Multiple Sites of Action Potential Initiation Increase Neuronal Firing Rate

Stephen A. Baccus,1 Christie L. Sahley,3 and Kenneth J. Muller1,2

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. The S cell chain and its mechanosensory input. The schematic diagram shows S cells in 5 ganglia of the chain that runs the animal's length; the S cell in each ganglion makes a strong electrical synapse with each neighboring S cell midway between ganglia. Also shown is a pressure (P) sensory neuron, 1 of 7 mechanosensory neurons including 2 P cells on each side of the ganglion. Each sensory neuron extends fine axons through adjacent ganglia to innervate regions of skin ("minor receptive fields") that are contiguous with the receptive area in each cell's own segment ("major receptive field"). Diagram not to scale; ganglia are typically ~0.5 mm diam and more than 5 mm apart.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2. Multiple initiation sites in the S cell chain. A: diagram schematically depicts the semi-intact preparation, consisting of a chain of 6 ganglia (here segmental ganglia 8-13) with all peripheral nerves intact on the left or right side. Top trace is a representative recording from the posterior electrode. Bottom trace is from the anterior electrode. Each S cell impulse appeared in both recordings; these impulse pairs are joined by dotted lines. For each impulse, the arrival time relative to the stimulus was determined as Tant and Tpost. The differences in arrival time, Tdif = Tpost - Tant, for the 1st 3 impulses and the last impulse were very similar, indicating that these impulses arose from the same initiation site. B and C: different preparation than for example traces in A, but using the same type of preparation. B: arrival time difference histogram produced by calculating Tdif for multiple impulses recorded during 35 trials. The data indicate 4 sites of impulse initiation. C: intracellular stimulation of the S cell somata in the 2 different middle ganglia produced 2 peaks. These peaks aligned within 1.5 ms with the 2 leftmost peaks in B, identifying the ganglia of origin for these impulses in the 2 peaks as particular middle ganglia as indicated by dotted vertical lines. The other 2 peaks correspond to the 2 anterior ganglia, as indicated by 2 additional dotted lines. The histogram in B was aligned with the diagram of the preparation in A by aligning the 2 peaks in B identified by S cell intracellular stimulation with the 2 ganglia containing these S cells (dotted lines in B).

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
<IT>c</IT>(<IT>t</IT>)<IT>=</IT><FR><NU><IT>1</IT></NU><DE><IT>T</IT></DE></FR> <LIM><OP>∫</OP></LIM><IT>d&tgr; </IT><FR><NU>[<IT>Sen</IT>(<IT>&tgr;</IT>)<IT>−&mgr;</IT><SUB><IT>Sen</IT></SUB>][<IT>S</IT>(<IT>&tgr;+</IT><IT>t</IT>)<IT>−&mgr;</IT><SUB><IT>S</IT></SUB>]</NU><DE><IT>&sfgr;</IT><SUB><IT>Sen</IT></SUB><IT>&sfgr;</IT><SUB><IT>S</IT></SUB></DE></FR>
where T is the duration of the recording, Sen(t) is the combined sensory cell firing rate at time t, S(t) is the S cell firing rate at time t, µSen and µS are the average sensory cell and S cell firing rates, and sigma Sen and sigma S are the standard deviations of the two firing rates.

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 Omega 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 3. Impulses typically do not collide in the S cell chain. Extracellular recordings were made from semi-intact preparations at the anterior and posterior ends of a chain of 6 ganglia, as well as at the midpoint along the chain. Same preparation as Fig. 2, B and C. A: arrival time difference histogram for the anterior and center electrodes, calculated from 35 trials. B: Tdif histogram for the outer electrode pair, which were the anterior and posterior electrodes. C: Tdif histogram for the center and posterior electrodes. Dotted line through A-C separates the impulses that initiated between the anterior pair of electrodes from those that arose between the posterior pair. The anterior Tdif histogram is shifted by 9.2 ms relative to the Tdif histogram of the outer electrode pair to account for the fact that a Tdif of 0 in the anterior electrode pair does not represent the same spatial location as a Tdif of 0 in the outer electrode pair. Likewise, the posterior Tdif histogram is shifted -9.2 ms. D: comparison of initiation sites estimated from the anterior pair of electrodes with those determined from the outer electrode pair. Initiation sites were calculated as (Tdif 1.6m/s)/2. The conversion to an actual distance in mm was made only to compare the 2 measurements, and this distance was not verified by actual measurements of distances between ganglia. For those impulses arising anterior of the center electrode, both electrode pairs localized the initiation to the same site, indicating that collisions were not occurring frequently. E: comparison of initiation sites determined from the posterior pair and outer pair of electrodes. Dotted line corresponds to the dotted line in A-C and indicates the position of the center electrode. Again, the 2 measures of initiation site did not show evidence of collisions.

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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Fig. 4. Initiation sites in single S cells. A: diagram of recording configuration showing 2 recordings from a single cell. B: Tdif histogram showing 3 distinct initiation sites (18 trials), 1 at each end of the cell presumably representing the propagation of impulses from adjacent S cells, and a 3rd apparently within the ganglion. C: after connectives were cut at the points marked by crosses in A, eliminating input from adjacent S cells, the 2 corresponding initiation sites were eliminated (17 trials).

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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Fig. 5. Different initiation sites produce activity at different times following a stimulus. A: schematic diagram similar to that in Fig. 2A of the intact segmental preparation consisting of segments 2-7. B: Tdif histogram showing activity initiating primarily in segments 2-6. C, top: raster plot showing activity during 46 trials. Each line represents a single trial. Dots mark the actual impulse initiation times, not the arrival times of impulses at the electrodes. Bottom: poststimulus time (PST) histogram showing the mean S-cell firing rate. Mean firing rate is calculated as the average number of impulses per bin divided by the binwidth, which was 5 ms in this case. D: activity evoked from ganglion 5 (G5), which was in the same segment as the stimulus, peaked at 55 ms after the stimulus. E: activity in G4, 1 segment adjacent to the stimulus, peaked at 85 ms. F: G3, peak activity at 115 ms. G: G2, peak activity at 195 ms.

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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Fig. 6. Impulse initiation at multiple sites produced by mechanical stimulation. The recording configuration was similar to that for Fig. 5, but a glass probe was lowered onto the skin as a stimulus, as described in METHODS. The chain included segmental ganglia 8 through 13. A: Tdif histogram of S cell activity showing multiple initiation sites produced by mechanical stimulation during 30 trials. B: raster plot and PST histogram of total activity in the S cell chain. C: PST histograms for different initiation sites.

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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Fig. 7. Sensory cell activity and S cell firing. A: S cell mean firing rate for each of 3 initiation sites. Same preparation as in Figs. 2, B and C, and 3. B: combined firing rate of P and T cells in each ganglion. For combined sensory cell activity, the mean firing rate was calculated for each cell separately, and then added together to represent the combined firing of multiple sensory cells. C: cross correlation between sensory cell activity and S cell activity. In the stimulated segment, the correlation between sensory activity and S cell impulse initiations in that segment had a peak at 45 ms, indicating that the greatest correlation for sensory cell activity was with S cell activity that was 45 ms later in time. One segment removed, the time of maximum correlation was at 65 ms. Two segments removed, the peak was at 115 ms.

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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Fig. 8. Schematic diagram of the increase in firing rate produced by the spatial and temporal separation of synaptic input. Two S cells in separate ganglia and a P cell extending to the next ganglion are depicted in each panel. A: a presynaptic impulse in a P cell activates the S cell closest to the stimulus, in the segment on the left. B: because of the faster conduction velocity of the S cell, this 1st S cell impulse passes the 2nd initiation site, in right segment, before the impulse in the presynaptic sensory neuron reaches that segment. C: the P cell impulse excites a 2nd postsynaptic impulse at the 2nd initiation site that does not collide with the 1st impulse in the S cell chain because of the rapid propagation along the S cell chain. Both regions of the S cell chain are consequently activated twice.

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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Fig. 9. Multiple initiation sites produce different activity patterns at different parts of the S cell chain. Stimuli were applied mechanically; same preparation as Fig. 6. A, top trace: recording from posterior connective in response to stimulation. Bottom trace: anterior recording. The last 2 impulses arose at different sites, as indicated by their difference in arrival time. This difference also produced a different interspike interval (ISI) in the anterior and posterior regions of the chain. B: histogram of the ratio between ISIs recorded at anterior and posterior sites for 30 trials. C: the ratio of ISIs at posterior and anterior electrodes as a function of time after a stimulus. Impulses were more closely spaced in the anterior of the chain because when the initiation site changed for 2 subsequent impulses, it most often moved toward the anterior (see Fig. 6).

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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Fig. 10. Compartmental model of sensory input to the S cell. A: diagram of model containing S cells, T cells, P cells, and their excitatory synaptic connections. For each mechanosensory cell, thick axons innervate the major sensory receptive field in the same segment as the soma, and thin axons innervate minor receptive fields in segments adjacent to the soma and major receptive field. A brief injection of current was applied to the peripheral axons that innervated segment 3 as indicated, generating a single impulse in each of these axons. B: activity in the S cell chain when conduction block occurred at P cell branch points in ganglia G2 and G4, reducing synaptic input to the S cells in those ganglia and eliminating it in G1 and G5. The trace shows the membrane potential at the anterior end of the S cell chain. Numbers indicate the segment of initiation for each impulse. The earliest impulses initiated in the same segment as the stimulus (3). C: reflection at P cell branch points (see text) was produced by decreasing the activity of the Na+-K+ ATPase uniformly across the P cell membrane. Reflection increased synaptic transmission to the S cell by activating P cell synapses twice in rapid succession. Even though facilitation, as is observed experimentally, was not included in the model, the increase in transmission due to summation of synaptic potentials was sufficient to increase the number of S cell impulses, as occurs in sensitization. The decreased activity of the pump slightly increased conduction velocity in P cells, producing a small change in the time of initiation for some S cell impulses. Impulses that initiated latest were produced by input from the slowest thin sensory axons innervating minor receptive fields. Diagram not to scale.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society