1Neuroscience Program and 2Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136; and 3Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907
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ABSTRACT |
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Baccus, Stephen A., Christie L. Sahley, and Kenneth J. Muller. Multiple Sites of Action Potential Initiation Increase Neuronal Firing Rate. J. Neurophysiol. 86: 1226-1236, 2001. Sensory input to an individual interneuron or motoneuron typically evokes activity at a single site, the initial segment, so that firing rate reflects the balance of excitation and inhibition there. In a network of cells that are electrically coupled, a sensory input produced by appropriate, localized stimulation can cause impulses to be initiated in several places. An example in the leech is the chain of S cells, which are critical for sensitization of reflex responses to mechanosensory stimulation. S cells, one per segment, form an electrically coupled chain extending the entire length of the CNS. Each S cell receives input from mechanosensory neurons in that segment. Because impulses can arise in any S cell and can reliably propagate throughout the chain, all the S cells behave like a single neuron with multiple initiation sites. In the present experiments, well-defined stimuli applied to a small area of skin evoked mechanosensory action potentials that propagated centrally to several segments, producing S cell impulses in those segments. Following pressure to the skin, impulses arose first in the S cell of the same segment as the stimulus, followed by impulses in S cells in other segments. Often four or five separate initiation sites were observed. This timing of impulse initiation played an important role in increasing the frequency of firing. Impulses arising at different sites did not usually collide but added to the total firing rate of the chain. A computational model is presented to illustrate how mechanosensory neurons distribute the effects of a single sensory stimulus into spatially and temporally separated synaptic input. The model predicts that changes in impulse propagation in mechanosensory neurons can alter S cell frequency of firing by changing the number of initiation sites.
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INTRODUCTION |
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Neurons that receive multiple
synaptic inputs must allow those located at distant sites to influence
impulse activity. This problem is solved in motoneurons and many
interneurons by the presence of a single region of low threshold for
action potentials at the initial segment. In certain other neurons,
multiple sites of action potential initiation occur. Thus neocortical
pyramidal cells in layer 5 (Schiller et al. 1997),
mitral cells of the mammalian olfactory bulb (Chen et al.
1997
), fish oculomotor neurons (Kriebel et al.
1969
), and locust sensory interneurons (O'Shea
1975
) are examples of cells that span a broad area of space and
have multiple sites of impulse initiation. This feature allows the
integration of inputs from separate locations, while still allowing
those inputs to be close enough to an initiation site to influence
activity. The property of multiple initiation sites that has been
addressed in the present experiments is whether the activity from
multiple sites can add together to increase the firing rate of a neuron.
A related problem is how networks of neurons that have multiple
initiation sites integrate synaptic input. While the operation of such
networks is less well characterized, the problem is of broad
significance, ranging from invertebrate neuronal circuits (Antic
et al. 2000; Calabrese 1980
) to mammalian
neocortex (Galarreta and Hestrin 1999
; Gibson et
al. 1999
).
The site of impulse initiation is defined as the first part of the cell
that reaches threshold. Although it is influenced by cellular
properties such as geometry, conductances, and depolarization of the
membrane, the site of initiation is not necessarily distinguished by
any specific molecular or cellular structure (Melinek and Muller 1996; Moore et al. 1983
).
In the leech, each segmental ganglion contains one S cell that is
necessary for sensitization of the shortening reflex (Sahley et
al. 1994) and participates in producing whole-body shortening (Shaw and Kristan 1995
, 1999
). The S cell
is also required for complete dishabituation, which increases reflexive
shortening. Each S cell receives sensory input from sensory cells that
span several segments (Baccus et al. 2000
; Muller
and Scott 1981
). Together the S cells form a chain because each
S cell makes a strong electrical synapse with the S cell in each
neighboring ganglion (Fig. 1). Impulses
that arise in one S cell reliably propagate to the ends of the chain.
The S cell chain therefore acts as a single neuron with multiple sites
of impulse initiation. It is critical to the function of the S cell
chain that it operate as a single unit, since breaking the chain by
severing one S cell axon eliminates sensitization (Modney et al.
1997
). Mechanosensory neurons in the leech make direct
excitatory connections with S cells that are either monosynaptic or,
through gap junctions, act as such (Baccus et al. 2000
;
Muller and Scott 1981
). The receptive field of each
sensory cell spans at least three segments in its innervation of skin
and in its central projection, and each produces a synaptic potential
in several S cells (Fig. 1). To investigate the problem of synaptic
integration across a wide spatial extent of several body segments,
action potential initiation in the S cell chain was examined by
measuring simultaneously the location and timing of impulse initiation
in response to single mechanosensory stimuli.
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METHODS |
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Preparations and electrophysiology
Leeches Hirudo medicinalis were obtained from a
supplier (Leeches USA, Westbury, NY) or raised in the laboratory. Two
different types of preparations were used. The first, an intact
segmental preparation, consisted of 6-12 intact segments with the
segmental nerve cord exposed on each end (Fig. 5A). The
second, a semi-intact preparation, consisted of a chain of six ganglia
with the skin and body wall attached on one side (Fig.
2A). The bath contained leech
saline, composed of (in mM) 115 NaCl, 4 KCl, 1.8 CaCl2, and 10 Tris maleate, pH 7.4 (Kuffler and Potter 1964). Experiments were conducted at
room temperature (20-22°C). Intracellular recordings were made using
sharp microelectrodes filled with 4 M potassium acetate. For
intracellular recording, touch (T), pressure (P), and nociceptive (N)
mechanosensory neurons were each identified by their distinctive size,
position, and action potential (Nicholls and Baylor
1968
). The S cell soma too was identified by its size, position, and action potential, as it is the only small (~20 µm) cell in the central glial packet that produces a fast, overshooting action potential in response to weak stimulation of the segmental nerve
cord (Frank et al. 1975
). Extracellular recordings were made using suction electrodes at anterior and posterior locations of
the segmental nerve cords, or connectives. In some experiments, a third
extracellular recording was added. Thus electrodes were placed either
at both ends of cut connectives or in en passant configuration. S cell
action potentials were identified in extracellular recordings as the
largest impulses in the recording. Skin was stimulated either
electrically or mechanically. Electrical stimuli (20-50 V,
approximately 3 ms) were applied using a suction electrode to a single
annulus other than those with sensillae. Mechanical stimuli were
applied by a fire-polished glass rod (1.2 mm diam) attached to a servo
(Futaba S3401). The servo was activated for a duration of 50 ms.
Because this time included the transit time for the servo, the actual
time of stimulation was <50 ms. Stimuli were applied every 2 min,
which is the rate of stimulation used for behavioral experiments
measuring sensitization and dishabituation (Modney et al.
1997
; Sahley et al. 1994
). More frequent
stimulation (once every 30 s) resulted in decreased S cell
activity. The time of impulse initiation was calculated by
(Tant + Tpost
Tcond)/2, with
Tant the arrival time at the anterior
extracellular electrode, Tpost the
arrival time at the posterior extracellular electrode, and
Tcond the conduction time between the
two electrodes, measured by stimulating the connectives with one
electrode and recording with the other. Values of n indicate
number of preparations, not measurements.
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Data analysis
The time at which each action potential arrived at a recording
electrode was measured using custom-designed software that allowed a
threshold to be set visually for each of two extracellular recordings
for each trace. Each trace was examined individually, lasted up to
1 s, and contained up to 15 S cell impulses. The arrival time of
action potentials was taken as the time of the largest positive peak
amplitude above the chosen threshold. Although polarity was not
controlled for different preparations, this amounted to a difference in
arrival time of approximately 1 ms, and affected arrival time equally
in all traces of an experiment. Each impulse could be inspected on slow
and fast time scales to verify that an impulse was an S cell impulse by
its waveform. The program verified that the number of threshold
crossings was equal in each channel. Only S cell impulses recorded at
both anterior and posterior extracellular electrodes were analyzed.
Impulses recorded by only one electrode and not both may represent
action potentials that failed or reflected (Baccus
1998), and amounted to fewer than 1% of the total recorded S
cell impulses. The cross-correlation c(t) between
sensory cell activity and S cell activity was determined as
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Compartmental modeling
The model, produced using the simulation program Neuron
(Hines 1989), included five S cells, five P cells, and
five T cells, as diagrammed in Fig. 10A. The integration
time step (dt) was 50 µs, axial resistivity was 200
cm,
and compartments were 2 µm in length.
MORPHOLOGY.
Segments were separated by 7.5 mm, and peripheral axons were 2 cm in
length. Peripheral axon diameters of sensory neurons were adjusted to
match experimentally measured conduction delays from the skin to the
soma. Major receptive field conduction delays were 11 ms for T cells
and 17 ms for P cells. Minor receptive field conduction delays were 45 ms for T cells and 88 ms for P cells. For P cells, to reproduce
conduction block and reflection, morphology of central major and minor
receptive field axons was modeled as described using experimentally
measured axon diameters (Baccus 1998). Conduction
velocity of the S cell chain was 1.6 m/s.
MEMBRANE PROPERTIES.
T cell and S cell membrane properties were modeled using the
Hodgkin-Huxley squid axon model, with temperature set to 20°C, as the
primary consideration for these cells was to reproduce experimentally
measured conduction delays and conduction velocities. For P cells, to
produce conduction block and reflection, membrane properties were
modeled as described (Baccus 1998) using
voltage-activated Na+, K+,
and Ca2+ conductances, a
Ca2+-dependent K+
conductance, and the Na+-K+
ATPase. However, because single brief stimuli were used here, the
activity of the Na+-K+
ATPase was varied not by prolonged electrical activity, but by changing
the maximum pump current between 0.1 and 5 µA/cm2 so as to change the membrane potential
approximately 3 mV. Activity in P cells can produce changes in resting
membrane potential equal to or greater than this amount. The change in
maximum pump activity implemented here represents neuromodulatory
effects, which can change conduction block in P cells (Mar and
Drapeau 1996
). Producing reflection and conduction block by
varying the maximum pump current differs slightly from the case of
electrical activity. During repetitive firing, thin and thick axons
accumulate Na+ and Ca2+ at
different rates, thereby producing spatially nonuniform membrane currents in the region of the branch point (Baccus
1998
). These nonuniform membrane properties can contribute to
reflection and conduction block, but are not essential, as reflection
and conduction block often occur at rest without activity.
SYNAPTIC CONDUCTANCES.
Direct monosynaptic inputs to the S cell chain were calculated as
Isyn = Gsyn(Vm Esyn),
Esyn = 0 mV. Each synaptic
conductance, Gsyn, was modeled using
an alpha function (Rall 1967
),
Gsyn = Gpeak[(t
ttrig)/tsyn]
exp{
[t
(ttrig + tsyn)]/tsyn},
for t
ttrig, where
Gpeak (peak conductance) and
tsyn (time until peak) are parameters
(see RESULTS for values), and
ttrig is the time that the presynaptic
membrane potential depolarized above a threshold of
20 mV. This
equation describing Gsyn is such that
at time t = ttrig + tsyn,
Gsyn = Gpeak. Peak conductances of synapses between sensory cells and S cells in adjacent segments were half as
large as those between sensory cells and S cells in the same segment.
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RESULTS |
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Multiple initiation sites in the S cell chain
Each action potential that initiates in the S cell chain propagates anteriorly and posteriorly. The speed with which it travels is the same no matter in which S cell it is initiated.
To determine in which ganglion action potentials were initiated, each
spike was recorded as it propagated anteriorly and posteriorly to two
extracellular electrodes at the ends of the chain (Fig. 2A).
In the figure, the pairs of impulses recorded by separate electrodes
and arising at individual sites are linked by dotted lines in Fig.
2A. The difference in arrival times of the impulses at the
two ends, Tdif, was calculated as
Tpost Tant, where
Tant was the arrival time at the
anterior electrode and Tpost the
arrival time at the posterior electrode. Impulses arising from the same site should have had approximately the same
Tdif.
Tdif was calculated from action
potentials in multiple trials, and a histogram of these values was
produced (Fig. 2B). The values clustered into peaks that
corresponded to distinct initiation sites in the S cell chain
(n = 14; Fig. 2B).
To confirm that the peaks calculated for action potentials arising from skin stimuli represented initiation sites in particular ganglia, S cells in those ganglia (the 2 middle ganglia shown in the diagram) were stimulated intracellularly at the soma, producing impulses in the extracellular recordings (n = 4, data not shown). The relative arrival times of the intracellularly evoked action potentials clustered into narrow peaks that aligned with the peaks in the skin-stimulus time-difference histogram (Fig. 2C). This indicated that the different peaks calculated from skin stimuli represented initiations in different S cells. Vertical dotted lines from Fig. 2A to Fig. 2B show the correspondence between particular ganglia in which impulses initiated and the peaks in the time-difference histogram. Impulses from intracellular stimulation were recorded after collecting impulses from skin stimulation for over 90 min. Because each peak from intracellular stimulation was less than 1 ms wide, the 1.5-ms shift of a peak may have represented a slow change in the preparation or slight movement of the connective in the suction electrode. Alternatively, the initiation site during intracellular stimulation may have been slightly different within a ganglion than that from skin stimulation.
Determination of whether impulses from different initiation sites collide
In time-difference histograms, some impulses appeared between the peaks. Such impulses might be explained as two action potentials that arose simultaneously in two different ganglia and then collided in between. To determine whether simultaneous initiations were occurring, a third electrode was placed halfway along chains of six ganglia. The three possible combinations of pairs of electrodes gave two independent measures of initiation site for each impulse (n = 3). Initiation sites were localized using the time difference histograms for the anterior electrode pair (Fig. 3A), the outer electrode pair (Fig. 3B), and the posterior electrode pair (Fig. 3C). Calculations were done as described in the figure legend. The outer electrode pair could localize initiations in any of the six ganglia, the anterior pair could localize initiations only in the anterior three ganglia (Fig. 3A, right of dotted line), and the posterior pair only in the posterior three ganglia (Fig. 3C). The results of these two independent measures confirmed that the anterior electrode pair (Fig. 3D, left of dotted line) did not localize impulses with a posterior initiation site, as determined by the outer electrode pair, but both the anterior and outer electrode pairs localized the anterior initiation sites (Fig. 3D, right of dotted line). Similarly, the posterior pair and the outer pair localized impulses to the same site if they initiated in posterior ganglia (Fig. 3E, left of dotted line). If simultaneous initiations had occurred on both sides of the center electrode, the anterior and posterior electrode pairs would have localized the initiations to two different sites, neither of which would have corresponded to the single site localized by the outer pair. Since this did not happen, collisions were infrequent if they occurred.
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Impulses that appeared between the histogram peaks as measured by the outer electrode pair also appeared between the peaks in the records of the anterior or posterior electrode pair (Fig. 3, A and C). Therefore either small changes in conduction velocity in the S cell chain changed the arrival times in the recordings, or impulses initiated between ganglia. Changes in conduction velocity would likely be due to synaptic input to the S cell chain, as during intracellular stimulation the relative arrival time of impulses at the two recordings changed very little (Fig. 2C). Only impulses that fell between a peak and 25% of the distance to an adjacent peak in Tdif histograms were assigned to an initiation site. Other impulses were excluded from further analysis, but these represented <5% of the data collected.
Multiple initiation sites in single S cells
Impulse initiation sites were examined at a higher spatial resolution within single S cells by recording extracellularly just anterior and posterior to a single ganglion as diagrammed in Fig. 4A. When the skin was stimulated, the Tdif histogram had three narrow peaks, indicating at least three distinct initiation sites (Fig. 4B). The connectives were then cut at the two points marked "x" in Fig. 4A, severing the axons of each adjacent S cell, but leaving the S cell recorded from intact. This left the central peak in the Tdif histogram but eliminated the two adjacent peaks. This indicates that impulses arising at the two adjacent sites were due to input from adjacent S cells through the electrical synapse, and that conduction along the chain is not saltatory between single S cell initiation sites. Furthermore, because the central peak was very narrow (<0.5 ms), this indicates that the site or region of initiation within a single ganglion varies little when examined at higher spatial resolution. Therefore under recording conditions when the electrodes are farther apart (e.g., Figs. 2 and 3), impulses that appear between peaks are most likely not due to shifting initiation sites, but due to small propagation delays along the S cell chain. The effect of propagation delays depends on the conduction time between the two electrodes, the conduction time between ganglia, and the point of initiation along the chain. For a 50-ms conduction time between electrodes and a 5-ms conduction time between ganglia, a minimum 10% change in conduction time is necessary to cause an error localizing the initiation site equal to half the distance between ganglia. Although fewer than 1% of impulses appeared at one electrode and not the other, representing either action potential failures or reflections, this suggests that some propagation delays may occur.
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Temporal separation of activity at different initiation sites
S cell activity was recorded from intact segmental preparations as diagrammed in Fig. 5A; in general, the less dissected the preparation, the greater the S cell activity. When a single site on the skin nearest ganglion 5 (G5) was stimulated electrically, impulses clustered into peaks in the Tdif histogram, showing that impulses initiated at multiple sites corresponding to separate ganglia (Fig. 5B). Figure 5C is a raster plot of multiple trials showing the total S cell activity following each trial (top part) and a poststimulus time (PST) histogram of that activity (bottom part). Initiation times were determined for S cells in different ganglia. Impulses were assigned to different initiation sites using the Tdif histogram. PST histograms were then produced for each separate initiation site (Fig. 5, D-G). Impulses initiated first in the same segment (G5) as the stimulus (Fig. 5D), while impulses arising from other segments tended to initiate later in time (Fig. 5, E-G). Because impulses arising in separate ganglia arose at different times, and because the S cell axon conduction velocity is the fastest in the leech (~1.6 m/s), impulses arising at separate sites did not collide but traveled past other initiation sites before those sites produced impulses. Although the PST histograms indicate that there was activity in different sites at the same time following a stimulus, this represents the average activity. In fact, analysis of individual trials (not shown) indicated that these different initiation sites were active during different trials.
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Mechanical stimulation
To test whether mechanical and electrical stimulation evoked similar patterns of activity at different sites in the S cell chain, intact segmental preparations were stimulated at a single point on the skin with a fire-polished glass rod. The chain included segmental ganglia 8 through 13. Figure 6A shows the Tdif histogram indicating that impulses initiated at multiple sites. Figure 6B shows a raster plot and PST histogram of activity following mechanical stimulation. In general, mechanical and electrical stimulation evoked similar patterns of initiation, with consistent individual responses from one stimulus to the next as shown by the raster plots. Impulses initiated first in the same segment as the stimulus and then later impulses initiated in other segments (n = 3). With mechanical stimulation, however, each initiation site appeared to be activated in a narrower window of time, and fired less outside this window (Fig. 6C).
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Mechanosensory cell activity
Sensory cell activity was measured using the semi-intact
preparation, which allowed access to the ganglia for intracellular recording. After measuring S cell activity, sensory cell activation was
measured intracellularly for each of the three touch (T), two pressure
(P), and two nociceptive (N) sensory neurons ipsilateral to the skin
stimulus (Fig. 7). Skin stimuli did not
activate contralateral sensory cells. Stimuli activated ipsilateral
sensory cells in three ganglia (n = 3), G9-11. These
ganglia contained the only S cells that produced substantial activity
in the semi-intact preparation. T and P cells produce direct excitatory
potentials in the S cell that are either monosynaptic or, in certain
cases, act as such via gap junctions (Baccus et al.
2000; Muller and Scott 1981
). Although N cells
were also activated, N cells were not included in the analysis;
preliminary results indicate that with the stimuli used, the N cells
excite S cells less than do the T and P cells. The time course of
combined T and P activity in response to a single stimulus is shown in
Fig. 7B. Because of conduction delays, sensory activity
tended to occur later in segments adjacent to the stimulus than in the
stimulated segment.
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Figure 7C shows the cross-correlation between sensory activity and S cell activity in the same segment for three different ganglia, calculated as described in METHODS. The cross-correlation shows the degree to which sensory cell activity correlates with S cell activity, offset by a certain interval of time. Because sensory cell and S cell activity were measured in different trials, this cross-correlation does not account for trial to trial variability in firing probability. The cross-correlation is near zero before 0 ms, indicating that sensory cell activity tended not to be correlated with past S cell activity, as expected. Average stimulus-evoked activity of sensory cells correlated tightly with average S cell activity in the stimulated segment (Fig. 7C). For adjacent ganglia, the peaks occurred later in time than in the stimulated segment. At each S cell initiation site, sensory activity in that segment correlated with S cell activity.
The mechanism by which the firing of S cells becomes prolonged and distributed as a result of the projections of sensory neurons is schematically depicted in Fig. 8, with A, B, and C occurring sequentially in time. It illustrates how a single stimulus produced sensory activity that was distributed in space and in time, leading to multiple firing in the S cell chain. In the diagram, an impulse in the P cell's own ganglion generates impulses traveling rapidly in both directions in the S cell in the same ganglion (A). That impulse passes through the S cell in the next ganglion (B) before the impulses in the P cell and other sensory cells arrive to excite a second impulse (C) in the S cell in that adjacent ganglion. The second impulse, which also propagated along the nerve cord in both directions, accounts for the increased firing produced by multiple initiation sites.
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Multiple initiation sites produce different activity patterns in different parts of the S cell chain
Although the S cell chain acts functionally as a single neuron
with multiple impulse initiation sites, different patterns of activity
were recorded at different locations within the chain (Fig.
9). When an impulse arose at a different
location than the previous impulse, a different interspike interval
(ISI) was recorded at the anterior and posterior ends of the chain
(Fig. 9A). For each impulse, the ratio of the ISI at the
posterior electrode relative to the ISI at the anterior electrode was
computed; this quantity is a measure of the difference in impulse
spacing in different regions of the S cell chain. Figure 9B
is a histogram of this ratio. Most impulses had shorter ISIs at the
anterior region of the chain, indicating subsequent initiations tended to progress toward the anterior of the chain for this preparation. Figure 9C plots the ratio of ISIs at the two ends of the
chain as a function of time following a stimulus. During trains of
activity, initiations at different sites produced different ISIs in
different regions of the S cell chain. At chemical synapses,
facilitation and depression depend strongly on the timing of recent
activity at presynaptic terminals, and at electrical synapses, closely spaced presynaptic impulses can sum postsynaptically (Zucker
1989). When impulses are more closely spaced at one end of the
S cell chain, S cell postsynaptic targets at the two ends of the chain may receive different levels of transmission.
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Compartmental model of the S cell chain and its mechanosensory synaptic inputs
To examine how the synaptic input from multiple mechanosensory
neurons is integrated at multiple sites in the S cell chain, a
compartmental model was created that included S, P, and T cells (Fig.
10). In the model, conduction delays
from the skin to the sensory cell somata were matched to experimental
results, as was the conduction velocity of the S cell chain. Direct
monosynaptic connections made by T and P cells were described by the
parameters of peak conductance (Gpeak)
and time course (tsyn). For the P cell
to S cell synaptic connection, multiple levels of transmission occur,
depending on action potential propagation at P cell central branch
points (Baccus et al. 2000). Impulses can undergo
conduction block, failing at branch points and thereby reducing
synaptic transmission by failing to activate all presynaptic terminals. Impulses can also propagate through all P cell central branch points,
activating each presynaptic terminal once. Additionally, it is common
for action potentials to reflect at P cell branch points. When
reflection occurs, impulses propagate through the branch point with a
delay and at the same time reverse direction from the branch point,
activating presynaptic terminals twice in rapid succession, thereby
increasing transmission by producing synaptic facilitation. Branch
point conduction state, and thus synaptic transmission, depends on the
membrane potential of the presynaptic cell, which can be changed by
activity of the Na+-K+
ATPase and a Ca2+-dependent
K+ conductance (Baccus 1998
).
Conduction state in P cells was varied in the model by changing
parameters for the activity of the
Na+-K+ ATPase.
Figure 10B shows the membrane potential in the anterior end
of the S cell chain when conduction block was present in P cells.
Following a stimulus to segment 3, the earliest S cell impulses
initiated in segment 3. Because of conduction delays in mechanosensory
axons, sensory input was delayed to the adjacent S cell in segment 2, causing impulses to initiate later in that segment, consistent with
experimental results shown in Fig. 7.
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Synaptic transmission from the P cells increased when reflection was produced in P cells by reducing the activity of the Na+-K+ ATPase. Consequently, the number of initiation sites and the number of impulses at some individual sites increased (Fig. 10C). The number of impulses and initiation sites was unchanged both for conduction block and reflection for a range of synaptic magnitudes and time courses (P cells: Gpeak = 0.08-0.20 µS, tsyn = 0.6-2.0 ms; T cells: Gpeak = 0.1-0.8 µS, tsyn = 0.1-5 ms). Synaptic magnitudes and time courses greater than these values increased the number of impulses equally for both conduction block and reflection (not shown).
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DISCUSSION |
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In the S cell chain, in order for impulses arising at multiple
sites to avoid collision and add together to increase the firing rate,
impulses that arise at separate sites must also arise at separate
times. Mechanosensory cells serve this function of spatially and
temporally separating input to the S cell chain. Each T and P cell
innervates three segments peripherally and forms synapses centrally in
those three segments. Thus a skin stimulus to one segment activates
sensory synapses in up to four additional segments, two anterior and
two posterior (Figs. 1 and 10). Because the conduction velocity of
sensory cell axons is much slower than that of the S cell, the time for
sensory impulses to propagate to adjacent segments and activate
adjacent S cells is far longer than the time to activate the S cell in
the stimulated segment and for the evoked impulses to propagate through
adjacent segments anteriorly and posteriorly. For example, when the P
cell peripheral axon is stimulated at the skin in an adjacent segment,
the conduction time from the skin to the soma can exceed 100 ms
(Yau 1976), compared with <20 ms when the skin in the
same segment as the soma is stimulated; the conduction delay between
ganglia for P cells exceeds 10 ms (Elliott and Muller
1983
; Wallace et al. 1977
), while for S cells it
is <5 ms (Muller and Carbonetto 1979
). Skin stimuli
activate sensory cells in the nearest ganglion for up to several
hundred milliseconds, and activity is progressively delayed in segments that are more distant. Thus there are multiple sensory pathways that
excite the S cell chain: a fast pathway that fires the S cell chain by
exciting the S cell in the same segment, and slower pathways that
excite S cells in adjacent segments, as demonstrated by a compartmental
model (Fig. 10).
In the model, experimentally measured conduction delays in sensory cell axons were used; these gave sufficient delays in synaptic input to adjacent segments that S cell impulses arising in the same segment as the stimulus propagated through adjacent segments before additional impulses arose from the delayed excitatory sensory input in those adjacent segments. Thus collisions did not occur, and the fast pathway to the S cell in the same segment did not interfere with the slower pathways to S cells in adjacent segments. The model was fairly insensitive to changes in the magnitude and time course of individual synapses, as each of these parameters could be varied by a factor of at least 2.5 without changing the number of initiation sites. The model was more sensitive to changes in conduction velocity of sensory cell axons, as the small change in P cell conduction velocity produced by changing the activity of the Na+-K+ ATPase noticeably altered the time of initiation of S cell impulses (Fig. 10).
Asynchronous input to the S cell chain also results from the time
course of individual synaptic inputs. Although P and T cells each make
a direct monosynaptic connection of short latency to the S cell chain,
both have excitatory polysynaptic components. T cell impulses can
depolarize the S cell for over 100 ms (Muller and Scott
1981), and P cell impulses for over 200 ms (Baccus et al. 2000
). Conduction time, prolonged sensory activity, and
prolonged synaptic potentials are thus mechanisms that allow
mechanosensory neurons to separate temporally input to the S cell
chain. This in turn is expected to prolong the S cell response and
provide an additional means for regulating its strength.
S cell activity in response to skin stimulation declines in habituation
and is elevated following sensitization of reflexive shortening
(Sahley et al. 1994). One contributor to these events might be that the number of initiation sites falls or rises,
respectively. This could occur by a change in strength of
mechanosensory transmission to different S cells or by changes in S
cell excitability (Burrell et al. 2001
) or both.
Conduction block and reflection change sensory transmission to the S
cell in segments adjacent to the one stimulated, but not in the S cell
in the same segment as the stimulus (Baccus et al. 2000
;
Muller and Scott 1981
). Experimental results have shown
that in P cells, when reflection occurs, the size of the S cell
synaptic potential increases to approximately four times that recorded
during conduction block (Baccus et al. 2000
). In the
model tested here, this change in synaptic transmission was sufficient
to produce additional S cell impulses. Therefore a change in
presynaptic conduction state may change the number of impulses that
initiate at multiple sites in the S-cell chain.
Because S cell activity increases with sensitization of reflexive
shortening (Sahley et al. 1994) and sensitization
requires an intact serotonergic system (Ehrlich et al.
1992
), it seems worthwhile to consider the effect of serotonin
on the number of initiation sites that are activated during reflexive
shortening. Serotonin can depolarize mechanosensory cells and relieve
conduction block (Mar and Drapeau 1996
), and can act as
a circulating hormone in the leech. Chemically lesioning
serotonin-containing neurons affects sensitization in a manner similar
to breaking the S cell chain (Ehrlich et al. 1992
),
although the S cell is not serotonergic. During sensitization, since
one of the effects of serotonin can be to change the branch point
conduction state of presynaptic cells, then S cell activity might be
increased by the initiation of impulses at additional sites.
Impulse initiation sites within cells can interact; for example,
initiations at one site can suppress those at another. A classic
example is the pacemaker system within the mammalian heart. In leech
heart interneurons, even though impulses can initiate in multiple
segments, activity at one segment acts as a pacemaker, controlling the
firing pattern of the cell and suppressing initiations elsewhere in the
cell (Calabrese 1980). In the S cell, although different
initiation sites can operate independently, they might also interact.
Sensory activity is more tightly correlated to S cell activity in the
stimulated segment than in other segments, indicating that in adjacent
segments, synaptic input may be less successful at evoking activity.
Because the stimulated segment fires an early burst of activity in
response to a stimulus, other S cells may be made somewhat refractory.
The S cell chain can support firing rates above 150 Hz. Multiple initiation sites allow the S cell chain to carry information from sensory cells in multiple segments, allowing the S cell chain to use more of its bandwidth to increase throughput of sensory signals.
In other neurons with multiple initiation sites, different sensory
modalities activate different initiation sites, as observed in the fish
oculomotor system and in the locust (Korn and Bennett 1975; O'Shea 1975
). This suggests that a
primary function of multiple initiation sites in these systems is to
allow a neuron to associate modalities. In contrast, the present
results show that multiple initiation sites in the S cell chain respond
to the same sensory modality, and in fact, to the same stimulus. This
demonstrates that when the same sensory information is distributed to
multiple initiation sites, those separate locations can perform the
function, not of association, but of amplification of sensory input.
Electrical coupling between neurons is common (Bennett
1966; McMahon et al. 1989
). In the vertebrate
retina, groups of neighboring ganglion cells can exhibit nearly
synchronous firing that persists when chemical transmission is
eliminated (Brivanlou et al. 1998
). Cell pairs can show
a strong tendency to fire within 1 ms of each other, with either cell
firing before the other, but less tendency to fire simultaneously,
suggesting that excitation can travel in more than one direction. This
general arrangement allows the integration of separate synaptic inputs,
while the resulting activity is quickly summed in multiple cells.
Electrical junctions therefore not only allow cells to fire with near
synchrony, but also may increase the overall firing rate. This
organization is analogous to the S cell chain in two dimensions.
Coincident activity in multiple presynaptic cells converging on the
same postsynaptic target can increase postsynaptic activity by summing
subthreshold synaptic inputs (Agmon-Snir and Segev 1993;
Cobb et al. 1995
). If each synaptic input is
suprathreshold, however, the postsynaptic response will be greatest
with asynchronous input, particularly if there are multiple initiation
sites. In the neocortex, electrically coupled networks are formed by
inhibitory interneurons that tend to fire within several milliseconds
of each other, suggesting that mutual excitation travels within these networks (Galarreta and Hestrin 1999
; Gibson et
al. 1999
). Because each neuron is a separate initiation site,
desynchronized input to these networks may result in greater inhibition
than synchronized input.
Multiple initiation sites in the S cell chain also provide a mechanism
for detecting direction of stimulus movement without relying on
synaptic inhibition. When consecutive impulses initiate at different
sites in the S cell chain, a different instantaneous firing frequency
is recorded in different regions of the chain (Fig. 9). In systems with
multiple initiation sites, this will generally be true for consecutive
impulses that arise at different sites and at different times. This
effect was observed regularly with fixed stimuli, and should be
enhanced by moving stimuli. A stimulus moving toward the anterior of
the leech would initiate impulses at successively more anterior sites.
This would produce more closely spaced impulses, giving rise to greater
synaptic facilitation in the anterior of the S cell chain than in the
posterior. Thus multiple initiation sites may increase synaptic
transmission preferentially from synapses at that end of the animal
that is in the direction of stimulus movement, operating as a mechanism to detect and encode motion. Significantly, sequential stimuli should
produce the same effect and are known to give the illusion of movement
(Livingstone and Hubel 1988).
Postsynaptic cells can process the spatial and temporal characteristics of synaptic input in different ways. By initiating impulses at multiple locations, postsynaptic neurons can integrate inputs separately, and in addition can increase output, increase throughput of synaptic information, and encode synaptic signals.
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ACKNOWLEDGMENTS |
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We thank Drs. J. Barrett, R. Bookman, B. Burrell, A. Chen, D. Johnston, W. Nonner, S. Roper, and R. Rotundo for helpful discussions.
This work was supported by National Institute of Neurological Disorders and Stroke Grant R01-NS-34927 to K. J. Muller and C. L. Sahley. S. A. Baccus was supported by a Howard Hughes Medical Institute Predoctoral Fellowship.
Present address of S. A. Baccus: Dept. of Molecular and Cell Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138.
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FOOTNOTES |
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Address for reprint requests: K. J. Muller, Dept. of Physiology and Biophysics, R-430 (RMSB 5092), University of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136 (E-mail: kmuller{at}newssun.med.miami.edu).
Received 18 December 2000; accepted in final form 1 June 2001.
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REFERENCES |
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