Departments of 1Neurosurgery and 2Applied Physics Laboratory, Johns Hopkins University, Baltimore, Maryland 21287
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ABSTRACT |
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Peng, Yuan B.,
Matthias Ringkamp,
James N. Campbell, and
Richard A. Meyer.
Electrophysiological Assessment of the Cutaneous Arborization of
A-Fiber Nociceptors.
J. Neurophysiol. 82: 1164-1177, 1999.
Little is known about the relationship
between the branching structure and function of physiologically
identified cutaneous nociceptor terminals. The axonal arborization
itself, however, has an impact on the afferent signal that is conveyed
along the parent axon to the CNS. We therefore developed
electrophysiological techniques to investigate the branching structure
of cutaneous nociceptors. Single-fiber recordings were obtained from
physiologically identified nociceptors that innervated the hairy skin
of the monkey. Electrodes for transcutaneous stimulation were fixed at
two separate locations inside the receptive field. For 32 A
-fiber
nociceptors, distinct steps in latency of the recorded action potential
were observed as the intensity of the transcutaneous electrical
stimulus increased, indicating discrete sites for action potential
initiation. The number of discrete latencies at each stimulation
location ranged from 1 to 9 (3.7 ± 0.2; mean ± SE)
and the mean size of the latency step was 9.9 ± 1.0 ms (range:
0.4-89.1 ms). For seven A
fibers, collision techniques were used to
locate the position of the branch point where the daughter fibers that
innervated the two locations within the receptive field join the parent
axon. To correct for changes in electrical excitability at the
peripheral terminals, collision experiments between the two skin
locations and between each skin location and a nerve trunk electrode
were necessary. Nine branch points were studied in the seven A
fibers; the mean propagation time from the action potential initiation site to the branch point was 31 ± 5 ms corresponding to a
distance of 54 ± 10 mm. Almost half of the daughter branches were
unmyelinated. These results demonstrate that collision techniques can
be used to study the functional anatomy of physiologically identified nociceptive afferent terminals. Furthermore these results indicate that
some nociceptive afferents branch quite proximal to their peripheral
receptive field. Occlusion of action potential activity can occur in
these long branches such that the shorter branches dominate in the
response to natural stimuli.
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INTRODUCTION |
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Several lines of evidence suggest that primary
afferent nociceptors can have multiple peripheral branches and may end
as a complex terminal arbor in their peripheral innervation territory. Multiple, punctate areas of high sensitivity to mechanical stimuli can
be found inside the receptive field of a single cutaneous nociceptor
(Perl 1968), arguing that these spots are innervated by
distinct axonal branches of the same afferent (Kruger et al. 1981
). Nociceptive cutaneous fibers with large peripheral
receptive fields up to several square centimeters have been described
in human and nonhuman primates (Meyer et al. 1991
;
Schmidt et al. 1997
), and innervation of such areas only
can be achieved by branching fibers. Even more, nociceptive fibers can
innervate multiple, isolated receptive fields in the same target tissue
(Meyer et al. 1991
; Michaelis et al.
1994
; Schmidt et al. 1997
) or the same fiber may
innervate anatomically distinct structures (Bove and Light
1995
; Mengel et al. 1996
). In these cases,
branching of fibers is self evident.
Electrophysiological recordings from unmyelinated dorsal root fibers or
dorsal root ganglion cells provided evidence that slowly conducting
peripheral axons can branch along the course of a peripheral nerve
(McCarthy et al. 1995; McMahon and Wall 1987
). In addition, electrical stimulation inside the receptive field of nociceptive afferent fibers revealed distinct steps in latencies with increasing stimulus intensity indicating discrete sites
for action potential initiation (Cervero and Sann 1989
; Jyväsjärvi and Kniffki 1989
; Matthews
1977
; Meyer et al. 1991
; Torebjörk
and Hallin 1974
). Collision experiments by Matthews (1977)
in tooth pulp suggested that these discrete latencies
originate from branches.
Peripheral branching of afferent fibers has been demonstrated by
anatomic studies (Heppelmann et al. 1990; Holland
1980
; Messlinger 1996
; Miller et al.
1960
; Reilly et al. 1997
), but such studies are
hampered by overlapping innervation between afferent fibers (Kruger 1988
; but see Ritter et al.
1998
) and reconstructing the peripheral terminal arbor of an
electrophysiologically identified fiber is almost an impossible task
especially for afferents with large receptive fields.
Despite the accumulating evidence for the existence of nociceptive
fibers that branch in the periphery, little is known about the
organization of the peripheral arborization of such fibers. Experimental findings indicate, however, that the axonal arborization influences the afferent signal that is conveyed along the parent axon
into the CNS. For low-threshold mechanoreceptors, it has been reported
that simultaneous activation of two terminal branches does not lead to
a simple summation of the neuronal activities in the parent axon
(Fukami 1980; Goldfinger 1990
;
Goldfinger and Fukami 1981
; Lindblom and Tapper
1966
). Action potentials generated in one branch may propagate
antidromically into other branches and collide with action potentials
generated there, leading to an occlusion of the action potential
signal. In addition, antidromic propagation of the action potential
into the sister branches may lead to a decrease in the excitability (or
"resetting") of the terminals (e.g., Eagles and Purple
1974
; Fukami 1981
; Lindblom and Tapper
1966
).
To allow a correlation between the peripheral structure and the
neuronal response in nociceptive fibers, information about the
peripheral arborization has to be obtained with methods that allow a
simultaneous investigation of the fiber's response properties. In the
present paper, we describe such a method. This method takes advantage
of the distinct steps in latency that occur after transcutaneous electrical stimulation that allow investigation of discrete action potential initiation sites in the peripheral arbor of the receptive field. Collision experiments were employed to test for the connectivity between these sites. Similar collision experiments have been used to
investigate the branching structure of CNS neurons (e.g., Lu and
Willis 1999; Shinoda et al. 1976
).
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METHODS |
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Animal preparation
Experiments were performed on nociceptive afferents in nonhuman
primates. Monkeys were sedated with ketamine and then anesthetized to a
level such that the corneal reflex was absent by intravenous administration of a mixture of pentobarbital sodium (3 mg · kg1 · h
1) and
morphine sulfate (0.5 mg · kg
1 · h
1 ). Animals were intubated, and peak expired
pCO2 was maintained at 35-40 torr using
mechanical ventilation. The animals were paralyzed with pancuronium
bromide (0.1 mg/kg) to minimize muscle artifacts during recordings as
well as to facilitate respiratory control. The rectal temperature was
controlled at 38°C by means of a circulating water heating pad. Five
percent dextrose in 0.9% normal saline was administered intravenously
in the course of the experiments to maintain hydration. Adequate depth
of anesthesia was ensured by continuous monitoring of the heart with an
electrocardiogram. The heart rate was maintained throughout the
experiment within 10% of the baseline heart rate prior to any surgical
stimulus. Any sudden increase in heart rate that was related temporally to a surgical or test stimulus was treated with an additional bolus of
the intravenous anesthetics. When it became apparent that the animal
was spontaneously breathing (~2-3 h after pancuronium bromide
administration), the absence of motor responses to noxious stimuli was
verified, and an additional bolus dose of pancuronium bromide
administered. All protocols were approved by the University Animal Care
and Use Committee. Animal housing conforms to federal regulations and
the facilities are accredited by the American Association for
Accreditation of Laboratory Animal Care.
Standard teased-fiber techniques (Campbell and Meyer
1983) were used to record from single primary afferents. Small
bundles of axons were cut away from the nerve trunk and dissected to
fine strands which were placed on the recording electrode (R in Fig. 1A). A nerve trunk electrode
(N in Fig. 1A) was placed on the nerve ~40 mm distal to
the recording electrode and used to identify strands containing one to
three A
-fibers at the recording electrode. The electrical threshold
and conduction delay (at twice threshold) of each afferent were
determined.
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Mapping of electrical receptive field (eRF)
For each A fiber, an electrocutaneous stimulation technique
(Meyer et al. 1991
) was used to locate the electrical
receptive field (eRF). A saline-soaked cotton-swab cathodic electrode
was placed on the skin (with a needle return electrode inserted several centimeters away) and used to electrically stimulate the afferent. The
cotton swab electrode was set at 150 V, 1.0-ms duration, and 0.2 Hz,
and moved around on the skin until spots were found where the fiber
could be activated. The eRF was identified as that area of the skin
where the electrical threshold was minimum and the conduction latency
was maximum. At these locations, suprathreshold stimulation led to
stepped decreases in conduction latency as described previously
(Meyer et al. 1991
). Once an initial estimate of the eRF
location was determined, the eRF border was remapped using several
return electrodes that surrounded the eRF to avoid artificial latency
steps caused by current spread across the eRF. For a given position
along the eRF border, the return electrode on that side of the eRF was used.
After mapping the eRF, the responses to mechanical and heat stimuli
were determined for some fibers. Von Frey probes were used to map the
mechanical receptive field and to identify spots within the receptive
field of highest mechanical sensitivity. A laser thermal stimulator
(Meyer et al. 1976) was used to test heat sensitivity.
A-fiber nociceptors were differentiated from low-threshold
mechanoreceptors by their lack of response to blunt pressure. In
addition, most nociceptors in this study had a high mechanical
threshold and/or a response to intense heat stimuli.
Collision techniques to investigate branching structure
Collision techniques were used to investigate the branching structure of the cutaneous arborization. Two electrodes (A and B in Fig. 1A) were placed inside the eRF. The distance between the electrodes ranged from 10 to 33 mm (mean separation = 18 ± 2 mm). Each electrode consisted of a 3-mm-diam plastic well (8 mm in height) that was glued to the skin and filled with electrolyte gel. A coiled silver wire was placed inside the well as a stimulating electrode. A separate needle return electrode was used for each well and was inserted in the skin several cm outside the eRF.
As illustrated in Fig. 1B, electrical stimulation at A (or B) initiates an action potential that propagates to the recording electrode with a conduction time of TAR (or TBR, respectively). Note that the subscripts denote the positions over which the conduction time is measured.
To determine the location of the branch point connecting locations A
and B, the conduction time (TCR) from
the branch point (C) to the recording electrode (R) needs to be
determined. From Fig. 1B, it is obvious that
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RESULTS |
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Thirty-two A fibers were studied. Their mean conduction
velocity was 11.8 ± 0.7 m/s (mean ± SE, range: 5.6-22.2
m/s) based on stimulation at the nerve trunk electrode at twice
threshold. Their mean electrical receptive field (eRF) area was
491 ± 68 mm2 (range: 63-1,476
mm2).
Stepped decreases in latency within the receptive field
Within the eRF, distinct steps in conduction latency from the skin to the recording electrode were observed when the intensity of the transcutaneous stimulus was increased. For most fibers, the number and the size of the latency steps were stable for hours, while different latency steps were observed at different stimulus locations in the receptive field. For the example shown in Fig. 2A,stimulation at location A in the receptive field led to three discrete latencies, whereas stimulation at location B (on the opposite side of the eRF, 14 mm away from location A) led to three different latencies.
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For all fibers in this study, distinct latency steps were observed at
most stimulus locations. Figure 2B shows the distribution of
latency increments that were observed. For the 32 A fibers, the
number of discrete latencies at each stimulation location ranged from
one to nine with a mean of 3.7 ± 0.2. The mean size of the
latency step was 9.9 ± 1.0 ms (range: 0.4-89.1 ms). The mean
threshold at the different AP initiation sites was 68 ± 2 V
(range: 6-148 V; 1-ms duration). The mean range in stimulus intensity
over which the latency remained constant was 16 ± 1 volts (range:
1-93 V).
For 27 A-fibers, latency data were obtained from at least two locations within the eRF. Although the stimulation electrodes usually were separated by >10 mm, stimulation at the two skin locations may induce AP initiation at identical axonal sites, leading to a common or shared latency between both sites. A common, shared latency (criterion = within 1 ms) was observed for <20 ± 4% (range: 0-80%) of the latencies at these locations. Seventeen of 27 A-delta fibers had one or more common latencies. A common latency is consistent with, but does not prove, a common AP initiation site. Because the majority of latencies were not shared between different locations, it is likely that most of these latencies originated from different AP initiation sites.
We hypothesize that these discrete latencies correspond to action potential (AP) initiation at different sites in the axonal arborization. Several different models for the possible sites of AP initiation are shown in Fig. 3. Model 1 assumes that all the AP initiation sites are in series along the same branch, whereas model 2 suggests that the AP initiation sites correspond to branching points in the peripheral arbor and model 3 positions the initiation sites at the terminals of different branches. Model 4 represents a combination of the other three models. The collision technique described in the next section was designed to investigate the connectivity between these AP initiation sites.
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Collision technique to measure conduction time between two AP initiation sites
As illustrated in Figs. 4 and 5, collision techniques were used to measure the AP propagation time between A and B. In this fiber, five and three AP initiation sites were detected by transcutaneous stimulation at location A and B (see Fig. 4). For simplicity, we will initially consider only one AP initiation site for each location. To accomplish this, the voltages at locations A and B are kept constant to obtain stable latencies. In this example, electrical stimulation at location B (45 V) led to an AP at the recording electrode with a shorter conduction time (21.6 ms, Fig. 4a) than after electrical stimulation at location A (30 V, 26.5 ms, Fig. 4b). When both locations were stimulated simultaneously (Fig. 4c), only the AP from site B was recorded. The AP initiated at site A collided with the AP from site B that had antidromically invaded into branch A (see cartoon associated with Fig. 4c).
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When electrical stimulation at site A was delayed (e.g., by 30 ms), the
site of collision moved up branch A toward site A (Fig. 4e
and cartoon associated with e). When stimulation at site A
was delayed long enough (33.8 ms), the AP initiated at site B had
(virtually) propagated beyond the AP initiation site A before stimulation at site A occurred. Consequently, two APs were recorded at
the recording electrode
one initiated from site B (first AP) and one
initiated from site A (second AP) (Fig. 4g). The minimum amount of time by which stimulation at site A had to be delayed before
two APs were seen (i.e., 33.8 ms in this example) is a first-order
measure of the AP propagation time from B to A (but see section that
follows on correction for refractoriness at AP initiation sites).
In a similar manner, the propagation time from A to B can be measured
(Fig. 5). As before, when site A and B were stimulated simultaneously
only one AP reached the recording electrode, and its conduction time
(21.6 ms) indicated that it originated from site B. After delaying
stimulation at site B by 2 ms with respect to stimulation at site A
(see Fig. 5d), a single AP was obtained at the recording
electrode with an apparent conduction time of 23.6 ms, i.e., the AP
originating from site B was delayed by 2 ms. With increasing delay of
the stimulation at site B, the apparent conduction time of the single
AP observed at the recording electrode increased (see Fig.
5e). However, when stimulation at B was delayed long enough
(5 ms), the single AP at the recording electrode became time-locked
at a latency of ~26.5 ms, i.e., the latency obtained for an AP
originating from site A (compare Fig. 5, b with
f-i ). Time-locking the AP at the recording electrode at a
latency characteristic for an AP initiated at site A reflects collision
of the delayed AP from B by the AP initiated at A (see cartoons
associated with Fig. 5, f and i). Again, when
site B was delayed sufficiently (i.e., delay
40.8 ms), two APs were seen at the recording electrode (Fig. 5j); the first AP was
initiated at site A and the second at site B. The minimum delay at B
and the minimum delay at A (from Fig. 4) needed to see two APs at the
recording electrode should be the same, if the minimum delay purely
reflects conduction between A and B. However, minimal delays were often
found to differ for the two directions, and for the given example, they
were 33.8 and 40.8 ms, respectively. Because the measured minimum delay
is the sum of the conduction time between A and B and the refractory
period at A or B, corrections for the refractoriness at the spike
initiation sites are necessary. For the example given, the refractory
time at site B was 7 ms longer than at site A.
Corrections for refractoriness at an AP initiation site
When an AP propagates past a spike initiation site (e.g., site B in Fig. 5), the excitability of the membrane at site B becomes refractory for a period of time. The duration of this relative refractory period is a function of the stimulus voltage at site B (i.e., the greater the voltage, the shorter the relative refractory period). This refractory period produces an error in the estimate of the propagation time from site A to site B. However, the refractory period at site B should be the same whether the AP is initiated from site A or from the nerve trunk electrode (N in Fig. 1). Therefore collision between an AP initiated from the nerve trunk electrode and an AP initiated from site B (or A) can be used to determine the relative refractory period at site B (or A).
Figure 6 shows the data that were collected to estimate the refractory interval at site B for a typical fiber. Electrical stimulation at the nerve trunk electrode produces an AP that propagates both toward the recording electrode and toward the periphery. If site B is stimulated at the same time as the nerve trunk electrode, only the AP from the nerve trunk electrode will be recorded because of collision. However, stimulation at site B can be delayed (in a manner similar to that shown in Fig. 5) until two APs are seen at the recording electrode. The minimum delay at site B to see two APs now equals the propagation time from the nerve trunk electrode to site B plus the refractory period at site B. The propagation time from site B to the nerve trunk electrode can be calculated because the propagation time from site B to the recording electrode and the propagation time from the nerve trunk electrode to the recording electrode can be measured. Thus the refractory period for site B can be determined by subtracting the propagation time between B and the nerve trunk electrode from the measured minimum delay to obtain two APs after stimulation at the nerve trunk and B. This refractory period then in turn is subtracted from the minimum delay at site B for two APs to occur after stimulation at site A and site B (as determined in Fig. 5) to determine the AP propagation time from site A to site B.
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If MDNB is the minimum delay of the
stimulus at site B to see two APs after stimulation at the nerve trunk
electrode, and RPB is the refractory period at B,
then
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In Fig. 6D, the propagation latency of the AP initiated at
site B is plotted as a function of the minimum delay. As the minimum delay decreased from 140 to 45 ms, the latency of the propagated AP
increased gradually from 46.3 to 48.6 ms. An increased latency is also
apparent for the second action potential in Figs. 4B, g and
h, and 5B, j and k. This increase in
latency of the second action potential at the recording electrode is
due to a slowing in conduction velocity associated with the propagation
of the preceding action potential. A supernormal conduction latency
(e.g., Swadlow et al. 1980) was not observed in these
experiments. The latency of the second action potential was monitored
(e.g., Fig. 6D) to verify that there was not a stepped
decrease which would indicate that the site of AP initiation had
changed. Refractory period calculations could be made using lower
voltages at site B. However, measurements made at low voltages were
usually less reproducible since small variations in threshold led to
large changes in delays. We therefore used the highest voltage at the skin site that still led to AP initiation from the same site (i.e., at
the same latency).
The excitability recovery function for a given fiber is a plot of the minimum electrical stimulus that can initiate an action potential as a function of time after the propagation of the action potential past the AP initiation site. A plot of this function can be obtained by reversing the axes of the minimum delay versus electrical stimulus intensity graph shown in Fig. 6B. The inset in Fig. 6B shows the excitability recovery function for this fiber for which the excitability at this AP initiation site recovered exponentially with a time constant of ~0.1 s. Similar excitability recovery functions were obtained from 19 locations in 10 nociceptive fibers. The mean time constant for recovery was 59 ± 9 ms (range: 2-135 ms, n = 19).
The plot of minimum delay versus voltage at site B after an AP initiated from the other skin location (site A) is shown in Fig. 7B. This plot looks the same as the plot obtained from stimulation at the nerve trunk electrode (Fig. 6B). At the maximum voltage (50 V), the minimum delay at site B to obtain two APs at the recording electrode was 61 ms. Subtracting the refractory period of 1.5 ms obtained at this voltage (from Fig. 6B), we obtain a propagation time from site A to site B of 59.5 ms. A similar propagation time was obtained when this procedure was repeated for propagation of the AP in the opposite direction (i.e., from site B to site A, data not shown). The conduction time between the branch point and the recording electrode (TCR) was determined from Eq. 4) to be 9.8 ms. Thus site B was 36.4 ms away from the branch point, and site A was 23.1 ms from the branch point.
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Calculation of branch point location
The preceding measurements provide an estimate for the conduction
time between sites A and B. Using Eq. 4, the conduction time
from the branch point C to the recording electrode
(TCR) can be determined. The actual
distance from the branch point to the recording electrode
(XCR) can be calculated as
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The conduction velocity VCR is unknown and has to be estimated. One estimate for this conduction velocity is given by the conduction velocity of the parent fiber obtained from stimulation at the nerve trunk electrode (i.e., VNR).
However, the conduction velocity between the branch point and recording
electrode is not uniform. In general, the distal segment of the parent
nerve (from C to N) has a slower conduction velocity than the proximal
segment (from N to R). Therefore a more accurate estimate of the branch
location is obtained if the conduction velocity of the distal segment
(VCN) is used to determine the distance from the nerve trunk electrode to the branch point. Now
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Maps of the receptive field architecture for a typical fiber are shown in Fig. 8. For this fiber, three latency steps were observed at location A, and seven latency steps were observed at location B (Fig. 8A). The connectivity between site A1 and site B3 was investigated using the collision techniques described in the preceding text, and the latency for the branch point was determined to be 15.5 ms. A conduction latency map for this fiber is shown in Fig. 8B. Dots correspond to the AP initiation sites at location A (left) or location B (right). The AP initiation site(s) investigated are indicated by filled circles (sites A1 and B3 in this example) and are connected by solid lines to the branch location between the two sites (labeled as C in the figure).
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The position of the branch point is plotted in Fig. 8, C-E, using the three estimates for the conduction velocity described in the preceding text. The most distal position for the branch point is obtained when the highest conduction velocity (i.e., the conduction velocity of the parent fiber obtained from stimulation at the nerve trunk electrode) is used (Fig. 8C). The most proximal position is obtained when the slowest conduction velocity is used, i.e., the distance and the latency obtained for site B3 (Fig. 8E). Perhaps the best estimate of the branch point location is obtained when the shortest latency from site B (i.e., from AP initiation site B7) is used (Fig. 8D).
Measurements of connectivity between multiple AP initiation sites in same fiber
So far we have only considered connectivity between a single AP initiation site at stimulus location A and a single AP initiation site at stimulus location B. In several fibers, we were able to investigate the connectivity between one AP initiation site at stimulation location A and several of the AP initiation sites associated with stimulus location B.
The technique to investigate multiple AP initiation sites in a single fiber is illustrated in Fig. 9. Collision experiments were done as described in the preceding text, but the latency of the AP elicited at the skin was recorded to verify from which AP initiation site it originated. For the example in Fig. 9, two AP initiation sites were found at location B (22.4 and 10.8 ms, respectively). Figure 9C illustrates the relationship between minimum delay and stimulus intensity for both initiation sites. As stimulus intensity increased, the minimum delay between stimulation at the nerve trunk electrode and site B to record two APs decreased. At a stimulus intensity of 26 V, the minimum delay was ~20.5 ms (see top dotted line in Fig. 9C). When the stimulus intensity was increased >26 V, the latency of the observed AP suddenly shifted to a short latency, characteristic for an AP initiated at site B2 (Fig. 9C). In addition, the minimum delay could now be reduced further to ~10 ms. As illustrated in Fig. 9A, 26 V was the threshold voltage for the initiation of the AP at site B2. Again, when the delay was decreased further, stimulus intensity had to be increased to elicit an AP at site B2. At the maximum stimulus intensity (38 V), the minimum delay was ~8.7 ms (see bottom dotted line in Fig. 9C). Together with the conduction time from B1 and B2 to the nerve trunk electrode (19.2 and 7.6 ms, respectively, Fig. 9B), the absolute refractory periods at B1 and B2 were determined to be 1.3 and 1.1 ms, respectively (see dashed lines in Fig. 9, C and D). A similar approach is used to obtain the minimum delays for propagation from skin site A to site B1 and site B2. Thus several AP initiation sites in one branch can be investigated.
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An example of the receptive field map for a fiber in which more than one AP initiation site was investigated is shown in Fig. 10. This fiber had three AP initiation sites from both the A location and the B location (Fig. 10A). The connectivity between site A2 and sites B1 and B2 was investigated using the collision technique described in the preceding text. As shown in Fig. 10B, the branch point between site A2 and site B1 had a longer conduction time (11.5 ms) than the branch point between site A2 and site B2 (7.9 ms). This example provides evidence that the multiple latencies obtained at location B were from different branches of the terminal arborization. The three estimates for the positions of the branch points are shown in Fig. 10, C-E. For this fiber, the two branch points were 75 and 60 mm from the receptive field based on the fastest conduction latency from the skin (Fig. 10D).
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Receptive field architecture for A-fiber nociceptors
The receptive field architectures for the seven A-fibers in
this study for which collision experiments were completed are shown in
Fig. 11. For this figure, the
architecture based on latency is shown in the top row, and
the corresponding position of the branch point(s) based on the
conduction velocity determined with the minimum conduction latency
(fastest conduction velocity) from the receptive field is shown in the
bottom row. For all fibers, the latency of the branch point
was shorter than the shortest latency obtained from stimulation at
either test location. The mean latency between the branch point and the
recording electrode was 10.8 ± 2.1 ms (range: 4.1-24 ms, 9 branch points in 7 A fibers). The mean propagation time in the daughter
branches from the branch point to the AP initiation site was 31 ± 5 ms (range: 3-75 ms).
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For the seven A fibers in this study, the propagation time from site A
to site B was the same as the propagation time from site B to site A. Thus there was no evidence for a unidirectional block at branch points,
because the action potentials were able to antidromically invade both
parts of the receptive field. Connectivity between these sites with
bidirectional propagation measurements is indicated in Fig. 11 (). In
some cases, the propagation time from A to B was determined in only one
propagation direction. The locations of the branch point for these
situations are indicated by a dotted line in Fig. 11 (· · ·).
For all fibers, the calculated position of the branch point was proximal to the receptive field. For one of the fibers (V53), the branch point was located 5 mm proximal to the receptive field. However, for the remaining six fibers, the branch point was quite proximal to the receptive field, and three fibers (V51, V52, V61) had one or more branch points >70 mm from the receptive field. The mean distance from the receptive field to the branch point was 54 ± 10 mm (range: 5-94 mm, for 9 branch points in 7 A fibers).
Once the position of the branch point is determined, the conduction velocity of the axon from the branch point to the AP initiation sites can be computed. Using the branch point locations shown in Fig. 11, the mean conduction velocity of the daughter branches was 2.6 ± 0.6 m/s (range: 0.3 to 11 m/s). As shown in Fig. 12, there was no correlation between the conduction velocity of the daughter and the parent axon. The daughter branches had conduction velocities that were significantly slower than the conduction velocity of the parent axon (13 ± 2 m/s, P < 0.001). About half of the daughter branches (9/19) had conduction velocities <2 m/s and therefore were probably unmyelinated. The remainder had very slow conduction velocities indicating that they were either thinly myelinated or unmyelinated over a portion of their length.
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For five fibers (V52, V61, V63, V68, and V71), the connectivity between multiple sites at each location was investigated. For four fibers (V61, V63, V68, V71), two or more sites at one location shared a common branch point with one or more sites at the other locations. For example, sites A1 and A3 in fiber V63 had a common branch location with sites B1 and B2. For two fibers (V52 and V61), AP initiation sites from A and B were not connected by a common branch point but by at least two branch points.
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DISCUSSION |
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These studies indicate that electrophysiological techniques can be used to assess the anatomic structure of physiologically identified nociceptors. Initial results with the technique demonstrate that A-fiber nociceptors branch quite proximal to the receptive field and that many of the daughter branches are unmyelinated. These long branches have an impact on how nociceptors spatially integrate information derived from stimulation at multiple sites within their receptive field.
Latency steps are due to action potential initiation at discrete sites
In previous studies, discrete latency shifts were observed when
electrical stimuli of increasing intensity were applied to the
peripheral target tissue (Jyväsjärvi and Kniffki
1989; Matthews 1977
; Mengel et al.
1996
; Meyer et al. 1991
; Torebjörk
and Hallin 1974
). This study corroborates these findings and
demonstrates in a systematic fashion that the latency of the recorded
action potential remains stable over a certain range of stimulus
voltage but then decreases abruptly to a shorter latency when the
stimulus is increased beyond a certain intensity. These findings
suggest that discrete sites for AP initiation exist in the peripheral arborization of nociceptive afferents. Because the action potential generated at one site is also propagated antidromically into other branches, collision occurs. When more than one AP initiation site is
activated, the AP from the shortest latency site reaches the parent
axon first and prevents the APs initiated at the other sites from
reaching the CNS.
We observed on average about four discrete latencies at each stimulation location (range: 1-9). The mean size of the latency step was 9.9 ± 1.0 ms (range: 0.4-89.1 ms). In addition, the latencies from each stimulus location were usually distinctive. Thus there are a large number of distinct AP initiation sites in the peripheral arborization of nociceptive afferents. The number of latencies and the size of the latency steps may provide an estimate of the complexity of the structure in the terminal arborization. For example, fibers with very few latencies and small latency steps may be less complex than those with many latencies.
The observation that the number and the size of the latency steps were reproducible in a given fiber for hours favors the concept that a stable anatomic configuration accounts for this phenomenon. There are several potential locations within the peripheral arborization that could represent anatomic correlates for AP initiation sites:
NONUNIFORM SENSITIVITY ALONG AXON.
Nonuniformities (model 1 in Fig. 3) of the axon membrane (e.g., changes
in myelination, changes in axon diameter, and changes in channel
densities) could be the sites for AP initiation. The axon diameter can
vary considerably due to axonal varicosities (Greenberg et al.
1990; Novotny and Gommert-Novotny 1988
).
Although variations in axon diameter could lead to low-threshold sites for AP initiation (Roth 1994
), the varicosities appear
to be ubiquitously and densely distributed along the axon, and
therefore it is unlikely that they account for distinct latency steps
in the range of several milliseconds.
BRANCH POINTS.
Axon branch points (model 2 in Fig. 3) may represent discrete impulse
initiation sites (Roth 1994; Struijk
1992
). The step-wise decreases in latency with increasing
stimulus intensity could be due to sequential excitation of more
proximal branch points.
TERMINALS.
The most peripheral cutaneous terminals (model 3 in Fig. 3) of an
afferent fiber may be the sites of AP initiation. Modeling studies have
shown that the terminal membrane has the lowest threshold for AP
initiation (e.g., Reilly 1998; Rubinstein
1994
). A different terminal ending is excited when the stimulus
intensity is increased beyond a certain strength. Because stepped
decrements in latency occur with voltage increments, the model requires
that the shorter latency ending terminates more deeply in the skin or,
alternatively, that it has a faster conduction velocity.
Branching structure
We provide evidence that A-fiber nociceptors have an extensive branching structure and that some of the branch points are quite proximal to the receptive field. For all fibers, the AP initiation site at location A was from a different branch than the AP initiation site at location B. For five fibers, the connectivity of multiple AP initiation sites was investigated. In three cases, the different AP initiation sites from one location had a common branch point with respect to the opposite location (e.g., fibers V63, V68, V71). However, in two fibers (V52, V61), a different branch point connected AP initiation sites from location B with location A. For these fibers, the discrete steps in latency observed as the stimulus voltage increases at one location is not due to a change in the site of AP initiation along the length of one axon but rather reflect an AP initiation site in a completely different branch of the fiber (e.g., model 4 in Fig. 3).
Surprisingly, there was no correlation between the conduction velocity of the daughter and the parent axon. However, the conduction velocities of the daughter branches were always slower than the conduction velocity of the parent axon. About half of the daughter branches had conduction velocities that were <2 m/s; these branches therefore probably were unmyelinated. The remainder had very slow conduction velocities, indicating that they were either thinly myelinated or unmyelinated over a portion of their length.
It is commonly thought that, for myelinated afferents, the generator
potentials from the afferent terminals summate at the first node of
Ranvier where the action potential is initiated (e.g., Kandel et
al. 1991; Mountcastle 1980
). Two lines of
evidence from our study suggest that this is not the site of AP
initiation for myelinated nociceptive afferents. First, for most of the
fibers, the parent myelinated axon was a long distance (>1 cm) from
the terminal structure, too long for electrotonic spread of generator potentials. Second, action potentials could be initiated in these unmyelinated branches with electrical stimulation. Thus it appears that
generator potential integration and action potential initiation occur
within the unmyelinated terminal branches of the myelinated nociceptor
and not at the first node of Ranvier.
Other authors also have demonstrated that afferent fibers branch along
their entire length from their central processes in the dorsal root, to
their course in peripheral nerves, and finally in their peripheral
target tissue. There are about twice as many dorsal root fibers and
peripheral nerve fibers as there are cells in the corresponding dorsal
root ganglion (DRG), suggesting that afferent neurons have more than
one central branch and more than one peripheral branch (Langford
and Coggeshall 1979, 1981
). In addition, some DRG cells can be
activated from two peripheral nerves (Devor et al. 1984
;
Pierau et al. 1982
). Double labeling studies with
retrograde tracers confirmed that DRG cells may have more than one
peripheral process (Taylor and Pierau 1982
). Peripheral branching close to the DRG likely represents the morphological correlate that accounts for somato-visceral convergence in some dorsal
root ganglion cells (Bahr et al. 1981
; Borges and
Moskowitz 1983
; Bove and Light 1995
;
Pierau et al. 1984
). Branches of the same afferent may
also be found within the same peripheral nerve (McCarthy et al.
1995
; McMahon and Wall 1987
).
It should be emphasized that the present study only allowed those branch points relatively close to the periphery to be located because the recordings were made in the peripheral nerve. Although the results provide evidence that branching indeed occurs along the course of the peripheral nerve, the incidence of such branching is clearly underestimated when recording from peripheral nerve fascicles. The method described in the present study, however, easily can be applied to dorsal root recordings, which should allow branching along the entire peripheral neuraxis of afferent fibers to be studied. If additional branches of the nerve exist more proximally, the receptive field size and architecture would be expected to be larger and more complex as the site of recording is moved proximally.
Functional implications of branching structure on activity in nociceptors
The interaction of impulse activity originating from different
branches in the terminal arbor of primary afferents has been investigated for many different classes of low-threshold receptors including slowly adapting and rapidly adapting mechanoreceptors, hair
afferents, and Golgi tendon organs (e.g., Fukami 1980;
Goldfinger 1990
; Goldfinger and Fukami
1981
; Lindblom and Tapper 1966
). Two modes of
interaction appear to predominate in these types of fibers: a
"resetting" of excitability in peripheral terminals and a
"mixing" of action potential activity between branches. In
nociceptors a third mode of interaction, which we term "occlusion,"
may occur and is described in the following text.
RESETTING IN NOCICEPTORS. A resetting of excitability in terminal branches occurs when an action potential from one branch antidromically propagates into the other branches resulting in an increased threshold of the other branches to initiate action potentials. Thus antidromic action potential propagation into the peripheral terminals of nociceptors leads to a decrease in excitability of the terminals to electrical stimulation. As part of the collision procedure in this study, we determined the excitability recovery function after antidromic propagation of a single action potential. The time constant for recovery ranged from 2 to 135 ms (mean = 59 ms). The time constant dictates the capacity of a given site to respond at high frequencies; branches with slow recovery times are not able to contribute to a high-frequency response of the nociceptor to natural stimuli. The wide range of recovery times observed for different branches indicates that some branches in the peripheral arbor are better able to maintain high discharge frequencies than others. Consequently, high discharge frequencies in nociceptors may be maintained by activity from a single branch.
MIXING IN NOCICEPTORS. Mixing refers to the situation where APs in the parent axon arise from different branches. A mixing of action potential activity between branches occurs when the frequency of discharge is slow enough that excitability of the terminal structure recovers. Furthermore, mixing can only occur if occlusion does not. For A-fiber nociceptors with long branches, as described in this study, mixing may only occur at relatively low discharge frequencies.
OCCLUSION IN NOCICEPTORS. The AP propagation times in the daughter branches were long (31 ± 5 ms, range: 3-75 ms). These long propagation times favor the presence of collision in the longer branch. For example, if two APs are initiated simultaneously at the receptor terminals, the AP from the terminal with the shortest conduction time to the branch point will propagate antidromically into the other daughter branch where it collides with the AP initiated in that branch. An AP from the longer branch will be seen only if it is initiated sufficiently before the AP from the first terminal so that collision does not occur. Thus terminals that are close to the branch point can dominate the response that is seen in the parent axon. This occlusion of action potential propagation may be important for nociceptors because the conduction time in the branches is long due to the slow conduction velocities and long distances in the branches. Antidromic invasion of a branch is required for occlusion to occur. Thus unidirectional branch points would protect the branch from occlusion. However, action potentials were able to antidromically invade both parts of the receptive field for the fibers in this study, and thus there was no evidence for a unidirectional block at branch points. Occlusion is not a major factor for low-threshold receptors because the conduction length of the branches is short (i.e., the conduction velocities are fast and conduction distances are short).
Summary
We describe an electrophysiological technique that allows the branching structure of physiologically identified nociceptors to be determined. This technique demonstrated that A-fiber nociceptors branch quite proximal to their termination in the skin and that the daughter branches can be unmyelinated over long distances. Interaction of action potential activity between different branches occurs. This interaction may favor the input of a particular branch when stimuli are applied concurrently to more than one spot in the receptive field. The techniques described here also should be useful to delineate the anatomy and signal processing properties of primary afferent C-fiber nociceptors.
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ACKNOWLEDGMENTS |
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Y. B. Peng and M. Ringkamp contributed equally to this work. The authors thank T. Hartke and S. Horasek for technical support, B. Turnquist for developing the customized data acquisition software, and S. Raja, G. Wu, and C. Roza for comments on an earlier draft of this manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-14447 and the J. B. Kempner Foundation.
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FOOTNOTES |
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Address for reprint requests: R. A. Meyer, Johns Hopkins Applied Physics Lab, Johns Hopkins Rd., Laurel, MD 20723.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 February 1999; accepted in final form 22 April 1999.
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