Jing W., Winfried Denk, Jorge Flores, and Alan Gelperin. Initiation and Propagation of Calcium-Dependent Action Potentials in a Coupled Network of Olfactory Interneurons. J. Neurophysiol. 85: 977-985, 2001. Coherent oscillatory electrical activity and apical-basal wave propagation have been described previously in the procerebral (PC) lobe, an olfactory center of the terrestrial slug Limax maximus. In this study, we investigate the physiological basis of oscillatory activity and wave propagation in the PC lobe. Calcium green dextran was locally deposited in the PC lobe; this led to cellular uptake and transport of dye by bursting and nonbursting neurons of the PC lobe. The change of intracellular calcium concentration was measured at several different positions in neurites of individual bursting neurons in the PC lobe with a two-photon laser-scanning microscope. Fluorescence measurements were also made from neurons intracellularly injected with calcium green-1. Two different morphological classes of bursting neurons were found, varicose (VB) and smooth (SB). Our results from concurrent optical and intracellular recordings suggest that Ca2+ is the major carrier for the inward current during action potentials of bursting neurons. Intracellular recordings from bursting neurons with nystatin perforated-patch electrodes made while simultaneously recording the local field potential (LFP) with extracellular electrodes indicate that the burster spikes are precisely phase-locked to the periodic LFP events. By referencing successive calcium measurements to the common LFP signal, we could therefore accurately determine the relative timing of calcium transients at different points along a neurite. Measuring the relation of temporal to spatial differences allowed us to estimate the velocity of action potential propagation, which was 4.3 ± 0.2 (SE) mm/s in VBs, and 1.3 ± 0.2 mm/s in SB.
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INTRODUCTION |
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Synchronous firing of cortical
principal neurons resulting from coupled inhibitory neuron activity may
be the basis for feature binding and pattern recognition (Gray
1999; Hopfield 1999
; Singer 1999
). Progress has been made in understanding how coupled
inhibitory networks can promote synchrony (Galarreta and Hestrin
1999
; Gibson et al. 1999
). However, the
mechanism by which action potential (AP) propagation contributes to the
properties of inhibitory networks is not well understood. We therefore
studied this problem using a network of coupled inhibitory interneurons
in the olfactory system of the terrestrial mollusk Limax
maximus.
The highly developed olfactory system of Limax is accessible
to physiological recordings and optical imaging (Delaney et al. 1994; Gelperin et al. 1996
; Kleinfeld et
al. 1994
). It provides a model system to study olfactory
information processing and learning (Gelperin 1999
).
Olfaction is the most important sense for Limax, which has
no auditory system and only a very primitive visual system. A
significant portion of the CNS is devoted to processing olfactory
information. The procerebral (PC) lobe, which is the olfactory
processing center, displays waves of cellular excitation that propagate
from the apex to the basal region of the PC lobe as shown by field
potential recordings and optical imaging with voltage-sensitive
fluorescent dye (Delaney et al. 1994
; Gelperin and Tank 1990
; Kawahara et al. 1997
;
Kleinfeld et al. 1994
). The oscillatory activity is
dependent on endogenous nitric oxide (Gelperin 1994
),
modified by odor stimulation (Delaney et al. 1994
;
Gervais et al. 1996
; Kimura et al. 1998
;
Kleinfeld et al. 1994
) and modulated by endogenous
neurotransmitters such as dopamine, serotonin, and glutamate
(Gelperin 1999
; Gelperin et al. 1993
).
Oscillatory activity in the PC lobe is required for discriminating
between closely related odor molecules in an isolated nose-brain
preparation (Teyke and Gelperin 1998
). The PC lobe
contains two cell types, classified as "bursting" and
"nonbursting," based on their physiological (Kleinfeld et
al. 1994
) and morphological (Watanabe et al.
1998
) properties. Based on these data, a minimal model of waves
and oscillations in the PC lobe has been proposed (Ermentrout et
al. 1998
).
A two-photon laser-scanning microscope (2PLSM), having the advantages
of reduced phototoxicity and deeper tissue penetration over confocal or
conventional fluorescence microscopy (Denk and Svoboda
1997; Denk et al. 1990
), is suitable for
measuring intracellular calcium dynamics of PC cells (Gelperin
et al. 1996
), which lie in a layer more than 100 µm in
thickness adjacent to a dense neuropil region. In this report, we
present results from simultaneous intracellular recording and
[Ca2+] imaging of bursting neurons that suggest
that APs in these cells are mediated in large part by
Ca2+ ions. By making use of a common local field
potential (LFP) signal as a reference, we were able to temporally align
optical recordings that were made at different times from multiple
points along a burster cell neurite. This allowed us to deduce
Ca2+AP arrival times at different locations and
thus determine the initiation point(s) and the conduction velocities of
APs along a neurite. Two distinct classes of bursting neurons were
found, clearly separated by their conduction velocity and morphology.
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METHODS |
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Animals (Limax maximus) were reared at 16°C with ad
libitum access to lab chow (Purina) supplemented with vitamins, sea
sand, and the fungicide Tegosept (methyl-p-hydroxybenzoate). PC
lobes were isolated from the cerebral ganglia by microdissection.
During dissection, synaptic activity was reduced by a sixfold increase of Mg2+ concentration to 27.6 mM with
Na+ concentration reduced accordingly to maintain
the same osmotic pressure. Before the start of recording, desheathed PC
lobes were embedded in 1-2% low gelling temperature agarose (A-0701,
Sigma, St. Louis, MO) in saline <38°C with the cell layer of the PC
lobe facing upward. Saline was subsequently added to cover the agarose gel for physiological recordings. Limax saline contained (in
mM) 55.4 Na+, 4.2 K+, 7.0 Ca2+, 4.6 Mg2+, 80.1 Cl, 0.2 H2PO4
, 2.5 HCO3
, 5.0 glucose, and 10 HEPES buffered to
pH 7.6 (Delaney and Gelperin 1990
). Isolated PC lobes
were used for experiments within 24 h.
The following procedure is used to label the PC neurons with calcium
green 10k-dextran (CGD, Molecular Probes, Eugene, OR) (Gelperin
and Flores 1997). A paste of CGD was made with distilled water
on a depression slide. The tip of a glass electrode was dipped into the
paste so that a small amount of dye adhered to the tip. The dye-coated
electrode was subsequently dried for 1 h in a stream of warm air.
The bulk solution of saline surrounding a PC lobe was removed, and the
dye-coated electrode was used to stab the PC lobe and deposit dye
densely in a restricted area of the cell layer. Fresh saline was added
after dye application. Cells were well labeled 6-12 h after dye application.
For intracellular injection of calcium green-1 (CG-1, Molecular
Probes), electrodes were fabricated from quartz capillary glass (1.0 mm
OD and 0.5 mm ID, Sutter Instrument, Novada, CA) and pulled with a
laser puller (P-2000, Sutter Instrument). Parameters of the laser
puller were: heat, 905; filament, 5; velocity, 50; delay, 140; pull,
175. Resistance of the electrodes was typically 400-600 M when
filled with 2 M KAc, 20 mM CG-1 (hexapotassium salt), 10 mM HEPES, 8 mM
KCl, 20 mM ATP, 2 mM GTP and 2 mM MgCl2. Neurobiotin was
iontophoresed by injecting depolarizing current (0.2-0.5 nA) for 5-20
min at a frequency of 1 Hz with 30% duty cycle from quartz electrodes
filled with 2 M KAc and 2% neurobiotin. Preparations were kept at
4°C overnight to allow the diffusion of neurobiotin throughout the
neurites. Tissues were fixed, processed using a DAB substrate kit
(Ratté and Chase 2000
), and sectioned by a
vibratome. Current injection and membrane potential recording were
carried out with an Axoclamp 2 amplifier (Axon Instrument, Foster City,
CA). The electrical signals were amplified, filtered, and digitized (8 bit) by a custom data-acquisition system and stored in one of the first
few pixels in the image file to ensure accurate temporal alignment with
optical recordings of
[Ca2+]i. Post hoc data
analysis was carried out using commercial software (Igor, Wavemetrics,
Lake Oswego, OR). The amplifier headstage (HS-2A, 0.1 gain) and
electrode holder were mounted on an MM33 manipulator (Fine Science
Tools, Foster City, CA), which was customized to incorporate a
motorizer (860A, Newport, Irvine, CA) in place of the micrometer on the
x axis. Cell penetration was achieved by passing a
high-frequency oscillating current (1 ms in duration) through the
electrode. The current oscillation was caused by a brief
overcompensation ("buzzing") of the pipette capacitance by the
negative capacitance circuit in the amplifier.
Calcium dynamics was measured with a custom-built 2PLSM as described
previously (Denk and Svoboda 1997). Briefly, sample
observation, two-photon laser scanning and fluorescence detection were
performed with a water-immersion lens (Achroplan, ×40, 0.75 NA,
Zeiss). Beam scanning was controlled by a pair of galvanometer mirrors (Model 6800HP, Cambridge Technology, Cambridge, MA) driven by custom
software (R. Stepnowski, Lucent Technologies). The light source was a
Ti:sapphire laser (Tsunami; Spectra-physics, Mountain View, CA), pumped
by a 5-W solid-state laser (Millenia, Spectra-Physics) that provided
pulses with width <200 fs and repetition rate at 100 MHz. A wavelength
of 830 nm was used to excite the calcium green dye. Fluorescence was
detected by an intensified photodiode (Intevac). All
Ca2+ dynamics data were acquired in line-scan
mode with 2-ms time resolution and were analyzed using Igor with custom
procedures. Data are presented as averages ± SD unless specified otherwise.
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RESULTS |
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Two types of bursting neurons in the Limax PC lobe
Previous experiments have shown two major classes of neurons in
the Limax PC lobe, bursting and nonbursting neurons
(Kleinfeld et al. 1994; Watanabe et al.
1998
). Nonbursting neurons receive periodic inhibitory inputs
and produce spikes that are not phase-locked to the ensemble LFP
recording. The bursting neurons show periodic bursts of APs that are
phase locked to the LFP (Delaney et al. 1994
;
Gelperin and Flores 1997
; Gelperin et al.
1996
; Kleinfeld et al. 1994
). In this study, we
employed 2PLSM to investigate the temporal dynamics of
Ca2+ transients in the bursting neurons.
CGD-coated microelectrodes (Gelperin and Flores 1997
)
were used to label the PC lobe. Bursting neurons have multiple
processes extending several hundred micrometers confined to the cell
layer while nonbursting neurons have a single neurite extending
directly into the adjacent neuropil (Ratté and Chase
2000
; Watanabe et al. 1998
). Thus any
CGD-labeled cell that is found in the cell layer 100 µm from the dye
deposition site can be assumed to be a bursting neuron. Indeed all
measurements from labeled cells >100 µm from the dye deposition site
showed Ca2+ transients phase locked to LFP
recordings as is typical of bursting PC neurons (see following text).
Based on their fine morphological properties, we discovered that there
are two different types of bursting neurons. One class, denoted as
smooth bursting neurons (SBs), has a soma diameter of 14.6 ± 2.3 µm (n = 11; all the soma size measurements refer to
the largest diameter unless indicated otherwise) and no varicosities in
their processes (Fig.
1A). SBs
are not evenly distributed throughout the PC lobe but occur with much
higher density near the base than in the apical region (data not
shown). The bursting neurons reported in Watanabe et al.
(1998) appear to have similar properties in both location and
soma size as the SBs in our study. The second class of bursting
neurons, which we denote as varicose bursting neurons (VBs), has
button-like structures along their neurites and a smaller soma diameter
(11.6 ± 0.9 µm, n = 3; Fig. 1B).
Figure 1B displays a VB that was identified by
sharp-electrode voltage recording and was subsequently filled with
neurobiotin by current injection. Typically the somata of VBs were not
labeled by CGD, perhaps due to the smaller diameter of their neurites
(as small as 0.1 µm, see Fig. 1D) or the existence of a
diffusion barrier at the initial segment of the neurite, as found in
cultured rat hippocampal neurons (Winckler et al. 1999
).
The distribution of CGD-labeled varicosities appears to be the same
throughout the PC lobe (data not shown). Figure 1C shows the
distribution of distances between consecutive varicosities for a sample
of VBs with an average distance 15.6 ± 7.5 µm (data pooled from
8 different PC lobes). The distribution histogram has peaks at 8, 16, 24, and 32 µm, which are multiples of 8 µm, the averaged somata
diameter of nonbursting neurons (Gelperin et al. 1993
).
Figure 1D shows a scanning electron micrograph of a branched
neurite of a VB apposed to the somata of nonbursting neurons in the
cell layer of the PC lobe. Several varicosities can be seen. The VB
shown in Fig. 1B has ~40 varicosities in all branches,
suggesting that this cell may contact 40 nonbursting neurons through
chemical synapses. However, synapses are not restricted to varicosities
as SB neurons lacking varicosities are synaptically coupled to the
network of bursting neurons.
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Concurrent optical and intracellular recordings in bursting neurons
To establish a temporal correlation between membrane potential
changes and calcium dynamics during AP production, we measured [Ca2+]i by monitoring the
time course of CGD fluorescence and performed simultaneous
intracellular recording from a bursting neuron (Fig. 2A). Once a bursting neuron
was identified and a stable resting potential was obtained, CG-1 was
injected into the soma using hyperpolarizing current. The 2PLSM line
scan technique (Helmchen et al. 1999; Svoboda et
al. 1997
; Yuste and Denk 1995
) was used to
obtain dynamics of Ca2+ transients with high
temporal resolution (2 ms). However, whether the recording is from a VB
or SB could not be determined in this case due to either slow diffusion
of the CG-1 dye in these cells or an intracellular diffusion barrier at
the neurite origin. Up to 3 h after dye injection, no neurites
were observed in three dye-injection experiments that were successful
as judged by the staining of the soma. The resting potentials were
64 ± 9 mV. The decay time constants of the fluorescent
transients ranged from 300 to 900 ms with peak amplitudes from 17 to
38%. It should be noted that the onset of the rising phase of
Ca2+ transients coincides with the onset of the
first AP in the burst of APs in the intracellular recording. In Fig.
2B, the first AP in each burst was used to align the
Ca2+ transients and averaged traces were
generated from the aligned [Ca2+] transients.
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We then compared the change of the membrane potential with
[Ca2+]i. In Fig.
2C, the differentiated dF/F, as a
measure of Ca2+ influx, is plotted together with
the membrane potential. Peaks of the first APs are remarkably well
aligned with peak of the (dF/F)/
t signal. Thus timing of
APs can be deduced with millisecond precision from the derivative of
fluorescence measurements,
(dF/F)/
t. We found that an
accurate way to obtain the times of the peaks of the
(dF/F)/
t signal is by fitting a
sum of multiple Gaussian curves, which is practical and numerically
stable (Fig. 2D). Even though the fluorescence signal per se
is noisier in neurites than in the soma, the surface-to-volume ratio
and consequently the relative changes are much larger in neurites and
better timing data can ultimately be obtained in neurites (see Fig. 4).
We believe that the APs in bursting neurons are mediated by
Ca2+ currents mainly because the duration of the
APs, ~10 ms, is typical of Ca2+ APs
(Baker et al. 1971
; Gelduldig and Junge
1968
). The presence of voltage-gated Ca channels is evident
from the close link between measured cellular depolarization and
Ca2+ influx.
LFP traces can be used to align the APs of bursting neurons
The extracellular LFP recording is a conveniently obtainable
signal, and it has been used for temporal alignment of nonsimultaneous intracellular electrical recordings (Kleinfeld et al.
1994). We therefore established that it can be used for
temporal alignment of nonsimultaneous optical and/or intracellular
electrical recordings. To this end, we performed simultaneous
nystatin-perforated patch recording (Delaney et al.
1994
; Kleinfeld et al. 1994
) from the soma of a
bursting neuron and LFP recording from the PC lobe (Fig. 3A). The intracellular
recording revealed a burst of three APs followed by a hyperpolarization
phase during each cycle of the oscillation. To explore the precision of
relative timing between the APs and the LFP signal, the time point of
each negative peak of the LFP was used to align the intracellular
traces. This resulted in the APs in successive bursts being well
aligned (Fig. 3B). Figure 3C shows the averaged
trace of the aligned APs in Fig. 3B. The half-width of the
APs in the averaged trace is almost the same as those before averaging.
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These results demonstrate that the negative peaks of the LFP can be
used to align successive optical (dF/F) signals
to allow accurate temporal comparison of optical signals from different sites with an accuracy of 2 ms. The application of such an alignment procedure to [Ca2+]i
recordings is presented in Fig. 4, where
the top panel shows dF/F of the
fluorescent signal and the bottom panel shows the LFP
traces. The superimposed traces (Fig. 4, B and E)
from the different oscillation cycles overlap well when the negative
peaks of the LFP are used for alignment of each oscillation cycle. The averaged dF/F traces display three peaks, which
correspond to the three APs per burst recorded in most bursting
neurons.
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Initiation and propagation of Ca2+APs in bursting neurons
We now used our ability to precisely align nonconcurrent
recordings to compare the arrival of AP at different locations in a
neuron. Figure 5A shows
Ca2+ imaging from five different points along the
neurite of a SB aligned by a common LFP recording. The
dF/F traces suggest that the earliest rise occurs
at location d and then spreads bilaterally to c-b-a and e,
respectively. This suggests an AP initiation zone at or near point d.
Similar results were also obtained from VBs. Figure 5B
presents the propagation speed of APs in both VBs and SBs. The timing
of APs was obtained from the first peak of
(dF/F)/
t by fitting with
Gaussian curves (see Fig. 2D). To avoid the erroneous estimates that result if an initiation zone was in between two measuring points, measurements from around apparent initiation zones
were excluded from Fig. 5B. We found consistently
that the conduction velocity was several-fold higher in VBs [4.3 ± 0.2 (SE) µm/ms, n = 9] than in SBs (1.3 ± 0.2 µm/ms, n = 6).
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A further test of our alignment procedure was provided by the following experiment where we performed simultaneous Ca2+ measurements from two different varicosities on the same neurite. Figure 6 shows two varicosities, 77 µm apart on the same neurite of a varicose bursting neuron that displayed a time difference of 18 ms. After the line-scan experiments, multiple images with higher resolution were taken at different focal planes and a three-dimensional reconstruction was performed to confirm that the two varicosities belong to the same neurite (data not shown). The calculated conduction velocity between these two varicosities is 4.3 µm/ms, consistent with measurements on VB neurites using the LFP for temporal registration.
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It has been shown previously that odor stimulation of the olfactory
epithelium in a nose-brain preparation causes a collapse of wave
propagation in the PC lobe and all regions of the PC lobe are excited
synchronously (Delaney et al. 1994; Kleinfeld et
al. 1994
). We therefore investigated the
Ca2+ dynamics of bursting neurons in a nose-brain
preparation. Figure 7A shows
the schematic arrangement. The LFP recording was obtained from the
apical region of the PC lobe. CGD-labeled bursting neurons in the basal
area of a PC lobe were again imaged with the line-scan technique to
monitor their Ca2+ dynamics. The odor response of
a smooth bursting neuron is presented in Fig. 7B;
points
to the extra peaks in the basal fluorescence signal that were absent
from the apical LFP recording. Similar results were obtained from two
other SBs and one VB in separate preparations. When the LFP electrode
was moved to the basal region of the PC lobe, it showed similar
supernumerary peaks as the dF/F signal.
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DISCUSSION |
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The coupled network of bursting neurons in the PC lobe of
Limax is responsible for the oscillation (0.7 Hz) in the LFP
and the activity wave which propagates from apex to base
(Delaney et al. 1994; Kleinfeld et al.
1994
; Watanabe et al. 1998
). The bursting
neurons are coupled by glutamatergic chemical synapses and by gap
junctions (Wang et al. 2000
). The bursters at the apical pole
of the PC lobe have a faster burst frequency than the bursters at the
basal end, which suggests a possible mechanism for wave propagation
(Ermentrout et al. 1998
). It remains to be determined, however, if VBs and SBs are equally involved in wave propagation. The
wave propagation speed measured optically with a voltage-sensitive dye
was 1.1 µm/ms (Kleinfeld et al. 1994
) as compared with
AP conduction velocity in neurites of SBs (1.3 ± 0.2 µm/ms) and
VBs (4.3 ± 0.2 µm/ms) obtained in our experiments. Oscillations
in LFP and apical-basal wave propagation have been recorded in several other terrestrial slug and snail species (Gelperin et al.
2000; Inoue et al. 1998
; Kawahara et al.
1997
; Kimura et al. 1998
; Nikitin and
Balaban 1999
). Odor-elicited oscillations were first described in hedgehog olfactory bulb (Adrian 1942
) and
subsequently in a variety of vertebrate (Adrian 1950
;
Delaney and Hall 1995
; Hughes and Mazurowski
1962
; Lam 2000
) and invertebrate
(Gelperin 1999
; Laurent and Naraghi 1994
)
species. Stimulus-induced wave propagation has been documented in
turtle olfactory bulb (Lam et al. 2000
) and visual
cortex (Prechtl et al. 2000
).
The conduction velocity of APs in the processes of individual bursting neurons would be expected to be faster than the propagation speed of the overall wave. Wave propagation results from the interaction of several processes, synaptic integration as well as neuritic conduction. There are also plausible conduction pathways from cell layer to neuropil sites and back again into the cell layer via the NB to B cell excitatory synapses that could be essential parts of the wave propagation mechanism. Also, the B cell neurites run at various angles relative to the apical-basal axis, as shown in Fig. 6. If the B cell neurites measured in our experiments were running at an angle to the apical-basal axis and required some chemical synaptic transmission to relay their excitation both along the cell layer and into the neuropil, one could have a plausible explanation for the difference between 1.3 mm/s for the slower B cell neurite propagation speed and the wave speed of 1.1 mm/s. The discrepancy between the faster B cell propagation speed (4.3 mm/s) and the overall wave propagation speed (1.1 mm/s) suggests that the faster B cells are doing something different from just propagating excitation for the wave along the apical-basal axis. For example, they may function to make sure that the wave occupies the full lateral extent of the PC as it moves along, i.e., they serve to propagate excitation in the axis transverse to the apical-basal axis to insure that the wave front occupies the full width of the PC lobe.
Several observations are consistent with the possibility that an influx of Ca2+ ions causes the membrane depolarization detected by intracellular recordings. The waveforms of electrically recorded spike activity are very similar to that of the differentiated dF/F signal (Fig. 3). The half-width of the spikes from intracellular recording is ~10 ms, also suggesting that this is a Ca2+ spike because the half-width of Na+ APs is ~1 ms in squid giant axon. Incubation of the PC lobes with 100 µM TTX for 1 h did not change the LFP oscillation nor the spike pattern of the bursting neurons measured by intracellular recordings. Replacement of extracellular Na+ with Tris did not prevent wave propagation in the PC lobes (Wang, Flores and Gelperin, unpublished data). Furthermore depolarization of the membrane by injecting current through the intracellular electrode increased the fluorescent intensity of a CG-1-labeled bursting neuron (Wang and Gelperin, unpublished data), suggesting that there are voltage-activated Ca2+ channels on the membrane. Future experiments with specific channel blockers and Ca2+ chelators will clarify this issue.
Each bursting neuron in the electrically and chemically coupled network
of bursting neurons inhibits a group of nonbursting neurons in the PC
lobe, producing inhibitory postsynaptic potentials (IPSPs, 7-9 mV in
amplitude) in each nonbursting neuron with each burst (Gelperin
1994; Kleinfeld et al. 1994
; Watanabe et
al. 1998
). The IPSP is thought to be glutamatergic
(Watanabe et al. 1998
). The inhibitory synaptic currents
driven in multiple nonbursting neurons coherently during each
presynaptic burst produce the extracellular current flow that is
recordable as the LFP (Gelperin et al. 1993
). Ultrastructural analysis of varicosities in the Helix PC
lobe indicates that these varicosities contain specialized structures indicative of presynaptic terminals (Ratté and Chase
2000
). Two-photon Ca2+ imaging of
varicosities of neurons in the pyloric network of spiny lobster
demonstrates that varicosities are sites of
[Ca2+]i accumulation
(Kloppenburg et al. 2000
). The ~40 varicosities seen
in all neurite branches of a carefully analyzed VB (Fig. 1B)
suggest that this neuron may innervate
40 different nonbursting neurons. This number is also consistent with the observation that <5%
of the PC neurons are bursting neurons.
The PC lobe has two modes of activity, waves and "blinking," the
latter a mode in which the apical-basal phase gradient is greatly
reduced, leading to near synchronous activity throughout the
apical-basal extent of the PC lobe (Ermentrout et al.
1998; Kleinfeld et al. 1994
). Mode switching
appears to occur within one cycle of the LFP oscillation because states
intermediate between waves and blinking are not observed (cf. Fig. 5 in
Ermentrout et al. 1998
). Similarly, bursting cortical
networks have been observed to show very short phase delays (<3 ms)
over distances of several millimeters (Gray et al.
1989
); these may be associated with spike doublet firing by
inhibitory interneurons (Traub et al. 1996
). When the
inhibitory burster neurons in the PC lobe of Limax produce
double bursts, the LFP recording shows double events and the
apical-basal latency is much reduced for the first LFP event of each
pair of LFP events (Ermentrout et al. 1998
; Kleinfeld et al. 1994
).
PC lobes receive direct input from the olfactory receptor neurons
(Chase and Tolloczko 1993). Odor stimulation on the nose epithelium is capable of changing the mode of wave propagation to
blinking in the PC lobe. In response to odor stimulation at the nose in
a nose-brain preparation, additional peaks were seen in
[Ca2+]i of bursting
neuron at the base different from the LFP recording at the apical
region (Fig. 7B). This result suggests that odor stimulation
may change the excitability of bursting neurons in the basal PC lobe
but not the apical PC lobe. Considering the fact that SBs are mostly
situated in the basal region and VBs are evenly distributed, we believe
that the observed odor response is mediated by the SBs. It is also
possible that a VB in the basal PC lobe, synaptically driven by a SB,
shows the supernumerary peaks in response to odor stimulation, which in
turn causes supernumerary peaks in LFP at the base because many
nonbursting neurons are innervated by VBs.
Networks of inhibitory interneurons in mammalian cortex have been shown
to be coupled with both chemical and electrical synapses (Galarreta and Hestrin 1999; Gibson et al.
1999
), both of which are believed to be critical to the ability
of coupled inhibitory interneurons to promote synchrony in activity of
groups of principal cells (Cobb et al. 1995
;
Michelson and Wong 1994
; Strata et al. 1997
). Temporal correlations among responding cortical
principal neurons resulting from coupled inhibitory neuron activity may be the basis for feature binding and pattern recognition (Gray 1999
; Hopfield 1999
; Singer
1999
).
The VBs have two or more neurites extending over several hundred
micrometers contacting several 10s of nonbursting neurons (cf. Fig.
1B) (Ratté and Chase 2000). The
conduction of activity in such highly branched small-diameter neuritic
networks could be subject to frequency-dependent block at branch points
or contain multiple sites of spike initiation as demonstrated in other
highly branched interneurons (Antic and Zecevic 1995
;
Baccus et al. 2000
; Gu 1991
;
Zecevic 1996
). Gap junctions between burster neurons must be located on their neurites where the change in
[Ca2+]i occurring during
AP production could cause an increase (Baux et al. 1978
)
or decrease (Pereda et al. 1998
) in conductance. The PC
lobe also contains symmetrical chemical synapses (McCarragher and Chase 1985
) although the identity of the PC cells forming the symmetrical synapses is not known. The use of symmetrical chemical
synapses and (nonrectifying) gap junctions to couple the burster
neurons could be related to the ability of the burster network to
conduct activity both from apex to base and from base to apex, the
latter direction of propagation seen after appropriate pharmacological
treatments in vitro (Kleinfeld et al. 1994
; Wang, Flores, and Gelperin, unpublished results). The modulation of burster-burster coupling may underlie the large changes in the amplitude of the LFP recorded with fine wire electrodes implanted in
the PC lobe in vivo (Cooke and Gelperin 2001
).
Electron micrographs of varicose bursting neurons in the
Helix PC lobe reveal postsynaptic specializations in
neuritic segments between varicosities and symmetrical chemical
synapses in varicosities (Ratté and Chase 2000).
The network of bursting neurons in the Limax PC lobe
(revealed by sulforhodamine labeling) suggests that bursting neurons
contact each other through their neurites (Wang, Flores, and Gelperin,
unpublished observations). These anatomical results are consistent with
our observation that APs are initiated in neurites rather than in
somata. Several classes of amacrine (Massey and Mills
1999
; Wright and Vaney 2000
) and horizontal (He et al. 2000
) cells in the vertebrate retina are
coupled by electrical and inhibitory chemical synapses (Becker
et al. 1998
; Vaney 1999
). The electrical
synapses contribute to the spread of activity from cells directly
activated by a visual stimulus while the inhibitory chemical synapses
counteract this spread of activation from the site of the direct visual
activation (Roska et al. 2000
). The coupling
strength between bursting neurons through chemical and electrical
synapses could be modulated independently by different factors such as
olfactory inputs or neuromodulatory inputs, reflecting physiological
variables such as the state of satiation or hydration. APs are used to
relay excitation from burster neuron to burster neuron presumably
because the long thin neurites of burster neurons would not sustain
electrotonic potentials over distance required for effective
burster-burster coupling. Furthermore refractory period of APs would
allow wave propagation in only one direction consistent with the
hypothesis that phase waves are used by the Limax PC lobe
(Ermentrout et al. 1998
).
Activity wave propagation like that recorded in the Limax PC
lobe has also been observed in turtle visual cortex in response to
visual stimuli (Prechtl et al. 1997). The propagating
activity in turtle visual cortex, as in Limax PC lobe, is most likely
due to local coupling in a network of coupled oscillators. Activity waves arising from stable phase differences in a network of coupled oscillators occur in Limax PC lobe (Ermentrout et al.
1998
) and in the swim circuit of the lamprey (Cohen et
al. 1992
; Deliagina et al. 2000
) and of the
leech (Brodfuehrer et al. 1995
). Activity waves can be
produced by other mechanisms, such as local pacemaker regions
periodically stimulating excitation along a coupled network, as in
developing retina (Butts et al. 1999
; Feller et
al. 1997
; Meister et al. 1991
). The
Limax PC lobe has some similarity to developing retina as
the PC lobe incorporates synaptic connections from new olfactory
receptors throughout life (Chase and Rieling 1986
) and
synaptic connections from new PC neurons born after hatching
(Zakharov et al. 1998
), as in the vertebrate analogue of
the PC lobe, the olfactory bulb (Gelperin 1999
).
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ACKNOWLEDGMENTS |
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Present address of W. Denk: Max-Planck Institute for Medical Research, Dept. of Biomedical Optics, Jahn-Str. 29, D-69120 Heidelberg, Germany.
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FOOTNOTES |
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Present address and address for reprint requests: J. W. Wang, Columbia University, Center for Neurobiology and Behavior, 701 West 168th St., Hammer Health Science Bldg., 10th Floor, New York, NY 10032 (E-mail: jw800{at}columbia.edu).
Received 24 May 2000; accepted in final form 18 October 2000.
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REFERENCES |
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