EXPERIMENTAL
NEUROLOGY
114,82-103
(1991)
Sensory Neurons of the Rat Sciatic Nerve JOHN
E. SWETT, YASUHIRO TORIGOE, VINCENT R. ELIE, CHARLES M. BOURASSA,* AND PETER G. MILLER Department
of Anatomy *Department
and Neurobiology, College of Medicine, UCI, Irvine, California 92717; of Psychology, University of Alberta, Edmonton, Alberta, Canada
data suggest that nearly 20% of all DRG neurons in the sciatic nerve supply muscle afferents. The vast majority of the remaining neurons are involved with innervation of the skin. The number of small DRG neurons in pure muscle nerves are few implying that a large proportion of the unmyelinated axons in these nerves are from postganglionic sympathetic neurons. The number of small DRG neurons (t29 pm in diameter) in cutaneous nerves outnumber the large ones by two- to threefold suggesting that neurons with unmyelinated axons dominate. 0 1991 Academic Press, Inc.
Experiments have been undertaken in this laboratory over recent years to accurately determine the numbers and sizes of somatic neurons which contribute to the normal sciatic nerve, at mid-thigh levels, of the adult, albino rat. This article is concerned with the dorsal root ganglion (DRG) neuron population of the sciatic nerve whose cell bodies were identified through retrograde labeling of cut branches of the sciatic with horseradish peroxidase (HRP) and/or its wheat germ conjugate (WGA-HRP). It is essential to understand the neuronal composition of the normal rat sciatic nerve if the consequences of aging, nerve injury, and surgical repair to improve functional regeneration are to be properly evaluated. Neuron counts were determined from camera-lucida paper drawings of all labeled profiles in DRGs L3L6 at 100X magnification. The profiles, obtained by labeling individual branches of the sciatic nerve (sural, lateral sural, tibial, peroneal, medial, and lateral gastrocnemiuslsoleus nerves) were traced from 40-pmthick, serial, frozen sections. The sizes of the perikarya, areas and diameters, were determined by tracing the perimeters of the drawn profiles on a digitizing tablet. The tablet’s output was inputted directly into a specially designed computer spreadsheet which contained a mathematical table for correcting the split-cell error inherent to the sectioning process. Afferents from any given branch of the sciatic normally occupied two to three adjacent ganglia. Sciatic DRG neurons were normally located in lumbar ganglia L3-L6. Nearly 9899% of all sciatic DRG perikarya resided in the L4 and L5 DRGs. The L6 DRG, traditionally regarded as an important contributor to the rat sciatic, contained merely 0.4% of its afferent neurons while the L3 ganglion, frequently overlooked as a contributor, contained 1.2% of the mid-thigh sciatic afferents. The mean size of rat DRG neurons was about 29 pm (550600 pm2). The corrected counts revealed that the normal sciatic nerve (at mid-thigh levels), in rats between 2 and 12 months of age, contained a mean, total DRG neuron population of about 10,500 neurons. This is probably an underestimate by 3-5% of the true number due to occasional unreliable labeling of some of the small DRG neurons. It is estimated that the normal, mean number of sciatic DRG neurons of young to middle-aged rats lies somewhere between 10,500 and 11,000 f 2000. The 0014-4&36/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
INTRODUCTION The rat sciatic nerve, at mid-thigh level, contains axons from about 2000 motoneurons (71). This finding was based on detailed reconstructions of motoneuron cell bodies retrogradely labeled with HRP reaction products from cut branches of the sciatic. The motoneurons resided in discrete compartments in the ventral horn of the spinal cord extending from the rostra1 third of spinal segment L6 into the caudal third of L3 (71). These experiments, and many since then, labeled large numbers of dorsal root ganglion (DRG) neurons (17,70). The labeling technique was perfected to assure that most, if not all, motoneuronal perikarya contributing axons to a given branch of the sciatic would be labeled without also labeling neurons in adjacent branches. Ganglia L3-L6 were routinely collected, sectioned serially at 40 pm, and processed for HRP histochemistry in a manner similar to that used for motoneuron populations. Reconstructions of DRG neuron populations were not attempted unless motoneurons from the same nerve were optimally labeled (for criteria see (71)). Reconstructions of the sciatic DRG neuron populations were undertaken because the rat sciatic nerve is the most widely used peripheral nerve in experimental studies involving nerve injury and regeneration. Despite this, quantitative descriptions of its neuronal populations are conflicting or incomplete. For example, on the basis of retrograde HRP-labeling, a total count of 5760 DRG neurons has been estimated for the tibia1 nerve 82
Inc. reserved.
and
RAT
DRG
(5). The sural nerve is reported to contain 3600 DRG neurons (5); others measured about 2550 DRG neurons (51). The DRG count of 530 has been reported for the medial (MG) and lateral gastrocnemius and soleus (LGSOL) branches of the sciatic (5). Unfortunately, no comparable information is available for the common peroneal and lateral sural branches. If it assumed that the common peroneal has a composition similar to that of the tibia1 nerve and two-thirds its size, a rough estimate of 3600 peroneal DRG neurons would not be unreasonable because it contains 632 motoneurons (71). The lateral sural nerve, being roughly half the size of the sural should have no more than about 1500 DRG neurons. If one assumes that there is little dichotomization of DRG axons into different tributary branches of the sciatic (13), the total DRG neuron population for the mid-thigh sciatic could contain as many as 14,000-15,000 DRG neurons. This estimate represents a maximum limit. If it is assumed, as will be confirmed later, that nearly all sciatic DRG neurons reside in two DRGs, L4 and L5, it is possible to extrapolate lower counts. For example, the L4 and L5 DRGs contain collectively about 23,300 DRG neurons (57). If 54% of these contribute to the sciatic (15) then the total number of DRG neurons should be in the range of 12,600. Using corrected nucleolar counts, it has been estimated that the sciatic of young rats contain about 6300 neurons while old ones contain as many as 9100 (14,15). From the data of Bondok and Sansone (6) the number of sciatic DRG neurons would be in the range of 5800. Axon counts can also be used for estimating numbers of DRG neurons. The sciatic nerve at mid-thigh levels contains between 23,700 and 27,000 axons (31, 56). Of this number 19,000 are thought to originate from DRG neurons (58). If each DRG neuron has on the average 2.3 axons (36), then the number of sciatic DRG neurons should be about 8300. The low value of 5800 DRG neurons or the high value of 14,500 may be correct, but not both. With motoneuron counts varying less than +5-6% among animals (71), it seems unreasonable that the normal DRG ‘neuron populations could vary so widely among animals of the same age group. The true number must lie somewhere between these extremes. The objective of the present study was to reconstruct as accurately as possible the number, ganglion location, and size distributions of the DRG neuron populations of the rat sciatic nerve as revealed by retrograde labeling of their cell bodies with HRP reaction products. It has long been recognized that the cell bodies of primary afferent neurons can be labeled from the periphery by retrograde transport of tracer substances (17, 80). In recent years this approach has been used by a number of investigators to obtain quantitative information about DRG neurons of the cat (8,29,38,42) and of the rat (4, 15, 20, 48-51, 59, 70, 75, 79).
NEURONS
83
One of the sources of variability in estimating numbers of sciatic DRG may be found in the assumption that the L6 DRG contributes significant numbers of neurons to the rat sciatic nerve at mid-thigh levels (5, 15,44,56-59, 76) when it does not. The L3 DRG, with some exceptions (5, 14), is overlooked as a source of sciatic DRG neurons. It contributes only 1.2% of the total, three times that of L6. Perhaps the greatest source of variability derives from the techniques used in counting neurons and the mathematical methods for correcting counts for splitcell error. Sampling, for example, can introduce large distortions in neuron counts because the strategy assumes a uniformity in the spatial packing of the cells being counted. Retrogradely HRP-labeled neurons, especially motoneurons and DRG neurons, are not uniformly distributed spatially. Tests on the error limits introduced by sampling every fifth section or less revealed a maximum error of +90% (74). To obtain the most accurate results, the count should include all labeled elements unless such an approach proves to be thoroughly impractical. When counting objects in tissue sections some mathematical correction of the raw counts is essential to subtract out the number of doubly counted cells (12, 34). Errors of underestimation or overestimation of cell counts will inevitably result (5,74). One can obtain accurate neuron counts and cell size distributions by reconstructing all neurons in a HRP-labeled population as was done with the sciatic motoneurons of the rat (71). However, the methods used for reconstructing motoneuron populations proved to be unworkable when applied to DRG neurons because of the small sizes and large numbers of profiles scattered unevenly across relatively large tissue sections. Attempts to match profiles belonging to the same cell in adjacent sections resulted in a severe underestimate of the actual amount of split-cell error. Attempts to construct an empirically derived correction factor, as others have attempted (5,42,59), were abandoned when it became evident that it resulted in inflated counts. A modified Abercrombie (1) method was also used for awhile (70) but abandoned, not because it underestimated the true neuron numbers by only 5-6%, but because it distorted neuron size distributions (74). Accuracy in numbers and sizes of sciatic DRG neurons was the primary objective of this study. It required a new and rigorous approach to the correction of splitcell error. This resulted in the development of a “fragment correction” procedure, a modified version of the Hendry method (24), that is described in detail elsewhere (74). When tested on neuron populations whose cellular profiles were completely known, this correction method produced a cell count that was on the average accurate to 98.6 + 3.4% of the true, known cell count.
84
SWETT MATERIALS
AND
METHODS
The experiments were conducted on 72 nerve preparations in Sprague-Dawley rats (60 females and 8 males) weighing 120-510 grams. Routinely, one nerve was labeled in each hindlimb. The nerves were exposed surgically under deep anesthesia (ketamine hydrochloride, 100 mg/kg and Rompun, 3 mg/kg). The nerve chosen for any given experiment was one of the following: sural, lateral sural, common peroneal, tibial, MG, and/or LG-SOL. The exposed nerve was prepared by gently stripping all visible blood vessels from the epineurium 2-3 mm on either side of the site of transection. This was done in order to minimize the incidence bleeding at the exposed nerve end that would impede axonal uptake of the labeling solution. The nerve was then sectioned and immediately immersed in a soft, low-melting-point wax while suspending the proximal cut nerve end by its epineurial sheath. Using a microsyringe, filled with distilled water, a droplet of water was placed over the nerve end just visible beneath surface of the wax. The wax is then excavated so that the water flooded the nerve end before it could be exposed to the air. More wax was added to build the depth of the reservoir over the nerve end to accommodate l-2 ~1 of labeling solution. The distilled water is left in place for about 10 min, removed by damp wick, and replaced by a 35-40% solution of HRP (Boehringer, Grad l), a 3-5% aqueous solution of its wheat-germ conjugate (WGA-HRP; Sigma), or, most commonly, a mixture of the two (MIX). The reservoirs were capped by wax to prevent drying. A mixture of HRP and WGA-HRP was employed in most preparations because of suggestive evidence that the number of small DRG neurons was below normal when labeled only with HRP. Conversely, the number of large DRG neurons appeared to be underrepresented when labeled only with WGA-HRP (66). Although this was not a consistent phenomenon, it occurred often enough to warrant use of a mixture of the two tracers to facilitate labeling of the maximum number of DRG neurons. The animals were maintained in a warm environment with additional supplements of anesthetic and dextrose-saline (ip) for 5-6 h. Removal of the label required three separate washings of the nerve ends by distilled water before the wax was gently removed from the wound in a manner to avoid stretching of the nerve. The wound was then flooded by warmed dextrose-saline solution for several minutes before closing it in layers with sutures. These steps were taken to avoid spurious labeling of peripheral neurons that belonged to nerves other than the one labeled. Spurious labeling was determined primarily by evaluating the positions of retrogradely labeled motoneuronal perikarya and the quality of their labeling (71). If there had been spread of the label, this
ET AL.
was disclosed by the presence of faintly labeled motoneurons in positions inappropriate for the nerve that had been labeled. If labeling criteria were satisfied the DRGs L2 through L6 were reconstructed. When it became apparent that the L2 DRGs never contained labeled profiles after labeling any of the sciatic branches at mid-thigh levels or beyond, they were eventually discarded. The ganglia were sectioned horizontally in 40-pmthick sections and the sections were reacted with TMB according to a slightly modified protocol of Mesulam (46, 71). All sections were retained and mounted in serial order on glass slides. The perimeters of all labeled profiles in all sections were traced with a 0.3-mm drafting pencil with the outer edge of the line coinciding with the perimeters of the profiles. This was done with the aid of a drawing tube at 100X magnification. All profiles were then numbered sequentially to assure that no profile was measured twice. The profiles were then traced on a digitizing tablet (Donsanto) and the numerical values for their areas were entered into a specially designed spreadsheet program (Symphony, Lotus Development Corp.). A separate computer file was maintained for each DRG so that it would be possible to determine the relative contributions of each nerve to each DRG. Another special spreadsheet file was created in which all ganglia in a given animal could be combined for the right and left sides, respectively. These files were used for developing XY-histogram plots of a nerve’s total DRG perikaryal size distributions. Because accuracy in neuron numbers was the highest priority of the study, the cell counts were based on the strategy of measuring the largest possible sample of labeled profiles. The least error will result if all profiles are measured. Because labeled DRG neurons are not distributed evenly throughout the tissue sections, sampling of all profiles in every second, third, or fifth section was rejected in favor of collecting data from all profiles in all sections to achieve maximum accuracy. With alternate sections the mean potential error was about +6-7% with a maximum potential error of +17%. With every third section the maximum potential error increased to *49%. When sampling was applied to every fifth or sixth section the maximum limit of error escalated to +89%. By counting every profile the mean error could be theoretically reduced to -1.4 + 3.5% with a maximum potential error of about 6-7% (74). The data from the digitizing tablet were imported into a 286 AT personal computer through a serial port using the Symphony (Lotus Development Corp.) communication protocol to directly insert morphometric data of the DRG neurons directly into worksheet columns. The worksheet contained formulae and macros for error checking, statistical functions, graphics, and the fragment correction table for split-cell error. The worksheet was errors could be flagged -~- desizned -~--~_~~~ so that innuttine: A -I
85
RAT DRG NEURONS and edited. The raw data consisted of a profile’s identification number, area, and diameter. The diameters were then sorted into l-pm-diameter bins. Area measurements were sorted into bins corresponding to the limits of the diameter bins. For illustrative and mathematical purposes the histograms were plotted with the data sorted to the centers of the bins; e.g., the 30-pm-diameter bin contains all corrected profiles representing neurons with diameters ranging from 29.51 to 30.50 pm. Areas were plotted linearly on the abscissa. The area and diameter histograms were plotted for all cases because the two sets of data emphasized different features of the neuron populations. The diameter histograms were most useful in visualizing the larger DRG neurons while the area histograms emphasize the dominance in numbers of the small DRG neurons, especially found in cutaneous and mixed nerves. To correct for split-cell error from tissue sectioning, an iterative procedure, the “fragment correction” procedure, was applied to the binned raw data of area measurements of all labeled profiles from all sections (74). This procedure is a refinement of a method described by others (24,55,60) and is capable of correcting split-cell error of spherically shaped, synthesized “neurons” with zero error (74). Briefly, this correction procedure estimates the number and size of labeled profiles in smaller bins that would be created by sectioning cells of larger diameters and then subtracts these profiles from their respective bins. The total count remaining should be equal on the average 98.6% of the true population (standard deviation = ?3.5%) providing that all neurons have been labeled and all labeled profiles were included in the initial uncorrected collection of profiles. Any profiles smaller than 10 pm in diameter were ignored because no DRG neurons of the size exist in the rat. A critical thickness limit was also incorporated into the mathematical correction procedure. It was assumed that any fragment of a labeled cell 2.0 pm or less in thickness would not be visible in the tissues and therefore not captured in the collection of sorted profiles to be mathematically corrected. The value for the critical thickness limit is important because, if it is inaccurately estimated, it could lead to a significant over- or underestimate in the number of neurons in the smaller bin sizes. This thickness limit was determined empirically using darkfield microscopy to resolve HRP-labeled neurons and curvefitting adjustments of the correction factor on known populations of neurons and their fragments (74). There are three assumptions inherent in the use of the fragment correction factor: (i) the largest neuron must be in the largest (top) bin (e.g., some of the profiles may be equal in size to the largest neuron), (ii) all neurons are assumed to be spherical although most are ellipsoidal in shape, and (iii) all measured profiles 10 pm or larger in all ganglia on one side of the animal repre-
sent the complete population of labeled DRG neurons many of which were cut into two or more fragments. The first condition is an assumption that is intuitively true if the neuronal population is pseudo-randomly arranged. In addition, given enough neurons (in the hundreds or thousands), there is a likelihood that most neurons would be cut in such a way as to show profiles representative of the neurons’ largest dimensions. The second condition, when tested on real, reconstructed motoneuron populations, was not met, but the error introduced by this resulted in only a small underestimate of true numbers (74). An addition of 1.4% of the corrected count would theoretically offset most of this error. The third condition did not appear to be consistently met. Some preparations, presumably typically well-labeled ones, exhibited high neuron counts with medium to small mean perikaryal sizes. Others, however, seemed to have abnormally low counts coupled with larger than average diameters, indicating that an unknown proportion of the smaller DRG neurons may have escaped labeling. Some of these preparations were included in the data presented in this article because there was no obvious criteria for excluding them. As a result the DRG neuron counts reported here will be an underestimate of the true numbers by approximately 3-5%. Cell counts in all the tables are corrected for split-cell error. RESULTS The DRG neuron populations are described individually for each labeled branch of the sciatic nerve.
The Sural Nerve (Fig. 1, Table 1) The vast majority of the sural nerve’s somatic components, about 96%, is composed of axons of DRG neurons which innervate the lateral, distal cutaneous aspect of the animal’s leg and foot (72). The remaining 4% is composed of motoneurons (71). The total mean DRG population of the sural nerve consists of 1675 f 316 neurons, 93% of which are located in the L5 DRG (Table 1). The L4 DRG contained a limited number of sural DRG neurons but this amounted to twice the total found in L6. The sural nerve’s contributions to L6 were frequently absent. Figure 1 illustrates DRG perikaryal size distributions of three sural nerve preparations listed in Table 1. The case in Fig. 1A was chosen as a typical example in terms of numbers and perikaryal dimensions. Figure 1B shows the case with the smallest mean for perikaryal size while Fig. 1C illustrates the case with the largest mean cell size. The most striking feature of the sural DRG perikaryal size distributions is that they are strongly skewed toward neurons of small sizes. The sural DRG neurons populations were among the smallest in mean perikar-
86
SWETT
ET
AL.
A 150 -I
1
0
B
0
CASE
0.4
0.8 1.2 1.6 AREA tjfm2) x
2 1000
R84
2.4
2.6 D~E~R
(mm)
150
CASE 1598
0
0
0.4
0.8
1.2
R94
CELLS
1.6
2
2.4
2.8
0
40
20 DIAMETER
CASE
1 0 0
1522
0.4
0.8 AREA
1.2 (ii&
1.6 x
60
(Cm)
B5 CELLS
2 2.4 1000
2.8 DLAMETER @m)
FIG. 1. DRG population profiles of the sural nerve. (A) A case with a mean perikaryal size close to the mean value (B) The case in Table (1) with perikarya of smallest mean size. (C) The case in Table 1 showing the largest perikaryal
yal dimension with a mean diameter of 26.5 pm (mean area = 520 pm2). The small neurons, those smaller than 29-30 pm, outnumbered the large by nearly 2 to 1 as may be seen in the population histograms of Figs. 1A and 1B.
for all cases in Table dimensions.
1.
The Lateral Sural Nerve (Fig. 2, Table 2) At mid-thigh levels the sciatic nerve gives off a small branch, the lateral sural nerve, which penetrates the biceps femoris to supply the skin region immediately
87
RAT DRG NEURONS TABLE Segmental Dist~bution
1
of Normal Sural DRG Neurons and Their Sizes
Animal
Label
L3
L4
L5
L6
Total
B3
MIX MIX HRP
-
52 16 213 16 4 58 26 151 110 84
1271 1505 1497 2322 1369 114% 1956 1479 1135 2161
0 1 0 0 173 277 0 0 1 12
1323 1522 1710 2338 1546 1483 1982 1630 1246 2257
96 120
1425 1478
0 0
1521 159%
1586
0
1612
36 2.1%
1675 i316
B5 H30 H31 H43 H44 H45 H49 R80 R83 R%4 R94
R95 N=
13
HRP
-
MIX MIX MIX
-
MIX
-
HRP HRP HRP HRP HRP
-
Mean total No. Mean %
0 0%
26
75 4.5%
1564 93.4%
proximal to that supplied by the sural nerve on the lateral aspect of the thigh (72). The lateral sural is a pure cutaneous nerve with a mean total of 886 DRG neurons nearly 96% of which are located in the L4 DRG. In the six preparations listed in Table 2 no ventral horn motoneurons were observed in the spinal cord after nerve labeling. This is the only major tributary branch of the main sciatic trunk to be so composed. The lateral sural DRG neurons also revealed population size distributions resembling sural DRG neurons with a mean size of 26 pm (480 pm’). A case representing values close to the mean perikaryal size for the lateral sural DRG neurons is shown in Fig. 2A. The contribution of small DRG neurons is again the dominant feature of these neurons. Figure 2B illustrates the case with the smallest. mean perikaryal size and Fig. 2C shows the case with the largest mean perikaryal size. An unusual feature of the lateral sural nerve, not clearly observed with any of the other tributary branches of the sciatic, is its tendency to show a quantal shift in size and nearly doubling its complement of DRG neurons from one animal to the next. This size difference in diameter is even detectable to the naked eye among preparations. Some cases contained about 570720 DRG neurons while others showed nearly double that number, 1090-1230. Although the sural nerve contains 70 motoneurons and perhaps an equal number of DRG neurons supplying afferent axons to muscle, its cutaneous components far outnumber its muscle components. For purposes of comparison with other tributary branches of the sciatic, the sural and lateral sural nerves serve as good examples of cutaneous nerves. The prominent group of small DRG neurons found in the perikaryal size histograms shown in Figs. 1 and 2 show distinct similarities.
Area 550 635 518 568 620 454 44% 474 639 438 520 430 468
+ + * f + + + I!z rt + k + k
365 413 355 372 429 333 317 371 51% 324 453 350 324
520 k 379
Diameter 28.6 30.0 26.8 29.1 29.4 25.5 25.3 25.6 28.3 23.6 25.6 23.1 24.2
+ ?I f +f + k ++ + + k ?
9 10 9 9 10 9 8 9 12 8 11 9 8
26.5 -c 9
The Peroneal Nerve (Fig. 3, Table 3)
The common peroneal nerve is always the second largest branch of the sciatic. It contains about one-third of all the sciatic motoneurons found at mid-thigh levels (71). The peroneal nerve contained axons from 2699 +: 557 DRG neurons. The L4 DRG was also the preferred location for 79% of them. The area and diameter distributions of this population resembles the sural nerve in being heavily skewed toward smaller-sized neurons, but the mean perikaryal size of 28.7 pm (Table 3) was nearly 3 pm larger than the cutaneous nerves (Tables 1 and 2) because the peroneal nerves contained a greater proportion of large diameter DRG neurons than the sural nerve (Fig. 3). This can be seen particularly well in Figs. 3A and 3B where the neurons larger than 700 pm’, or ~27 pm in diameter, are noticeably greater in number than in Figs. 1A and 2A. Figure 3C shows the extreme case in which the peroneal DRG neurons were unusually large. The Tibia1 Nerve
(Fig. 4, Table 4)
The tibia1 nerve is the largest tributary branch of the sciatic trunk; it contains the axons of nearly half of all the ventral horn motoneurons found in the sciatic at mid-thigh levels (71). It also contains the largest number of DRG neurons, 4748 4 800 (Table 4) accounting for about 45% of all sciatic DRG neurons (Table 7). The typical population profile of the tibia1 nerve (Fig. 4A) is essentially identical to that of the peroneal nerve (Fig. 3A) in perikaryal dimensions although it contains nearly 75% more DRG neurons than the peroneal nerve. The L4 and L5 DRGs contained 99% of the tibia1 DRG
88
SWETT
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CASE T19
z 23
1089
100
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E B 5 B z
50
0
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2 1000
2.4
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40 DIAMETER
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CASE T36 1102
CELIS
0 0
C
AREA @rn’)
150
20
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x
40 (ym)
60
DIAMETER
40 (jun)
60
DIAMETER
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CASE Ti' z E
719
3 100 2
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8 3B 50 ii 0
FIG. 2. cases in Table
0
0.4
0.8 1.2 1.6 AREA (pm') x
DRG population profiles 2. (B) The case in Table
2 1000
of the lateral sural 2 with the smallest
2.4
2.8
0
20
nerve. (A) A case with mean perikaryal dimensions close to the mean mean perikaryal size. (C) The case in Table 2 with the largest perikaryal
neurons with the L4 DRG containing more than twice the number of tibia1 neurons than the L5 DRG. The peroneal and tibia1 nerves are mixed nerves. They collectively account for about 81% of the sciatic motoneurons (71) and 70% of the DRG neurons (Table 7). The peroneal nerve contains about 4.3 DRG neurons
value for all dimensions.
for each somatic motoneuron and the tibia1 nerve contains about 4.7 DRG neurons/motoneuron.
Muscle Nerve Branches (Figs. 5 and 6; Tables 5 and 6) Two small tributary branches of the sciatic nerve leave the main trunk of the sciatic near the nerve’s ma-
RAT
DRG
89
NEURONS
TABLE
2
Segmental Distribution of Normal Lateral Sural DRG Neurons and Their Sizes Animal H50 H51 T7 T19 T36 T42
N=6
Label
L3
L4
L5
L6
Total
MIX MIX MIX MIX
2 12 8 20
MIX MIX
18
1201 556 678 973 1095 584
30 0 33 96 7 1
0 0 0 0 0 0
1233 568 719 1089 1102 603
404 493 587 438 403 557
Meantotal No. Mean %
10 1.1%
848 95.7%
28 3.2%
0 0%
886 +288
480 CL 346
0
Area k f 4 zk * k
268 423 431 252 300 399
Diameter 23.9 25.3 28.8 26.1 23.7 27.4
+ ? + -rIL +
26.0 -t
8 10 10 7 8 10 9
jor division into its tibia1 and common peroneal branches. The smaller medial one innervates the MG; the larger lateral branch supplies the LG-SOL muscles. The MG nerve (Fig. 5, Table 5), although sometimes appearing nearly the same size as the lateral sural nerve, contains only 111 ~fr 16 DRG neurons, 93% of which are located in the L5 DRG. In only one-third of the cases will DRG neurons from the MG nerve appear in the L6 DRG. This mean number of 111 DRG neurons (Table 5) is only about 20-30 less than the total number of ventral horn motoneurons in the same nerve (71). Mean perikaryal size of MG DRG neurons, 35.5 pm, was significantly larger than that found in any other nerve except for the other muscle nerve, LG-SOL (Table 6). Comparison of Fig. 5 with the preceding figures shows a conspicuous lack of skewing of the populations toward the smaller cell sizes as seen in Figs. l-4. The population histograms, as in Figs. 5A and 5B, tend to be symmetrical and unimodal in shape with DRG neurons 3040 pm in diameter dominating the distributions. The LG-SOL nerve is very similar to the MG nerve in mean size of its DRG neurons (Fig. 6, Table 6) with only slight skewing of the histogram profiles to smaller sized neurons (Fig. 6A and 6B). The mean number of DRG neurons in the LG-SOL nerve (230 + 44) is slightly more than double that found in the MG nerve and its neurons are nearly equally distributed between the L4 and L5 DRGs (Table 7). It is of interest to note that the mean areas and diameters of the DRG neurons of the MG and LG-SOL nerves are nearly identical (Tables 5 and 6). These neurons are significantly larger than any of the previously described DRG neuron populations. Like the MG nerve, the LG-SOL nerve contained relatively few neurons of small sizes (~29-30 Km).
ble 7). However, nearly 99% of the sciatic DRG neurons have their cell bodies located in only two of these DRGs, L4 and L5, with L4 containing almost two-thirds of the total (62%). Of the four DRGs that contributed afferent axons to the sciatic nerve, L6 contributed the least. The L6 DRG accounted for only 0.4% of the sciatic afferents. By comparison, the L3 DRG contributed three times the number of afferent neurons than L6. Although there are exceptions to the rule, every tributary branch of the sciatic nerve, on the average, contained axons from three adjacent DRGs (Table 7). Usually one DRG contained the majority of the afferent perikarya for any given nerve. The nerves supplying the distal skin of the hindleg, the sural, peroneal, and especially the tibia1 nerve which contained about 46% of all sciatic afferents, contained the largest numbers of small DRG neurons. One of the most striking features of the data summary presented in Table 7 is the small numbers of DRG neurons associated with the muscle afferents. The total number of sciatic sensory neurons reported in Table 7, 10,349, represents the count corrected for split-cell error and does not include the additional correction to offset the -1.4% error introduced by the “fragment correction” procedure (74). When this error is corrected the total adjusted DRG count for the rat sciatic nerve equals in round numbers about 10,500 f 2000 DRG neurons. As will be discussed later this number may still underestimate the true mean number of sciatic DRG neurons by 3-5%. The true mean number of DRG neurons probably lies between 10,500 and 11,000 f 2000.
Segmental Distributions and Total Numbers of Sciatic DRG Neurons
Segmental Distribution
The sciatic nerve of the rat receives somatic motor and sensory axons from neurons of the spinal cord and DRGs of four spinal segments, L3, L4, L5, and L6 (Ta-
DISCUSSION of Sciatic Afferents
It is widely assumed that axons of the sciatic nerve of the albino rat originate from spinal segments L4, L5, and L6 (15, 19, 33, 57, 58, 76). Segment L3 must be added to this list because it consistently supplied moto-
90
SWETT
CASE 2635
0 0
0.4
ET
AL.
W6 CELLS
0.6 1.2 1.6 2 2.4 AREA (pm’) x 1000
2.6
0
20 ~IAM~ER
40 (pm)
60
40
60
B300 3
CASE
CJl240 f3 5 E 160
3076
W51 CELLS
0
0 AREA
C 300
em’)
x
2922
ri
120 -
52 2
60-
FIG. 3. DRG all cases in Table dimensions.
DIAMETER
(lm)
-
CASE
0
20
1000
0
0.4
0.6 1.2 1.6 AREA (rm’) x
H19 CELLS
2 2.4 1000
2.6
0
20
40 DIAMETER
60
@ m)
population profiles for the common peroneal nerve. (A) A case with mean perikaryal dimensions close to the mean value for 3 (B) The case in Table 3 with the smallest mean perikaryal size. (C) The case in Table 3 with the largest perikaryal
neurons to the peroneal nerve (71) and received axonal projections from a small number of L3 DRG neurons whose distal processes usually contributed to the lateral
sural and peroneal nerves and often to the tibia1 nerve (Table 7). The small contribution of L3 to the sciatic has been noted before by others (5,15). Although the affer-
RAT
DRG
TABLE Segmental Animal
Label
GlRl GlR4
MIX MIX MIX MIX MIX HRP HRP HRP HRP HRP HRP HRP MIX HRP HRP HRP WGA-HRP WGA-HRP MIX HRP
G2R2 G2R3 G4R4 I2 Rlll
w3 WS
w7 Wll w14 HlQ
w22 w23 W46 w49
w51 Xl
x2
N==
20
“-Dash
Mean Mean
total %
in the L3 column
Distribution
L3 1 21
176 5
266 -
indicates
that
Neurons
L5
L6
1480 2218
881 580
2812 2123 1820
806 752 161 647 270 257 60 676 362 210
2547 2254
382 743 497
2661 2924
269 152
1880
1081 737
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
501 18.6%
0 0%
2775 2775 2407
-
1495
30 2
data were
DRG
L4
2790 1592
65 2.4%
3
Personal
1129 1924 1626
19
No.
of Normal
91
NEURONS
496
1419
2133 79.0% absent.
This
sum represents
ent fiber contributions to the sciatic nerve from the L3 DRG were few, they were more numerous than the contributions from the L6 DRG. Contributions from the latter to the rat sciatic were negligible. In the Sprague-Dawley strain of rats used in the present study (obtained from Charles River or Simonson Laboratories), nearly 99% of all the sciatic DRG neurons were located in the L4 and L5 DRGs. The relative unimportance of the L6 DRG to the sciatic afferent population strongly influences the interpretation of data in which the loss of neurons due to nerve injury is determined from counts of surviving DRG neurons (58, 76). The L3 motoneuron contributions to the sciatic (71) appeared to be slightly greater than the corresponding proportion for DRG neurons, providing weak ad~tional evidence in support of a general pattern that efferents from a given distal structure are staggered rostrally to a small degree in comparison to the segmental level of the afferents that supply it (67). The fact that neurons in DRGs L3-L6 contribute to the sciatic has been recognized for several years (l&20). The relative contributions of each DRG to the sciatic in the material of Devor et al. (15) differed from the data of Table 7 in that about 9% of the sciatic afferents were found in L3 and L6 with the latter containing three times the number of labeled DRG neurons than L3 as if their animals had a more postfixed organization than the ones used in the present study. In keeping with this,
the horizontal
and
Their
Sizes
Total
2362
Area
Diameter
2819 3794
595
667 4 350 Ltr446 6711430
30.8 f 8 28.8 k 11 30.4 f 10
2880 2247 1776 2194 1883 2835 3466
609 It. 391
2158
525+- 374 599F377 652 4408 666f 473 595 + 358 603 f 445 7082 429 490 2373 818~~ 433 597 + 471 572 + 417 515~429 441* 350 395 rt330 5482329 730~~349
28.9 + 26.6 k 31.1 f 30.9 t 29.8 k 28.0 + 27.5 + 31.9 + 26.3 k 33.1 f 28.0 zk 27.4 + 25.6 f 24.4 + 25.4 + 27.7 + 32.0 i
2699”
600+ 398
28.7 + 9
1954 2985 2922
1877 3290
2751 2930 3076 2991
9 9 10 10 11
8 10 10 10
8 11 10 11 9 7
8 7
+557 total.
they also found that the L5 DRG contained the majority of the sciatic DRG neurons while our data indicated L4 as being the dominant one. The reasons for these subtle differences are unknown but could be related to the different strains of albino rats used by the two laboratories or possibly to the difference in counting methods. In most DRGs, particularly those with the most labeled profiles, there was no clear evidence of any somatotopic distribution of cell bodies. This feature has been noted by others (51, 75). Labeled profiles were found scattered randomly everywhere throughout the neuronal compartments of unlabeled, counterstained DRG perikarya. Occasionally, those DRGs which contained a very small number of profiles at the extreme rostra1 or caudal limits of a nerve’s afferent projection boundaries showed a preferential position within the ganglion as been described for visceral afferents (35). These labeled profiles appeared to be spatially confined to roughly one quadrant of the DRG giving tenuous evidence for poorly defined somatotopic localization of perikarya. In unreported double-labeling experiments in this laboratory with the HRP/WGA-HRP mixture and Fluoro-Gold applied separately to pairs of sciatic nerve branches (see (71)), labeled profiles of all sizes appeared to be randomly intermingled spatially in sharp contrast to the high degree of somatotopic localization of their respective motoneuron populations in the spinal cord.
92
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ET AL.
A 400 , CASE
R112
5633 CELLS
0
6
0
0.4
0.6 1.2 1.6 AREA (pm’) x
2 2.4 iOO0
2.6
0
20 D~AM~ER
40 (pm)
80
400 1
CASE
z” 0300
3874 CELLS
5 22 &ZOO
j
0
c
W31
400
0.4
0.8 1.2 1.6 AREA (pd) x
2 2.4 1000
2.8
20
0
DIAMETER
40 o(m)
60
40 (pm)
60
‘1
CASE $300
-
4321
H13 CELL5
5 E
$200
-
i3 g100 2
-
0
0
0.4
0.8 1.2 1.6 AREA (Il$) x
2 1000
2.4
2.8
0
20 DIAMETER
FIG. 4. DRG population profiles for the tibia1 nerve. (A) A case with mean perikaryal dimensions close to the mean value for all cases in Table 4. (B) The case in Table 4 with the smallest mean perikaryal size. (C) The case in Table 4 with the largest perikaryal dimensions.
RAT
DRG
93
NEURONS
TABLE
4
Segmental Distribution of Normal Tibia1 DRG Neurons and Their Sizes Animal
Label
L3
L4
L5
H13 H14 H29 RlllL R112L w30 w31
MIX MIX MIX HRP HRP HRP HRP
0 54 280
0 0
2136 2778 4326 3836 3821 3186 2552
2185 734 856 1828 1806 1534 1322
48 1.0%
3234 68.1%
1466 30.9%
N=l
Mean Mean
total %
No.
In our experience, maximal cell counts are related to conditions favoring dense perikaryal labeling that usually obscures the nuclei and nucleoli. Emphasis on dense labeling helped to expose maximal numbers of the smallest DRG neurons, which are thought to be more difficult to label retrogradely with HRP than large ones (59).
Differences between Cutaneous and Muscle Afferents The DRG neuron population of the sciatic nerve can be subdivided into two basic groups: (i) neurons supplying skin, and (ii) neurons supplying muscles and joints. Cutaneous DRG neurons far outnumbered those supplying muscle. Afferents supplying other structures such as joints are presumed to be present in relatively small numbers among both types. The MG and LGSOL nerves at the point of labeling contained no joint afferents; the only DRG neurons that would be labeled in those cases would be muscle afferents. These pure muscle nerves collectively contain about 334 f 10 motoneurons ((71) plus recent data) and 341+_ 60 DRG neurons (Figs. 5 and 6; Table 7). There is approximately a one to one ratio between the number of DRG neurons and the number of motoneurons. Somewhat smaller ratios of numbers of DRG neurons to motoneurons has been described by others (51). What numerical ratios exist between muscle afferents and motoneurons in large mixed nerves, such as the tibia1 and peroneal nerves, is unknown, but it would not be unreasonable to assume that the one-to-one ratio observed in the triceps surae group may represent a general pattern. If this hypothesis is correct, there should be a total of approximately 2000 muscle afferents in the distal branches of the sciatic because the documented number of motoneurons in the nerve, including the small y-motoneurons, average about 2000 (71). The proportion of all the sciatic DRG neurons involved with innervating muscle tissue must therefore be about 19% of the total. Ignoring the few engaged in innervating
L6
0 0%
Total
Area 2 k f + + f k
Diameter
4321 3566 5462 5664 5633 4720 3874
160 652 544 572 621 652 536
381 339 383 343 354 490 326
4148 &SO0
620 f 373
32.3 30.5 27.3 28.4 29.2 30.5 28.7
k 8 + 8 XL 9 + 8 + 8 * 12 + 9
29.6 +
9
joints, perhaps t5%, the remaining 76% must be primarily engaged in innervating cutaneous receptive fields (72). For every DRG neuron supplying muscle there are at least four others supplying the skin.
Perikaryal
Size in Relation
to Nerve Type
The DRG neuron populations of cutaneous, mixed, and muscle nerves were fundamentally different in cellsize characteristics one from the other. These differences are illustrated in the normalized plots of Fig. 7. Each plot represents pooled data from six nerves, three nerves from each of the Tables l-4, that were close to the mean perikaryal size for each table. The solid-line plot illustrates the typical DRG neuron population profile of pure cutaneous nerves. It was derived from about 7800 DRG neurons of three sural and three lateral sural nerves listed in Tables 1 and 2 including the cases shown in Figs. IA and 2A and two other cases in each group with similar dimensions. The dashed-line curve is composed in similar fashion of data from six mixed nerves, three cases each from Tables 3 and 4 representing those in Figs. 3A and 4A plus two others in each group of similar dimensions. This curve is the normalized distribution of about 21,000 DRG neurons from the peroneal and tibia1 nerves. The dot-dashed line is the normalized plot of about 1000 neurons from six cases of pure muscle nerves, three each from the MG and LGSOL nerves (Tables 5 and 61, two of which are shown in Figs. 5A and 6A. The maximum and minimum size limits for the DRG neurons is similar for all three nerve types shown in Fig. 7, pure cutaneous, pure muscle, and mixed nerves. This size range does not differ significantly from that described by others (50,51,57,78). DRG perikarya smaller than 13-14 pm proved to be very rare. Perikarya larger than 65 pm were also seldom found, but, within these limits, the population profiles of the DRG neurons of these three nerve types showed consistent differences. Nerves with large cutaneous components revealed his-
94
SWETT
ET
AI,.
A 10
CASE
O,,,,r,,,,,,,,,,, 0 0.4
B
0.8 1.2 AREA Qrn
1.6 ) x
RllOL
2 1000
2.4
2.8
0
20 DIAMETER
40 (rm)
60
10
CASE z” g
140
AREA
(jun’)
x
RllOR CELLS
1000
CASE
DIAMETER
Cm)
II15
0 0
0.4
0.8 1.2 1.6 AREA (gmr ) x
2 1000
2.4
2.8
0
20 DIAMETER
40 (jun)
60
FIG. 5. DRG population profiles of the MG nerve. (A) A case with mean perikaryal dimensions close to the mean value Table 5. (B) The case in Table 5 with the smallest mean perikaryal size. (C) The case in Table 5 with the largest perikaryal Irregularity of the plots is a result of the small numbers of DRG neurons found in the MG nerve.
togram profiles strongly skewed toward large numbers of small DRG neurons. This same principle has been observed in the cat (42). Muscle afferent populations,
for all cases in dimensions.
on the other hand, revealed histogram profiles that tended to be unimodal, symmetrical and of much larger mean size than cutaneous nerves (51). Interestingly,
RAT
CASE g 2
15
270
DRG
NEURONS
’ zle
’
95
H41
CELLS
r2 “d
10
z g2
5
0
0,
I 0.41
1 0.8I 1 1.2 , r 1:s T 3 ’ 214 AREA (J&) x 1000
CASE 239
1
0.4
0.8 1.2 1.6 AREA @nZ) x
0
20
40 DIAMETER
60
(pm)
R78
CELLS
2 1000
2.4
CASE
2.6
0
20
40 (am)
60
DIAMETER
40 (km)
60
DIAMETER
H16
188 CELLS
0
0
0.4
0.8 1.2 1.6 AREA (pm’) x
2 1000
2.4
2.6
20
FIG. 6. DRG population profiles of the LG-SOL nerve. (A) A case with mean perikaryal dimensions close to the mean value Table 4. (B) The case in Table 4 with the smallest mean perikaryal size. (C) The case in Table 4 with the largest perikaryal Irregularity of the plots is a result of the small numbers of DRG neurons found in the LG-SOL nerve.
this may be a feature unique to rodents because muscle afferents in the cat appear to have strongly skewed size distribution similar to that found for cell bodies of cutaneous afferents (42). This fundamental difference be-
tween cutaneous and muscle DRG shown to exist in the radial nerve of A feature of interest in Fig. 7 is the the three population histograms that
for all cases in dimensions.
neurons has been the rat (77). cross-over point of occurs at about 29
96
SWETT
ET
TABLE Segmental Distribution
AL.
5
of Normal MG DRG Neurons and Their Sizes
Animal
Label
L3
L4
L5
L6
Total
R71L R72L R86 R105 R109 RllOL RllOR Hll H15 H27 FG5L FG5R
HRP HRP HRP HRP HRP HRP HRP MIX MIX MIX MIX MIX
-
-
0 7 3 5 17 3 14 0 0 0 0 0
96 80 97 120 112 103 85 111 101 125 96 116
0 0 0 1 0 2 41 0 0 0 0 0
96 87 100 126 129 108 140 111 101 125 96 116
0 0%
4 3.6%
103 92.8%
4 3.6%
111 +16
N=
12
Mean Mean
total %
-
No.
Variability
Animal
Label
R78 HlO H12 H16 H17 H40 H41 W28 w29 FG2
WGA-HRP MIX MIX MIX MIX MIX MIX HRP HRP MIX
N=
10
Mean Mean
total %
in DRG Neuron
LG-SOL
DRG
Neurons
and
Their
L4
L5
L6
Total
-
71 98 101 35 91 58 166 122 221 67
163 91 104 153 105 216 104 146 82 101
5 0 0 0 0 0 0 0 0 0
239 189 205 188 196 274 270 268 303 168
103 44.8%
126 55.0%
tl 0.2%
230 +44
0 0%
627 519 423 431 449 411 357 446 455 450 527 491
925 f 466
40.0 38.0 33.4 33.4 33.0 34.4 30.4 39.2 42.3 33.2 37.1 31.4
+ + k k k f + + + + + k
11 10 9 10 9 8 8 8 9 10 11 11
35.5 + 10
Counts and Perikmyal
Sizes
6
L3
-
No.
of Normal
f + f +k + + + + + + +
How accurate is the estimate of 10,500 DRG neurons for the rat sciatic nerve? For reasons listed below this mean value may be an underestimate of the true count by as much as 3-5%. Perikaryal sizes are underestimated by no more than 3-6%. There are six potential sources of error (or variability) that could theoretically introduce significant distor-
TABLE Distribution
1219 1057 816 769 831 910 711 1105 1161 819 969 759
Diameter
from muscles are fundamentally different in their axonal transport properties than those from the skin, the conclusion that may be drawn from Fig. 7 is that the number of unmyelinated afferent fibers supplying rat muscle must be relatively modest (2, 45, 65). The large numbers of unmyelinated axons reported in muscle nerves (61) would then appear to be primarily from postganglionic sympathetic neurons rather than from DRG neurons (10,42,51).
pm. Below this limit pure cutaneous nerves show an overwhelming preponderance, more than two-thirds (69%), of small diameter DRG neurons. In mixed nerves, with about 20% of their afferents from muscle, the proportion of neurons smaller than 29 pm dropped to nearly half (55%). Finally, in pure muscle nerves, the proportion of neurons smaller than 29 pm was only about 25% of the total. Cutaneous nerves and muscle nerves differ fundamentally in the size distributions of their DRG neurons. While there are exceptions (40), it is widely assumed that the vast majority of the perikarya of the slowly conducting, unmyelinated, primary afferent fibers belong to the small “B’‘-type DRG neurons that have perikaryal dimensions smaller than 29-30 pm (22,27,39-41, 63,64). Cutaneous nerves in the rat have large numbers of unmyelinated axons most of which presumably derive from small DRG neurons (5,9). Unless small axons
Segmental
Area
Sizes Area 667 1113 1021 1257 1091 672 794 797 765 819
-+ + + k f + k + f. f
Diameter 488 455 390 497 489 415 444 427 485 607
900 + 470
28.8 40.1 38.0 42.0 39.4 31.7 34.3 33.0 32.0 33.0
+ 11 + 8 + 7 rt 9 + 9 AZ 10 + 10 f 9 + 10 + 12
35.2 f 10
RAT
DRG
TABLE Total Distribution Nerve
L4
0 (0) 0 (0) 0 (0) 48 (1)
65 (2) 10 (1)
Actual Count % of total
123 1.2%
4 75 103 3234 2133 848
L5 (4) (5) (45) (68) (79) (96)
6397 61.8%
tions in the size representations of cell bodies and numbers of DRG neurons. These potential sources of error, not listed in the order of importance, are: (i) spurious labeling due to uncontrolled spread of the tracer substances; (ii) errors in reconstruction and data processing; (iii) incomplete labeling of a population of neurons whose axons were exposed to the tracer substances; (iv) inaccuracies in the split-cell error correction, (v) duplicate counting of DRG neurons with bifurcating axons that project into separate branches of the sciatic, and (vi) natural variability in numbers of neurons contribut-
------FEPOlEAL TIBIAL
-.-.-.-.-.-.-. Ala ‘1ER”ES
MC A,40
7
of Sciatic Afferent Neurons According to Segmental Level and Tributary L3
Med. gastroc. Sural LG and soleus Tibia1 Peroneal Lateral sural
97
NEURONS
LiGSOL
P‘ERVES
LATERAL SURAL AND SURN NERb!S
FIG. 7. Normalized histograms of DRG neuron populations of cutaneous, muscle, and mixed nerves. The diameter profiles of six pooled cutaneous nerves, three each from Tables 1 and 2 for the sural and lateral sural nerves. These nerves contain few (0%) muscle afferents. The histogram profile for typical mixed nerves, representing pooled cases of three peroneal and three tibia1 nerves, has a larger mean diameter; roughly 20% of its afferents innervate muscle. In the case of muscle afferents, three cases each from Tables 5 and 6, all of which innervate muscle; the mean size is the largest. The proportion of small neurons (t29 mm in diameter) in muscle nerves is greatly reduced in comparison to mixed and pure cutaneous nerves.
103 1564 126 1466 501 28
L6 (92) (93) (55) (31) (19) (3)
3788 36.6%
4
(4)
36 (2) 0.5 0 0 0
(tl) (0) (0) (0)
41 0.4%
Branch Total neurons (% of total) 111 1675 230 4748 2699 886
(1.1) (16.2) (2.2) (45.9) (26.1) (8.5)
10,349 100%
ing to a given tributary branch due to developmental or aging factors. (i) Unintentional or spurious labeling of neurons, other than those whose cut axons were exposed to the tracer solutions, could produce excess numbers of labeled profiles leading to an overestimate in the corrected numbers of neurons. The wax reservoir technique of labeling cut axons was developed specifically to render insignificant the incidence of such labeling errors (71,72). It is difficult to know with certainty from DRG material alone whether spurious labeling is present or not. Fortunately, with the exception of the lateral sural nerve, all branches of the sciatic used in this study also contained axons of motoneurons. If weakly labeled motoneurons had occurred in inappropriate ventral horn locations for the nerve labeled, spread of the labeling solution was assumed to have occurred. Such preparations were discarded. By thorough washing of the nerve ends and surgical wounds at the time of closure, and strict adherence to labeling criteria of motoneurons, spurious labeling of motoneurons was usually absent, or so limited as to be insignificant (71). Under these conditions it was assumed that spurious labeling among DRG neurons would be no greater than that observed for motoneurons. As a source of error in the present study, spurious labeling can be excluded. (ii) Errors in counting due to tissue reconstruction and data processing are inevitable when dealing with large quantities of numerical data as in the present study. The most common source of error in this category was the oversight of some labeled profiles in the tissue slices and subsequent failure to plot them on paper with the aid of the drawing tube. Such an error would lead to underestimation of neuron counts. This was circumvented by using a standard spot-check in about 25% of the sections to confirm that all labeled profiles in the sections were correctly transferred to paper in both numbers and sizes. Furthermore, every section for each DRG was double-checked to verify that it had a corresponding drawing and that the data for
98
SWETT
each had been appropriately entered into a spreadsheet file. Errors were caught and corrected in the raw data of several preparations in the early stages of the study but they seldom exceeded l-Z% of the profiles. The accuracy limit in drawing of the profiles too large or too small was probably in the range of +1.5-2 pm; distortions produced by this type of error are unknown but probably self-canceling for practical purposes. Error check protocols were also embedded in the spreadsheets to trap entry errors which could be flagged for editing before fragment correction procedures were applied for estimating true neuron counts. Error checks and corrections consumed about 15% of the total effort required to complete each reconstruction. Errors of omission eventually became rare or absent. No reconstruction was attempted of any nerve preparation unless all sections of every ganglion, especially DRGs L4 and L5, were captured and properly processed. Sometimes, if only one section was known to be missing from an L3 or L6 DRG, reconstructions were carried out as the loss of a section in these ganglia would not have had any impact on the total profile count. Thus, while the process of drawing, measuring, and inputting labeled profiles all introduced unavoidable errors and distortions, these proved to be negligible and can be discounted as having any significant impact on the results. (iii) Incomplete labeling of a neuron population is probably the most serious potential source of error in the study. This error is difficult to identify, circumvent, correct, or quantify. Even though nerve labeling procedures were geared to produce maximal retrograde filling and density of reaction products, the smallest DRG neurons associated with unmyelinated axons did not seem to be labeled as reliably as the large DRG neurons. Because motoneuron counts labeled with the same technique reached “ceiling” levels with small standard deviations (71), it was assumed that all myelinated DRG axons would transport and label their respective perikarya with comparable efficiency. As Sittiracha and McLachlan (59) pointed out, small unmyelinated axons may be unable to take up and transport enough tracer substance to be detected in some cases. A hint of this, they pointed out, may be gauged from cases in which lower mean cell counts are coupled with larger mean perikaryal diameters. There are a few HRP-labeled cases in Tables l-4 that show this type of pattern. They were admitted into the database because they could not be excluded by the criteria for labeling quality. It cannot be excluded that these cases reflect accurately the true compositions of these nerves, but such variability as seen in these tables must be attributable to other factors as well. One such factor, for example, may be attributed to the use of HRP or WGA-HRP alone as the labeling agent. It was reported earlier that HRP alone does not label unmyelinated axons as well as myelinated axons. Conversely, WGA-HRP appeared to label unmyelin-
7” I51
AL. a-
ated axons better than myelinated ones (66). If a mixture of these tracers is not used there is a risk that some neurons may escape labeling. For example, 2 HRP-labeled MG nerve cases (Table 5) showed fewer than 100 neurons and they also showed some of the largest mean perikaryal sizes. This suggests that a few small DRG neurons may have escaped labeling. The same phenomenon appears in the data for sural nerve case R80. In five peroneal cases (12, W3, Wll, W22, X2) low cell counts were again coupled with normal or larger than normal mean perikaryal ~mensions suggesting an underrepresentation of its small DRG neuron components. In many preparations there was an impression that a natural “ceiling” had been reached and that labeling of the available population had been essentially complete. These have large counts and medium to smaller mean cell sizes. If the few low-count, large-sized cases were excluded from Tables l-4, it is evident that the mean cell counts in each nerve would be increased by roughly 3-5%. There is another type of potential error in labeling that could also lead to an underestimate in neuron counts. If postlabeling survival times are not properly controlled, some elements will fail to be labeled. When postlabeling survival times are less than 24 h, large neurons may be incompletely labeled. Conversely, small neurons may be underrepresented if postlabeling survival times extend beyond 72-96 h (l&62). Such differential labeling has been claimed for motoneurons as well (47). This source of error does not apply to the present study. All experiments involved labeling for 4872 h when cell counts appeared to be maximal and stable. (iv) Another potential source of unavoidable error lies in the use of a mathematical “fragment correction” factor to cancel out the split-cell error. It is nearly impossible to arrive at any accurate estimate of DRG neuron counts without the use of one, but, as correctly pointed out by Schmalbruch (58), the use or misuse of such a tool can introduce errors of enormous proportions. It is probably one of the major causes underlying the lack of agreement in the literature over the number of DRG neurons or axons related to a given nerve. It became essential for the present study to develop a mathematical correction factor with a prouen margin of accuracy. A parallel series of experiments were undertaken to perfect an iterative mathematical technique for correction of split-cell error originally developed by Hendry (24) and further improved by others (55, 60). The procedure used in the present study, described in detail elsewhere (74), can reconstruct computer simulated sectioned populations of spherical “cells,,’ similar in numbers and size to those seen in Fig. 4, with 0% error in number and size. When applied to real motoneuron populations, whose perikaryal tend to be elliptical in shape, the result was a mean underestimate of true neu-
99
RAT DRG NEURONS ron numbers by 1.4 f 3.5%. When the total observed count in Table 7 is adjusted for this mean correction error, the total DRG neuron count is close to 10,500. Mean perikaryal sizes were reduced by about 2-3% due to the slight tendency of the correction procedure to underestimate the numbers of large cells and overestimate the numbers of small ones. (v) Another potential source of error lies in the knowledge that individual DRG neurons may have more than one axon projecting distally (28,37,43). It has been claimed that these dichotomizing axons project in large numbers into different nerve branches to innervate even widely separated peripheral structures (4, 52-54, 73). If such branching of DRG axons existed to any significant extent among the tributary branches of the sciatic nerve, it would produce an overestimate in DRG neuron numbers in the present work due to the nervelabeling strategy used. To control for this possibility double-labeling experiments were performed on pairs of sciatic nerve branches with a sensitive HRP and Fluoro-Gold combination (71). Although still in progress, these studies show that double-labeled neurons exist but they are so few in number that the potential error introduced by this would be tl% and of no significance to this study. This observation supports the conclusion of others; dichotomizing primary afferent axons exist but so few of them occupy adjacent branches of the sciatic that they are a doubtful source of error in the present study (5,13,X, 16). How can this be reconciled with reports that many primary afferent axons dichotomize (37) and that many of them are unmyelinated (26)? The most logical explanations are that they (i) do not extend into the periphery far enough to be labeled (see (26)) or (ii) remain in such close proximity to each other that they normally occupy the same labeled tributary branch. Axonal branching can be safely discounted as a significant source of error in the present study. (vi) Variability in the DRG counts for individual branches of the sciatic can be attributed in part to factors related to development and aging. It has been claimed that DRG neurons of the rat do not remain stable in numbers or perikaryal dimensions during aging; they reportedly increase in number and decrease in size with increasing age (14,15,23). If the animals in the present study had widely differing ages, the variability seen in the DRG counts could be partly attributed to such an age-related factor. This cannot apply to the present work. The mean DRG count for the sciatic nerve young to middle-aged rats in the present study was significantly higher than the count reported for old rats (14, 15). This observation, coupled with data from the cat (3), suggests that neurogenesis does not occur among DRG neurons as a normal feature of aging as has been claimed by others (14,15). Over 90% of the experiments were carried out on young, sexually mature female rats weighing 120-240 g. Less than 6 months dif-
ference, in the extreme, separated them in age. Age-related neurogenesis of DRG neurons would not have had any effect on the total counts of neurons. In any event, there is no evidence that such neurons could extend their axons far enough into the periphery to be labeled (14). Only a few animals in the study were male and 6 months older than the oldest female. These cases did not differ noticeably from the others so there was nothing to suggest that the variability seen could be attributed to sex or age. Variability in DRG counts for sciatic tributary branches could be partly related to factors governing pathfinding of growth cones of DRG axons which determine the boundaries andinnervation densities ofperiphera1 receptive fields in early development. Motoneurons and DRG neurons may differ in this respect. The mean variability of rat motoneurons is about +6% (71), nearly a quarter of that for DRG neurons (22%) with a range of 14% for the MG nerve to 33% for the lateral sural (Tables l-6). While muscle innervation is by nature compartmentalized, the components destined to innervate a cutaneous receptive field probably vary more widely from one animal to another due to factors that determine the branching patterns of nerve trunks during development and the number of neurons that succumb to normal, massive cell death near the time of birth (11, 21). The lateral sural nerve has the curious property of nearly doubling the number of DRG neurons from one animal to another. The small version contains a mean of about 630 DRG neurons while the larger one contains about 1140 DRG neurons, an apparent incremental difference of nearly 500 neurons (Table 2). The total surface area of their respective cutaneous receptive fields was not measured but would presumably differ in size accordingly. From its population profile (Fig. 2), the vast majority of its neurons are small and presumably supply unmyelinated axons. Assuming that homologous skin fields have a fairly constant innervation density from one animal to the next, the small lateral sural, in comparison with the large, must have relinquished 500 DRG neurons to one or more of the nerves supplying adjacent skin areas such as the saphenous or sural(72). Although this point was not discussed by Sittiracha and McLachlan (59), examination of their data in Table 1 for the rat MG and sural nerves shows comparable degrees of variability of DRG counts found in the present study. In view of this, it is interesting that axonal counts of the lateral sural by Schmalbruch (56) do not show the degree of variability reported here. Sciatic Nerve DRG Neurons-Accuracy
in Numbers
The results of the present study reveal that the normal rat sciatic nerve contains approximately 10,500 DRG neurons, the total value of Table 7 being increased
100
SWETT
by 1.4% to offset the known error of the fragment correction procedure. Assuming that this is an underestimate by 35% due to incomplete labeling in some cases, the true, mean count should be somewhere in the range of 10,500 and 11,000. This number is greater than that reported by investigators who used thinner sections, nucleolar counts, section sampling, and correction procedures of unproven accuracy to obtain their results (6, 14, 15). This number, however, is noticeably smaller than what can be calculated from the results of others who counted DRG neurons retrogradely labeled from tributary branches of the sciatic in a manner similar to the one reported here. Baron et al. (5) counted a total of 9900 DRG neurons for the tibial, sural, and muscle nerves, a number that is 30% greater than the sum of the same nerves listed in Table 7. Assuming that they would have reported a comparable difference for the peroneal and lateral sural branches, their sciatic nerve would presumably be supplied by a mean number of about 14,500 DRG neurons. The disparity between this number and the 10,500 figure reported in this work can be mostly explained by the different mathematical correction methods used in the two studies to compensate for split-cell error. Baron et al. (5) counted every labeled profile in every section, as done in this study, but their counts were made from sections 30-brn thick and reduced by 30% to correct for split-cell error. This resulted in an inflated cell count by at least 20% in their work because the mean diameter of rat sciatic DRG neurons is close to 30 pm (Tables l-4), the same as their section thickness. Nearly every cell on the average would have been cut in two to produce two labeled profiles; all neurons larger than 34 pm, a significant proportion of the total, would have produced three profiles. Their raw profile counts should have been reduced by at least 50% instead of 30%. If the mean, raw profile counts of six tibia1 nerve cases (containing more than 6000 profiles) from Table 1 of Baron et al. (5) are corrected according to the procedures used in the present study, i.e., by 50%, their count for tibia1 DRG neurons would be 3950, or 1 standard deviation below the results reported in Table 4. The latter difference could also be partly explained by the fact that Baron et al. (5) labeled the tibia1 1 cm above the ankle instead of near the popliteal fossa as done in this study. There would be more afferent neurons present at the more proximal labeling site. The data from Baron et al. (5) are compatible with our results when raw profiles are “fragment corrected” (74). Using the same rationale and correcting the mean count of the four sural casesfrom Table 1 of Baron et al. (5) by 50%, the count would be 2220 in contrast to our estimate of 1675 neurons (Table l), a difference of about 2 standard deviations. However, a better match for our sural count was found with a similar calculation
l!J'I‘AL. -- ‘-
from the data of Sittiracha and McLachlan (59) in which they must have had a raw profile count of about 3500. When corrected by 50% according to our requirements, their data would produce a count of about 1750 DRG, a value close to the mean total shown in Table 1. An almost perfect match is also found in recalculating the four largest muscle nerve cases in Table 1 of Baron et al. (5). Because of the larger mean size of these neurons, their raw profile counts must be reduced by at least 55%. The result is an recalculated estimate of 342 DRG neurons, a value nearly identical to the sum of the MG and LG-Sol nerves in Tables 5 and 6. These examples collectively suggest that the completeness of retrograde labeling of DRG neurons in the present study was comparable to that of others (5, 59) and that the estimates for neuron counts differ because of the alternate strategies used for correcting split-cell error (5,59). Given the evidence that the corrected data in Tables l-6 show relatively stable “ceiling” counts, the data from these other investigators would seem to reinforce our contention that the data summarized in Table 7 is very close to the true population count. There is some additional, indirect evidence from axonal counts that the data in Tables l-7 may also accurately reflect the sciatic DRG neuron population. Take, for example, the common peroneal nerve. The axon counts for this nerve from Jenq et al. (32) and Schmalbruch (56) are similar with a mean of about 1930 myelinated axons and 3900 unmyelinated ones. From the latter it is necessary to subtract 1100 unmyelinated postganglionic sympathetic axons (56) to yield a total of 2800 unmyelinated sensory (afferent) axons. According to our most recent data, there are about 630 motoneurons in the nerve (68,69,71). This corresponds well with the number of myelinated motor axons in the nerve (56). When the motoneuron axons are subtracted from the myelinated group the net result is a total count of 1300 myelinated afferent fibers. The total number of afferent fibers in the peroneal nerve must then be about 4100 axons. If DRG neurons on the average provide 2.3 axons to peripheral nerve (36, 37) then the total DRG count for the peroneal nerve would be close to 1800, significantly below the total estimated in Table 3. However, if only those DRG neurons giving rise to unmyelinated fibers give rise to 2.3 axons, then a total of 2700 DRG neurons should contribute to the common peroneal nerve. Interestingly, this is the same count derived from reconstruction of labeled DRG neurons listed in Table 3. The results of the above comparisons are obviously highly speculative and perhaps merely coincidental. It is instructive then to examine the sural nerve. It contains about 70 motoneurons (51,59,71), almost exactly 1000 myelinated axons, and 3800 unmyelinated axons (30, 32, 49, 51, 56). One study calculated that 28% of all axons in the sural were postganglionic sympathetic neu-
RAT
DRG
rons (56). If this number is correct, the total number of afferent axons, 930 myelinated plus 2460 unmyelinated, would equal 3390. If it is again assumed that all DRG neurons give rise to 2.3 axons, a total of 1470 DRG neurons would be estimated for the sural; if such branching is primarily a property of small DRG neurons then the total sural DRG population would be closer to 2000. These estimates are 1 standard deviation above and below the mean number of sural DRG neurons shown in Table 1. A third example is the MG nerve which has a mean of about 660 axons (30, 32, 51). Muscle nerves are rich in unmyelinated axons (61) and there is evidence that about 50% of all axons in muscle nerves may originate from postganglionic sympathetic neurons, 55% in cat (42), and 44% in rat (5). This leaves about 330 somatic motor and sensory axons in the MG nerve. If one assumes that myelinated axons in muscle nerves remain unbranched until they are intramuscular (7), 140 myelinated motoneuron axons (71) may be subtracted to obtain 190 sensory axons of all types. If rat muscle nerve is similar to cat muscle nerve (7), about three quarters of these (140) will be myelinated afferent axons (30,32, 51). Assuming that the remaining 50 are branched unmyelinated afferent axons, a grand total of about 160 DRG neurons should be associated with the MG nerve. Although the match in Table 7 is not as good as in the preceding two examples, the example nevertheless augments the evidence presented earlier that the numbers of DRG neurons in pure muscle nerves are few and that the numbers of small DRG neurons are very few (Tables 5 and 6; Figs. 5-7). The tibia1 nerve is reported to have about 13,000 axons (32, 56). About 1000 of them are myelinated axons of somatic motoneurons (56, 71). Of the remaining 12,000 axons, 27% of which are presumably from autonomic (sympathetic) postganglionic neurons (56). The total number of sensory axons would therefore equal 8800. Nothing is known about branching of tibia1 DRG neurons, but if it is assumed that there are roughly at least 1000 muscle afferents that do not branch and the remainder give rise to about two axons each, the tibia1 nerve should contain about 4900 DRG neurons, a value remarkably close to that found through retrograde labeling. The comparisons above, while speculative, nevertheless emphasize that the numbers of DRG neurons reported in Table 7 cannot be far from the true numbers of DRG neurons that contribute to the rat sciatic nerve.
able to contribute to the project thanks to the help of the Natural Sciences and Engineering Council of Canada. This work was made possible by a research grant from the National Institutes of Health (NS-25707).
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TORIGOE, Y., C. M. BOURASSA, C. PRAKASH, AND J. E. Swan. 1991. “Fragment correction” of split-cell error for accurate determination of the numbers and size distributions of neuron cell bodies labeled with horseradish peroxidase. J. Comp. Neural., submitted.
75.
YGGE, J. 1984. On the organization of the thoracic spinal glion and nerve in the rat. Exp. Brain Res. 55: 395-401.
76.
YGGE, J. 1989. Neuronal loss in lumbar dorsal root proximal compared to distal sciatic nerve resection: tive study in the rat. Brain Res. 478: 193-195.
77.
YGGE, J. 1989. Central projections of the rat radial nerve investigated with transganglionic degeneration and transganglionic transport of horseradish peroxidase. J. Comp. Neurol. 279: 199211.
78.
YGGE, J. AND H. ALDSKOGIUS. 1984. Intercostal nerve transection and its effects on the dorsal root ganglion. A quantitative study on thoracic ganglion cell numbers and sizes in the rat. Exp. Bruin Res. 55: 402-408.
79.
YIP, H. K., K. M. RICH, P. A. LAMPE, AND E. M. JOHNSON, JR. 1984. Effects of nerve growth factor and its antiserum on the postnatal development and survival after injury of sensory neurons in rat dorsal root ganglia. J. Neurosci. 4: 2986-2992.
80.
ZENKER, W., A. MYSICKA, AND W. NEUHUBER. of the transganglionic movement of horseradish primary afferent sensory neurons. Cell Tissue 489.
gan-
ganglia after a quantita-
1980. Dynamics peroxidase in Res. 207: 479-
NEUROLOGY
114,82-103
(1991)
Sensory Neurons of the Rat Sciatic Nerve JOHN
E. SWETT, YASUHIRO TORIGOE, VINCENT R. ELIE, CHARLES M. BOURASSA,* AND PETER G. MILLER Department
of Anatomy *Department
and Neurobiology, College of Medicine, UCI, Irvine, California 92717; of Psychology, University of Alberta, Edmonton, Alberta, Canada
data suggest that nearly 20% of all DRG neurons in the sciatic nerve supply muscle afferents. The vast majority of the remaining neurons are involved with innervation of the skin. The number of small DRG neurons in pure muscle nerves are few implying that a large proportion of the unmyelinated axons in these nerves are from postganglionic sympathetic neurons. The number of small DRG neurons (t29 pm in diameter) in cutaneous nerves outnumber the large ones by two- to threefold suggesting that neurons with unmyelinated axons dominate. 0 1991 Academic Press, Inc.
Experiments have been undertaken in this laboratory over recent years to accurately determine the numbers and sizes of somatic neurons which contribute to the normal sciatic nerve, at mid-thigh levels, of the adult, albino rat. This article is concerned with the dorsal root ganglion (DRG) neuron population of the sciatic nerve whose cell bodies were identified through retrograde labeling of cut branches of the sciatic with horseradish peroxidase (HRP) and/or its wheat germ conjugate (WGA-HRP). It is essential to understand the neuronal composition of the normal rat sciatic nerve if the consequences of aging, nerve injury, and surgical repair to improve functional regeneration are to be properly evaluated. Neuron counts were determined from camera-lucida paper drawings of all labeled profiles in DRGs L3L6 at 100X magnification. The profiles, obtained by labeling individual branches of the sciatic nerve (sural, lateral sural, tibial, peroneal, medial, and lateral gastrocnemiuslsoleus nerves) were traced from 40-pmthick, serial, frozen sections. The sizes of the perikarya, areas and diameters, were determined by tracing the perimeters of the drawn profiles on a digitizing tablet. The tablet’s output was inputted directly into a specially designed computer spreadsheet which contained a mathematical table for correcting the split-cell error inherent to the sectioning process. Afferents from any given branch of the sciatic normally occupied two to three adjacent ganglia. Sciatic DRG neurons were normally located in lumbar ganglia L3-L6. Nearly 9899% of all sciatic DRG perikarya resided in the L4 and L5 DRGs. The L6 DRG, traditionally regarded as an important contributor to the rat sciatic, contained merely 0.4% of its afferent neurons while the L3 ganglion, frequently overlooked as a contributor, contained 1.2% of the mid-thigh sciatic afferents. The mean size of rat DRG neurons was about 29 pm (550600 pm2). The corrected counts revealed that the normal sciatic nerve (at mid-thigh levels), in rats between 2 and 12 months of age, contained a mean, total DRG neuron population of about 10,500 neurons. This is probably an underestimate by 3-5% of the true number due to occasional unreliable labeling of some of the small DRG neurons. It is estimated that the normal, mean number of sciatic DRG neurons of young to middle-aged rats lies somewhere between 10,500 and 11,000 f 2000. The 0014-4&36/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
INTRODUCTION The rat sciatic nerve, at mid-thigh level, contains axons from about 2000 motoneurons (71). This finding was based on detailed reconstructions of motoneuron cell bodies retrogradely labeled with HRP reaction products from cut branches of the sciatic. The motoneurons resided in discrete compartments in the ventral horn of the spinal cord extending from the rostra1 third of spinal segment L6 into the caudal third of L3 (71). These experiments, and many since then, labeled large numbers of dorsal root ganglion (DRG) neurons (17,70). The labeling technique was perfected to assure that most, if not all, motoneuronal perikarya contributing axons to a given branch of the sciatic would be labeled without also labeling neurons in adjacent branches. Ganglia L3-L6 were routinely collected, sectioned serially at 40 pm, and processed for HRP histochemistry in a manner similar to that used for motoneuron populations. Reconstructions of DRG neuron populations were not attempted unless motoneurons from the same nerve were optimally labeled (for criteria see (71)). Reconstructions of the sciatic DRG neuron populations were undertaken because the rat sciatic nerve is the most widely used peripheral nerve in experimental studies involving nerve injury and regeneration. Despite this, quantitative descriptions of its neuronal populations are conflicting or incomplete. For example, on the basis of retrograde HRP-labeling, a total count of 5760 DRG neurons has been estimated for the tibia1 nerve 82
Inc. reserved.
and
RAT
DRG
(5). The sural nerve is reported to contain 3600 DRG neurons (5); others measured about 2550 DRG neurons (51). The DRG count of 530 has been reported for the medial (MG) and lateral gastrocnemius and soleus (LGSOL) branches of the sciatic (5). Unfortunately, no comparable information is available for the common peroneal and lateral sural branches. If it assumed that the common peroneal has a composition similar to that of the tibia1 nerve and two-thirds its size, a rough estimate of 3600 peroneal DRG neurons would not be unreasonable because it contains 632 motoneurons (71). The lateral sural nerve, being roughly half the size of the sural should have no more than about 1500 DRG neurons. If one assumes that there is little dichotomization of DRG axons into different tributary branches of the sciatic (13), the total DRG neuron population for the mid-thigh sciatic could contain as many as 14,000-15,000 DRG neurons. This estimate represents a maximum limit. If it is assumed, as will be confirmed later, that nearly all sciatic DRG neurons reside in two DRGs, L4 and L5, it is possible to extrapolate lower counts. For example, the L4 and L5 DRGs contain collectively about 23,300 DRG neurons (57). If 54% of these contribute to the sciatic (15) then the total number of DRG neurons should be in the range of 12,600. Using corrected nucleolar counts, it has been estimated that the sciatic of young rats contain about 6300 neurons while old ones contain as many as 9100 (14,15). From the data of Bondok and Sansone (6) the number of sciatic DRG neurons would be in the range of 5800. Axon counts can also be used for estimating numbers of DRG neurons. The sciatic nerve at mid-thigh levels contains between 23,700 and 27,000 axons (31, 56). Of this number 19,000 are thought to originate from DRG neurons (58). If each DRG neuron has on the average 2.3 axons (36), then the number of sciatic DRG neurons should be about 8300. The low value of 5800 DRG neurons or the high value of 14,500 may be correct, but not both. With motoneuron counts varying less than +5-6% among animals (71), it seems unreasonable that the normal DRG ‘neuron populations could vary so widely among animals of the same age group. The true number must lie somewhere between these extremes. The objective of the present study was to reconstruct as accurately as possible the number, ganglion location, and size distributions of the DRG neuron populations of the rat sciatic nerve as revealed by retrograde labeling of their cell bodies with HRP reaction products. It has long been recognized that the cell bodies of primary afferent neurons can be labeled from the periphery by retrograde transport of tracer substances (17, 80). In recent years this approach has been used by a number of investigators to obtain quantitative information about DRG neurons of the cat (8,29,38,42) and of the rat (4, 15, 20, 48-51, 59, 70, 75, 79).
NEURONS
83
One of the sources of variability in estimating numbers of sciatic DRG may be found in the assumption that the L6 DRG contributes significant numbers of neurons to the rat sciatic nerve at mid-thigh levels (5, 15,44,56-59, 76) when it does not. The L3 DRG, with some exceptions (5, 14), is overlooked as a source of sciatic DRG neurons. It contributes only 1.2% of the total, three times that of L6. Perhaps the greatest source of variability derives from the techniques used in counting neurons and the mathematical methods for correcting counts for splitcell error. Sampling, for example, can introduce large distortions in neuron counts because the strategy assumes a uniformity in the spatial packing of the cells being counted. Retrogradely HRP-labeled neurons, especially motoneurons and DRG neurons, are not uniformly distributed spatially. Tests on the error limits introduced by sampling every fifth section or less revealed a maximum error of +90% (74). To obtain the most accurate results, the count should include all labeled elements unless such an approach proves to be thoroughly impractical. When counting objects in tissue sections some mathematical correction of the raw counts is essential to subtract out the number of doubly counted cells (12, 34). Errors of underestimation or overestimation of cell counts will inevitably result (5,74). One can obtain accurate neuron counts and cell size distributions by reconstructing all neurons in a HRP-labeled population as was done with the sciatic motoneurons of the rat (71). However, the methods used for reconstructing motoneuron populations proved to be unworkable when applied to DRG neurons because of the small sizes and large numbers of profiles scattered unevenly across relatively large tissue sections. Attempts to match profiles belonging to the same cell in adjacent sections resulted in a severe underestimate of the actual amount of split-cell error. Attempts to construct an empirically derived correction factor, as others have attempted (5,42,59), were abandoned when it became evident that it resulted in inflated counts. A modified Abercrombie (1) method was also used for awhile (70) but abandoned, not because it underestimated the true neuron numbers by only 5-6%, but because it distorted neuron size distributions (74). Accuracy in numbers and sizes of sciatic DRG neurons was the primary objective of this study. It required a new and rigorous approach to the correction of splitcell error. This resulted in the development of a “fragment correction” procedure, a modified version of the Hendry method (24), that is described in detail elsewhere (74). When tested on neuron populations whose cellular profiles were completely known, this correction method produced a cell count that was on the average accurate to 98.6 + 3.4% of the true, known cell count.
84
SWETT MATERIALS
AND
METHODS
The experiments were conducted on 72 nerve preparations in Sprague-Dawley rats (60 females and 8 males) weighing 120-510 grams. Routinely, one nerve was labeled in each hindlimb. The nerves were exposed surgically under deep anesthesia (ketamine hydrochloride, 100 mg/kg and Rompun, 3 mg/kg). The nerve chosen for any given experiment was one of the following: sural, lateral sural, common peroneal, tibial, MG, and/or LG-SOL. The exposed nerve was prepared by gently stripping all visible blood vessels from the epineurium 2-3 mm on either side of the site of transection. This was done in order to minimize the incidence bleeding at the exposed nerve end that would impede axonal uptake of the labeling solution. The nerve was then sectioned and immediately immersed in a soft, low-melting-point wax while suspending the proximal cut nerve end by its epineurial sheath. Using a microsyringe, filled with distilled water, a droplet of water was placed over the nerve end just visible beneath surface of the wax. The wax is then excavated so that the water flooded the nerve end before it could be exposed to the air. More wax was added to build the depth of the reservoir over the nerve end to accommodate l-2 ~1 of labeling solution. The distilled water is left in place for about 10 min, removed by damp wick, and replaced by a 35-40% solution of HRP (Boehringer, Grad l), a 3-5% aqueous solution of its wheat-germ conjugate (WGA-HRP; Sigma), or, most commonly, a mixture of the two (MIX). The reservoirs were capped by wax to prevent drying. A mixture of HRP and WGA-HRP was employed in most preparations because of suggestive evidence that the number of small DRG neurons was below normal when labeled only with HRP. Conversely, the number of large DRG neurons appeared to be underrepresented when labeled only with WGA-HRP (66). Although this was not a consistent phenomenon, it occurred often enough to warrant use of a mixture of the two tracers to facilitate labeling of the maximum number of DRG neurons. The animals were maintained in a warm environment with additional supplements of anesthetic and dextrose-saline (ip) for 5-6 h. Removal of the label required three separate washings of the nerve ends by distilled water before the wax was gently removed from the wound in a manner to avoid stretching of the nerve. The wound was then flooded by warmed dextrose-saline solution for several minutes before closing it in layers with sutures. These steps were taken to avoid spurious labeling of peripheral neurons that belonged to nerves other than the one labeled. Spurious labeling was determined primarily by evaluating the positions of retrogradely labeled motoneuronal perikarya and the quality of their labeling (71). If there had been spread of the label, this
ET AL.
was disclosed by the presence of faintly labeled motoneurons in positions inappropriate for the nerve that had been labeled. If labeling criteria were satisfied the DRGs L2 through L6 were reconstructed. When it became apparent that the L2 DRGs never contained labeled profiles after labeling any of the sciatic branches at mid-thigh levels or beyond, they were eventually discarded. The ganglia were sectioned horizontally in 40-pmthick sections and the sections were reacted with TMB according to a slightly modified protocol of Mesulam (46, 71). All sections were retained and mounted in serial order on glass slides. The perimeters of all labeled profiles in all sections were traced with a 0.3-mm drafting pencil with the outer edge of the line coinciding with the perimeters of the profiles. This was done with the aid of a drawing tube at 100X magnification. All profiles were then numbered sequentially to assure that no profile was measured twice. The profiles were then traced on a digitizing tablet (Donsanto) and the numerical values for their areas were entered into a specially designed spreadsheet program (Symphony, Lotus Development Corp.). A separate computer file was maintained for each DRG so that it would be possible to determine the relative contributions of each nerve to each DRG. Another special spreadsheet file was created in which all ganglia in a given animal could be combined for the right and left sides, respectively. These files were used for developing XY-histogram plots of a nerve’s total DRG perikaryal size distributions. Because accuracy in neuron numbers was the highest priority of the study, the cell counts were based on the strategy of measuring the largest possible sample of labeled profiles. The least error will result if all profiles are measured. Because labeled DRG neurons are not distributed evenly throughout the tissue sections, sampling of all profiles in every second, third, or fifth section was rejected in favor of collecting data from all profiles in all sections to achieve maximum accuracy. With alternate sections the mean potential error was about +6-7% with a maximum potential error of +17%. With every third section the maximum potential error increased to *49%. When sampling was applied to every fifth or sixth section the maximum limit of error escalated to +89%. By counting every profile the mean error could be theoretically reduced to -1.4 + 3.5% with a maximum potential error of about 6-7% (74). The data from the digitizing tablet were imported into a 286 AT personal computer through a serial port using the Symphony (Lotus Development Corp.) communication protocol to directly insert morphometric data of the DRG neurons directly into worksheet columns. The worksheet contained formulae and macros for error checking, statistical functions, graphics, and the fragment correction table for split-cell error. The worksheet was errors could be flagged -~- desizned -~--~_~~~ so that innuttine: A -I
85
RAT DRG NEURONS and edited. The raw data consisted of a profile’s identification number, area, and diameter. The diameters were then sorted into l-pm-diameter bins. Area measurements were sorted into bins corresponding to the limits of the diameter bins. For illustrative and mathematical purposes the histograms were plotted with the data sorted to the centers of the bins; e.g., the 30-pm-diameter bin contains all corrected profiles representing neurons with diameters ranging from 29.51 to 30.50 pm. Areas were plotted linearly on the abscissa. The area and diameter histograms were plotted for all cases because the two sets of data emphasized different features of the neuron populations. The diameter histograms were most useful in visualizing the larger DRG neurons while the area histograms emphasize the dominance in numbers of the small DRG neurons, especially found in cutaneous and mixed nerves. To correct for split-cell error from tissue sectioning, an iterative procedure, the “fragment correction” procedure, was applied to the binned raw data of area measurements of all labeled profiles from all sections (74). This procedure is a refinement of a method described by others (24,55,60) and is capable of correcting split-cell error of spherically shaped, synthesized “neurons” with zero error (74). Briefly, this correction procedure estimates the number and size of labeled profiles in smaller bins that would be created by sectioning cells of larger diameters and then subtracts these profiles from their respective bins. The total count remaining should be equal on the average 98.6% of the true population (standard deviation = ?3.5%) providing that all neurons have been labeled and all labeled profiles were included in the initial uncorrected collection of profiles. Any profiles smaller than 10 pm in diameter were ignored because no DRG neurons of the size exist in the rat. A critical thickness limit was also incorporated into the mathematical correction procedure. It was assumed that any fragment of a labeled cell 2.0 pm or less in thickness would not be visible in the tissues and therefore not captured in the collection of sorted profiles to be mathematically corrected. The value for the critical thickness limit is important because, if it is inaccurately estimated, it could lead to a significant over- or underestimate in the number of neurons in the smaller bin sizes. This thickness limit was determined empirically using darkfield microscopy to resolve HRP-labeled neurons and curvefitting adjustments of the correction factor on known populations of neurons and their fragments (74). There are three assumptions inherent in the use of the fragment correction factor: (i) the largest neuron must be in the largest (top) bin (e.g., some of the profiles may be equal in size to the largest neuron), (ii) all neurons are assumed to be spherical although most are ellipsoidal in shape, and (iii) all measured profiles 10 pm or larger in all ganglia on one side of the animal repre-
sent the complete population of labeled DRG neurons many of which were cut into two or more fragments. The first condition is an assumption that is intuitively true if the neuronal population is pseudo-randomly arranged. In addition, given enough neurons (in the hundreds or thousands), there is a likelihood that most neurons would be cut in such a way as to show profiles representative of the neurons’ largest dimensions. The second condition, when tested on real, reconstructed motoneuron populations, was not met, but the error introduced by this resulted in only a small underestimate of true numbers (74). An addition of 1.4% of the corrected count would theoretically offset most of this error. The third condition did not appear to be consistently met. Some preparations, presumably typically well-labeled ones, exhibited high neuron counts with medium to small mean perikaryal sizes. Others, however, seemed to have abnormally low counts coupled with larger than average diameters, indicating that an unknown proportion of the smaller DRG neurons may have escaped labeling. Some of these preparations were included in the data presented in this article because there was no obvious criteria for excluding them. As a result the DRG neuron counts reported here will be an underestimate of the true numbers by approximately 3-5%. Cell counts in all the tables are corrected for split-cell error. RESULTS The DRG neuron populations are described individually for each labeled branch of the sciatic nerve.
The Sural Nerve (Fig. 1, Table 1) The vast majority of the sural nerve’s somatic components, about 96%, is composed of axons of DRG neurons which innervate the lateral, distal cutaneous aspect of the animal’s leg and foot (72). The remaining 4% is composed of motoneurons (71). The total mean DRG population of the sural nerve consists of 1675 f 316 neurons, 93% of which are located in the L5 DRG (Table 1). The L4 DRG contained a limited number of sural DRG neurons but this amounted to twice the total found in L6. The sural nerve’s contributions to L6 were frequently absent. Figure 1 illustrates DRG perikaryal size distributions of three sural nerve preparations listed in Table 1. The case in Fig. 1A was chosen as a typical example in terms of numbers and perikaryal dimensions. Figure 1B shows the case with the smallest mean for perikaryal size while Fig. 1C illustrates the case with the largest mean cell size. The most striking feature of the sural DRG perikaryal size distributions is that they are strongly skewed toward neurons of small sizes. The sural DRG neurons populations were among the smallest in mean perikar-
86
SWETT
ET
AL.
A 150 -I
1
0
B
0
CASE
0.4
0.8 1.2 1.6 AREA tjfm2) x
2 1000
R84
2.4
2.6 D~E~R
(mm)
150
CASE 1598
0
0
0.4
0.8
1.2
R94
CELLS
1.6
2
2.4
2.8
0
40
20 DIAMETER
CASE
1 0 0
1522
0.4
0.8 AREA
1.2 (ii&
1.6 x
60
(Cm)
B5 CELLS
2 2.4 1000
2.8 DLAMETER @m)
FIG. 1. DRG population profiles of the sural nerve. (A) A case with a mean perikaryal size close to the mean value (B) The case in Table (1) with perikarya of smallest mean size. (C) The case in Table 1 showing the largest perikaryal
yal dimension with a mean diameter of 26.5 pm (mean area = 520 pm2). The small neurons, those smaller than 29-30 pm, outnumbered the large by nearly 2 to 1 as may be seen in the population histograms of Figs. 1A and 1B.
for all cases in Table dimensions.
1.
The Lateral Sural Nerve (Fig. 2, Table 2) At mid-thigh levels the sciatic nerve gives off a small branch, the lateral sural nerve, which penetrates the biceps femoris to supply the skin region immediately
87
RAT DRG NEURONS TABLE Segmental Dist~bution
1
of Normal Sural DRG Neurons and Their Sizes
Animal
Label
L3
L4
L5
L6
Total
B3
MIX MIX HRP
-
52 16 213 16 4 58 26 151 110 84
1271 1505 1497 2322 1369 114% 1956 1479 1135 2161
0 1 0 0 173 277 0 0 1 12
1323 1522 1710 2338 1546 1483 1982 1630 1246 2257
96 120
1425 1478
0 0
1521 159%
1586
0
1612
36 2.1%
1675 i316
B5 H30 H31 H43 H44 H45 H49 R80 R83 R%4 R94
R95 N=
13
HRP
-
MIX MIX MIX
-
MIX
-
HRP HRP HRP HRP HRP
-
Mean total No. Mean %
0 0%
26
75 4.5%
1564 93.4%
proximal to that supplied by the sural nerve on the lateral aspect of the thigh (72). The lateral sural is a pure cutaneous nerve with a mean total of 886 DRG neurons nearly 96% of which are located in the L4 DRG. In the six preparations listed in Table 2 no ventral horn motoneurons were observed in the spinal cord after nerve labeling. This is the only major tributary branch of the main sciatic trunk to be so composed. The lateral sural DRG neurons also revealed population size distributions resembling sural DRG neurons with a mean size of 26 pm (480 pm’). A case representing values close to the mean perikaryal size for the lateral sural DRG neurons is shown in Fig. 2A. The contribution of small DRG neurons is again the dominant feature of these neurons. Figure 2B illustrates the case with the smallest. mean perikaryal size and Fig. 2C shows the case with the largest mean perikaryal size. An unusual feature of the lateral sural nerve, not clearly observed with any of the other tributary branches of the sciatic, is its tendency to show a quantal shift in size and nearly doubling its complement of DRG neurons from one animal to the next. This size difference in diameter is even detectable to the naked eye among preparations. Some cases contained about 570720 DRG neurons while others showed nearly double that number, 1090-1230. Although the sural nerve contains 70 motoneurons and perhaps an equal number of DRG neurons supplying afferent axons to muscle, its cutaneous components far outnumber its muscle components. For purposes of comparison with other tributary branches of the sciatic, the sural and lateral sural nerves serve as good examples of cutaneous nerves. The prominent group of small DRG neurons found in the perikaryal size histograms shown in Figs. 1 and 2 show distinct similarities.
Area 550 635 518 568 620 454 44% 474 639 438 520 430 468
+ + * f + + + I!z rt + k + k
365 413 355 372 429 333 317 371 51% 324 453 350 324
520 k 379
Diameter 28.6 30.0 26.8 29.1 29.4 25.5 25.3 25.6 28.3 23.6 25.6 23.1 24.2
+ ?I f +f + k ++ + + k ?
9 10 9 9 10 9 8 9 12 8 11 9 8
26.5 -c 9
The Peroneal Nerve (Fig. 3, Table 3)
The common peroneal nerve is always the second largest branch of the sciatic. It contains about one-third of all the sciatic motoneurons found at mid-thigh levels (71). The peroneal nerve contained axons from 2699 +: 557 DRG neurons. The L4 DRG was also the preferred location for 79% of them. The area and diameter distributions of this population resembles the sural nerve in being heavily skewed toward smaller-sized neurons, but the mean perikaryal size of 28.7 pm (Table 3) was nearly 3 pm larger than the cutaneous nerves (Tables 1 and 2) because the peroneal nerves contained a greater proportion of large diameter DRG neurons than the sural nerve (Fig. 3). This can be seen particularly well in Figs. 3A and 3B where the neurons larger than 700 pm’, or ~27 pm in diameter, are noticeably greater in number than in Figs. 1A and 2A. Figure 3C shows the extreme case in which the peroneal DRG neurons were unusually large. The Tibia1 Nerve
(Fig. 4, Table 4)
The tibia1 nerve is the largest tributary branch of the sciatic trunk; it contains the axons of nearly half of all the ventral horn motoneurons found in the sciatic at mid-thigh levels (71). It also contains the largest number of DRG neurons, 4748 4 800 (Table 4) accounting for about 45% of all sciatic DRG neurons (Table 7). The typical population profile of the tibia1 nerve (Fig. 4A) is essentially identical to that of the peroneal nerve (Fig. 3A) in perikaryal dimensions although it contains nearly 75% more DRG neurons than the peroneal nerve. The L4 and L5 DRGs contained 99% of the tibia1 DRG
88
SWETT
A
ET
AL.
150
CASE T19
z 23
1089
100
CELLS
E B 5 B z
50
0
0
0.4
0.8 1.2 1.6 AREA (pm') x
2 1000
2.4
2.6
20
0
40 DIAMETER
60
(pm)
CASE T36 1102
CELIS
0 0
C
AREA @rn’)
150
20
1000
x
40 (ym)
60
DIAMETER
40 (jun)
60
DIAMETER
7
CASE Ti' z E
719
3 100 2
CELLS
8 3B 50 ii 0
FIG. 2. cases in Table
0
0.4
0.8 1.2 1.6 AREA (pm') x
DRG population profiles 2. (B) The case in Table
2 1000
of the lateral sural 2 with the smallest
2.4
2.8
0
20
nerve. (A) A case with mean perikaryal dimensions close to the mean mean perikaryal size. (C) The case in Table 2 with the largest perikaryal
neurons with the L4 DRG containing more than twice the number of tibia1 neurons than the L5 DRG. The peroneal and tibia1 nerves are mixed nerves. They collectively account for about 81% of the sciatic motoneurons (71) and 70% of the DRG neurons (Table 7). The peroneal nerve contains about 4.3 DRG neurons
value for all dimensions.
for each somatic motoneuron and the tibia1 nerve contains about 4.7 DRG neurons/motoneuron.
Muscle Nerve Branches (Figs. 5 and 6; Tables 5 and 6) Two small tributary branches of the sciatic nerve leave the main trunk of the sciatic near the nerve’s ma-
RAT
DRG
89
NEURONS
TABLE
2
Segmental Distribution of Normal Lateral Sural DRG Neurons and Their Sizes Animal H50 H51 T7 T19 T36 T42
N=6
Label
L3
L4
L5
L6
Total
MIX MIX MIX MIX
2 12 8 20
MIX MIX
18
1201 556 678 973 1095 584
30 0 33 96 7 1
0 0 0 0 0 0
1233 568 719 1089 1102 603
404 493 587 438 403 557
Meantotal No. Mean %
10 1.1%
848 95.7%
28 3.2%
0 0%
886 +288
480 CL 346
0
Area k f 4 zk * k
268 423 431 252 300 399
Diameter 23.9 25.3 28.8 26.1 23.7 27.4
+ ? + -rIL +
26.0 -t
8 10 10 7 8 10 9
jor division into its tibia1 and common peroneal branches. The smaller medial one innervates the MG; the larger lateral branch supplies the LG-SOL muscles. The MG nerve (Fig. 5, Table 5), although sometimes appearing nearly the same size as the lateral sural nerve, contains only 111 ~fr 16 DRG neurons, 93% of which are located in the L5 DRG. In only one-third of the cases will DRG neurons from the MG nerve appear in the L6 DRG. This mean number of 111 DRG neurons (Table 5) is only about 20-30 less than the total number of ventral horn motoneurons in the same nerve (71). Mean perikaryal size of MG DRG neurons, 35.5 pm, was significantly larger than that found in any other nerve except for the other muscle nerve, LG-SOL (Table 6). Comparison of Fig. 5 with the preceding figures shows a conspicuous lack of skewing of the populations toward the smaller cell sizes as seen in Figs. l-4. The population histograms, as in Figs. 5A and 5B, tend to be symmetrical and unimodal in shape with DRG neurons 3040 pm in diameter dominating the distributions. The LG-SOL nerve is very similar to the MG nerve in mean size of its DRG neurons (Fig. 6, Table 6) with only slight skewing of the histogram profiles to smaller sized neurons (Fig. 6A and 6B). The mean number of DRG neurons in the LG-SOL nerve (230 + 44) is slightly more than double that found in the MG nerve and its neurons are nearly equally distributed between the L4 and L5 DRGs (Table 7). It is of interest to note that the mean areas and diameters of the DRG neurons of the MG and LG-SOL nerves are nearly identical (Tables 5 and 6). These neurons are significantly larger than any of the previously described DRG neuron populations. Like the MG nerve, the LG-SOL nerve contained relatively few neurons of small sizes (~29-30 Km).
ble 7). However, nearly 99% of the sciatic DRG neurons have their cell bodies located in only two of these DRGs, L4 and L5, with L4 containing almost two-thirds of the total (62%). Of the four DRGs that contributed afferent axons to the sciatic nerve, L6 contributed the least. The L6 DRG accounted for only 0.4% of the sciatic afferents. By comparison, the L3 DRG contributed three times the number of afferent neurons than L6. Although there are exceptions to the rule, every tributary branch of the sciatic nerve, on the average, contained axons from three adjacent DRGs (Table 7). Usually one DRG contained the majority of the afferent perikarya for any given nerve. The nerves supplying the distal skin of the hindleg, the sural, peroneal, and especially the tibia1 nerve which contained about 46% of all sciatic afferents, contained the largest numbers of small DRG neurons. One of the most striking features of the data summary presented in Table 7 is the small numbers of DRG neurons associated with the muscle afferents. The total number of sciatic sensory neurons reported in Table 7, 10,349, represents the count corrected for split-cell error and does not include the additional correction to offset the -1.4% error introduced by the “fragment correction” procedure (74). When this error is corrected the total adjusted DRG count for the rat sciatic nerve equals in round numbers about 10,500 f 2000 DRG neurons. As will be discussed later this number may still underestimate the true mean number of sciatic DRG neurons by 3-5%. The true mean number of DRG neurons probably lies between 10,500 and 11,000 f 2000.
Segmental Distributions and Total Numbers of Sciatic DRG Neurons
Segmental Distribution
The sciatic nerve of the rat receives somatic motor and sensory axons from neurons of the spinal cord and DRGs of four spinal segments, L3, L4, L5, and L6 (Ta-
DISCUSSION of Sciatic Afferents
It is widely assumed that axons of the sciatic nerve of the albino rat originate from spinal segments L4, L5, and L6 (15, 19, 33, 57, 58, 76). Segment L3 must be added to this list because it consistently supplied moto-
90
SWETT
CASE 2635
0 0
0.4
ET
AL.
W6 CELLS
0.6 1.2 1.6 2 2.4 AREA (pm’) x 1000
2.6
0
20 ~IAM~ER
40 (pm)
60
40
60
B300 3
CASE
CJl240 f3 5 E 160
3076
W51 CELLS
0
0 AREA
C 300
em’)
x
2922
ri
120 -
52 2
60-
FIG. 3. DRG all cases in Table dimensions.
DIAMETER
(lm)
-
CASE
0
20
1000
0
0.4
0.6 1.2 1.6 AREA (rm’) x
H19 CELLS
2 2.4 1000
2.6
0
20
40 DIAMETER
60
@ m)
population profiles for the common peroneal nerve. (A) A case with mean perikaryal dimensions close to the mean value for 3 (B) The case in Table 3 with the smallest mean perikaryal size. (C) The case in Table 3 with the largest perikaryal
neurons to the peroneal nerve (71) and received axonal projections from a small number of L3 DRG neurons whose distal processes usually contributed to the lateral
sural and peroneal nerves and often to the tibia1 nerve (Table 7). The small contribution of L3 to the sciatic has been noted before by others (5,15). Although the affer-
RAT
DRG
TABLE Segmental Animal
Label
GlRl GlR4
MIX MIX MIX MIX MIX HRP HRP HRP HRP HRP HRP HRP MIX HRP HRP HRP WGA-HRP WGA-HRP MIX HRP
G2R2 G2R3 G4R4 I2 Rlll
w3 WS
w7 Wll w14 HlQ
w22 w23 W46 w49
w51 Xl
x2
N==
20
“-Dash
Mean Mean
total %
in the L3 column
Distribution
L3 1 21
176 5
266 -
indicates
that
Neurons
L5
L6
1480 2218
881 580
2812 2123 1820
806 752 161 647 270 257 60 676 362 210
2547 2254
382 743 497
2661 2924
269 152
1880
1081 737
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
501 18.6%
0 0%
2775 2775 2407
-
1495
30 2
data were
DRG
L4
2790 1592
65 2.4%
3
Personal
1129 1924 1626
19
No.
of Normal
91
NEURONS
496
1419
2133 79.0% absent.
This
sum represents
ent fiber contributions to the sciatic nerve from the L3 DRG were few, they were more numerous than the contributions from the L6 DRG. Contributions from the latter to the rat sciatic were negligible. In the Sprague-Dawley strain of rats used in the present study (obtained from Charles River or Simonson Laboratories), nearly 99% of all the sciatic DRG neurons were located in the L4 and L5 DRGs. The relative unimportance of the L6 DRG to the sciatic afferent population strongly influences the interpretation of data in which the loss of neurons due to nerve injury is determined from counts of surviving DRG neurons (58, 76). The L3 motoneuron contributions to the sciatic (71) appeared to be slightly greater than the corresponding proportion for DRG neurons, providing weak ad~tional evidence in support of a general pattern that efferents from a given distal structure are staggered rostrally to a small degree in comparison to the segmental level of the afferents that supply it (67). The fact that neurons in DRGs L3-L6 contribute to the sciatic has been recognized for several years (l&20). The relative contributions of each DRG to the sciatic in the material of Devor et al. (15) differed from the data of Table 7 in that about 9% of the sciatic afferents were found in L3 and L6 with the latter containing three times the number of labeled DRG neurons than L3 as if their animals had a more postfixed organization than the ones used in the present study. In keeping with this,
the horizontal
and
Their
Sizes
Total
2362
Area
Diameter
2819 3794
595
667 4 350 Ltr446 6711430
30.8 f 8 28.8 k 11 30.4 f 10
2880 2247 1776 2194 1883 2835 3466
609 It. 391
2158
525+- 374 599F377 652 4408 666f 473 595 + 358 603 f 445 7082 429 490 2373 818~~ 433 597 + 471 572 + 417 515~429 441* 350 395 rt330 5482329 730~~349
28.9 + 26.6 k 31.1 f 30.9 t 29.8 k 28.0 + 27.5 + 31.9 + 26.3 k 33.1 f 28.0 zk 27.4 + 25.6 f 24.4 + 25.4 + 27.7 + 32.0 i
2699”
600+ 398
28.7 + 9
1954 2985 2922
1877 3290
2751 2930 3076 2991
9 9 10 10 11
8 10 10 10
8 11 10 11 9 7
8 7
+557 total.
they also found that the L5 DRG contained the majority of the sciatic DRG neurons while our data indicated L4 as being the dominant one. The reasons for these subtle differences are unknown but could be related to the different strains of albino rats used by the two laboratories or possibly to the difference in counting methods. In most DRGs, particularly those with the most labeled profiles, there was no clear evidence of any somatotopic distribution of cell bodies. This feature has been noted by others (51, 75). Labeled profiles were found scattered randomly everywhere throughout the neuronal compartments of unlabeled, counterstained DRG perikarya. Occasionally, those DRGs which contained a very small number of profiles at the extreme rostra1 or caudal limits of a nerve’s afferent projection boundaries showed a preferential position within the ganglion as been described for visceral afferents (35). These labeled profiles appeared to be spatially confined to roughly one quadrant of the DRG giving tenuous evidence for poorly defined somatotopic localization of perikarya. In unreported double-labeling experiments in this laboratory with the HRP/WGA-HRP mixture and Fluoro-Gold applied separately to pairs of sciatic nerve branches (see (71)), labeled profiles of all sizes appeared to be randomly intermingled spatially in sharp contrast to the high degree of somatotopic localization of their respective motoneuron populations in the spinal cord.
92
SWETT
ET AL.
A 400 , CASE
R112
5633 CELLS
0
6
0
0.4
0.6 1.2 1.6 AREA (pm’) x
2 2.4 iOO0
2.6
0
20 D~AM~ER
40 (pm)
80
400 1
CASE
z” 0300
3874 CELLS
5 22 &ZOO
j
0
c
W31
400
0.4
0.8 1.2 1.6 AREA (pd) x
2 2.4 1000
2.8
20
0
DIAMETER
40 o(m)
60
40 (pm)
60
‘1
CASE $300
-
4321
H13 CELL5
5 E
$200
-
i3 g100 2
-
0
0
0.4
0.8 1.2 1.6 AREA (Il$) x
2 1000
2.4
2.8
0
20 DIAMETER
FIG. 4. DRG population profiles for the tibia1 nerve. (A) A case with mean perikaryal dimensions close to the mean value for all cases in Table 4. (B) The case in Table 4 with the smallest mean perikaryal size. (C) The case in Table 4 with the largest perikaryal dimensions.
RAT
DRG
93
NEURONS
TABLE
4
Segmental Distribution of Normal Tibia1 DRG Neurons and Their Sizes Animal
Label
L3
L4
L5
H13 H14 H29 RlllL R112L w30 w31
MIX MIX MIX HRP HRP HRP HRP
0 54 280
0 0
2136 2778 4326 3836 3821 3186 2552
2185 734 856 1828 1806 1534 1322
48 1.0%
3234 68.1%
1466 30.9%
N=l
Mean Mean
total %
No.
In our experience, maximal cell counts are related to conditions favoring dense perikaryal labeling that usually obscures the nuclei and nucleoli. Emphasis on dense labeling helped to expose maximal numbers of the smallest DRG neurons, which are thought to be more difficult to label retrogradely with HRP than large ones (59).
Differences between Cutaneous and Muscle Afferents The DRG neuron population of the sciatic nerve can be subdivided into two basic groups: (i) neurons supplying skin, and (ii) neurons supplying muscles and joints. Cutaneous DRG neurons far outnumbered those supplying muscle. Afferents supplying other structures such as joints are presumed to be present in relatively small numbers among both types. The MG and LGSOL nerves at the point of labeling contained no joint afferents; the only DRG neurons that would be labeled in those cases would be muscle afferents. These pure muscle nerves collectively contain about 334 f 10 motoneurons ((71) plus recent data) and 341+_ 60 DRG neurons (Figs. 5 and 6; Table 7). There is approximately a one to one ratio between the number of DRG neurons and the number of motoneurons. Somewhat smaller ratios of numbers of DRG neurons to motoneurons has been described by others (51). What numerical ratios exist between muscle afferents and motoneurons in large mixed nerves, such as the tibia1 and peroneal nerves, is unknown, but it would not be unreasonable to assume that the one-to-one ratio observed in the triceps surae group may represent a general pattern. If this hypothesis is correct, there should be a total of approximately 2000 muscle afferents in the distal branches of the sciatic because the documented number of motoneurons in the nerve, including the small y-motoneurons, average about 2000 (71). The proportion of all the sciatic DRG neurons involved with innervating muscle tissue must therefore be about 19% of the total. Ignoring the few engaged in innervating
L6
0 0%
Total
Area 2 k f + + f k
Diameter
4321 3566 5462 5664 5633 4720 3874
160 652 544 572 621 652 536
381 339 383 343 354 490 326
4148 &SO0
620 f 373
32.3 30.5 27.3 28.4 29.2 30.5 28.7
k 8 + 8 XL 9 + 8 + 8 * 12 + 9
29.6 +
9
joints, perhaps t5%, the remaining 76% must be primarily engaged in innervating cutaneous receptive fields (72). For every DRG neuron supplying muscle there are at least four others supplying the skin.
Perikaryal
Size in Relation
to Nerve Type
The DRG neuron populations of cutaneous, mixed, and muscle nerves were fundamentally different in cellsize characteristics one from the other. These differences are illustrated in the normalized plots of Fig. 7. Each plot represents pooled data from six nerves, three nerves from each of the Tables l-4, that were close to the mean perikaryal size for each table. The solid-line plot illustrates the typical DRG neuron population profile of pure cutaneous nerves. It was derived from about 7800 DRG neurons of three sural and three lateral sural nerves listed in Tables 1 and 2 including the cases shown in Figs. IA and 2A and two other cases in each group with similar dimensions. The dashed-line curve is composed in similar fashion of data from six mixed nerves, three cases each from Tables 3 and 4 representing those in Figs. 3A and 4A plus two others in each group of similar dimensions. This curve is the normalized distribution of about 21,000 DRG neurons from the peroneal and tibia1 nerves. The dot-dashed line is the normalized plot of about 1000 neurons from six cases of pure muscle nerves, three each from the MG and LGSOL nerves (Tables 5 and 61, two of which are shown in Figs. 5A and 6A. The maximum and minimum size limits for the DRG neurons is similar for all three nerve types shown in Fig. 7, pure cutaneous, pure muscle, and mixed nerves. This size range does not differ significantly from that described by others (50,51,57,78). DRG perikarya smaller than 13-14 pm proved to be very rare. Perikarya larger than 65 pm were also seldom found, but, within these limits, the population profiles of the DRG neurons of these three nerve types showed consistent differences. Nerves with large cutaneous components revealed his-
94
SWETT
ET
AI,.
A 10
CASE
O,,,,r,,,,,,,,,,, 0 0.4
B
0.8 1.2 AREA Qrn
1.6 ) x
RllOL
2 1000
2.4
2.8
0
20 DIAMETER
40 (rm)
60
10
CASE z” g
140
AREA
(jun’)
x
RllOR CELLS
1000
CASE
DIAMETER
Cm)
II15
0 0
0.4
0.8 1.2 1.6 AREA (gmr ) x
2 1000
2.4
2.8
0
20 DIAMETER
40 (jun)
60
FIG. 5. DRG population profiles of the MG nerve. (A) A case with mean perikaryal dimensions close to the mean value Table 5. (B) The case in Table 5 with the smallest mean perikaryal size. (C) The case in Table 5 with the largest perikaryal Irregularity of the plots is a result of the small numbers of DRG neurons found in the MG nerve.
togram profiles strongly skewed toward large numbers of small DRG neurons. This same principle has been observed in the cat (42). Muscle afferent populations,
for all cases in dimensions.
on the other hand, revealed histogram profiles that tended to be unimodal, symmetrical and of much larger mean size than cutaneous nerves (51). Interestingly,
RAT
CASE g 2
15
270
DRG
NEURONS
’ zle
’
95
H41
CELLS
r2 “d
10
z g2
5
0
0,
I 0.41
1 0.8I 1 1.2 , r 1:s T 3 ’ 214 AREA (J&) x 1000
CASE 239
1
0.4
0.8 1.2 1.6 AREA @nZ) x
0
20
40 DIAMETER
60
(pm)
R78
CELLS
2 1000
2.4
CASE
2.6
0
20
40 (am)
60
DIAMETER
40 (km)
60
DIAMETER
H16
188 CELLS
0
0
0.4
0.8 1.2 1.6 AREA (pm’) x
2 1000
2.4
2.6
20
FIG. 6. DRG population profiles of the LG-SOL nerve. (A) A case with mean perikaryal dimensions close to the mean value Table 4. (B) The case in Table 4 with the smallest mean perikaryal size. (C) The case in Table 4 with the largest perikaryal Irregularity of the plots is a result of the small numbers of DRG neurons found in the LG-SOL nerve.
this may be a feature unique to rodents because muscle afferents in the cat appear to have strongly skewed size distribution similar to that found for cell bodies of cutaneous afferents (42). This fundamental difference be-
tween cutaneous and muscle DRG shown to exist in the radial nerve of A feature of interest in Fig. 7 is the the three population histograms that
for all cases in dimensions.
neurons has been the rat (77). cross-over point of occurs at about 29
96
SWETT
ET
TABLE Segmental Distribution
AL.
5
of Normal MG DRG Neurons and Their Sizes
Animal
Label
L3
L4
L5
L6
Total
R71L R72L R86 R105 R109 RllOL RllOR Hll H15 H27 FG5L FG5R
HRP HRP HRP HRP HRP HRP HRP MIX MIX MIX MIX MIX
-
-
0 7 3 5 17 3 14 0 0 0 0 0
96 80 97 120 112 103 85 111 101 125 96 116
0 0 0 1 0 2 41 0 0 0 0 0
96 87 100 126 129 108 140 111 101 125 96 116
0 0%
4 3.6%
103 92.8%
4 3.6%
111 +16
N=
12
Mean Mean
total %
-
No.
Variability
Animal
Label
R78 HlO H12 H16 H17 H40 H41 W28 w29 FG2
WGA-HRP MIX MIX MIX MIX MIX MIX HRP HRP MIX
N=
10
Mean Mean
total %
in DRG Neuron
LG-SOL
DRG
Neurons
and
Their
L4
L5
L6
Total
-
71 98 101 35 91 58 166 122 221 67
163 91 104 153 105 216 104 146 82 101
5 0 0 0 0 0 0 0 0 0
239 189 205 188 196 274 270 268 303 168
103 44.8%
126 55.0%
tl 0.2%
230 +44
0 0%
627 519 423 431 449 411 357 446 455 450 527 491
925 f 466
40.0 38.0 33.4 33.4 33.0 34.4 30.4 39.2 42.3 33.2 37.1 31.4
+ + k k k f + + + + + k
11 10 9 10 9 8 8 8 9 10 11 11
35.5 + 10
Counts and Perikmyal
Sizes
6
L3
-
No.
of Normal
f + f +k + + + + + + +
How accurate is the estimate of 10,500 DRG neurons for the rat sciatic nerve? For reasons listed below this mean value may be an underestimate of the true count by as much as 3-5%. Perikaryal sizes are underestimated by no more than 3-6%. There are six potential sources of error (or variability) that could theoretically introduce significant distor-
TABLE Distribution
1219 1057 816 769 831 910 711 1105 1161 819 969 759
Diameter
from muscles are fundamentally different in their axonal transport properties than those from the skin, the conclusion that may be drawn from Fig. 7 is that the number of unmyelinated afferent fibers supplying rat muscle must be relatively modest (2, 45, 65). The large numbers of unmyelinated axons reported in muscle nerves (61) would then appear to be primarily from postganglionic sympathetic neurons rather than from DRG neurons (10,42,51).
pm. Below this limit pure cutaneous nerves show an overwhelming preponderance, more than two-thirds (69%), of small diameter DRG neurons. In mixed nerves, with about 20% of their afferents from muscle, the proportion of neurons smaller than 29 pm dropped to nearly half (55%). Finally, in pure muscle nerves, the proportion of neurons smaller than 29 pm was only about 25% of the total. Cutaneous nerves and muscle nerves differ fundamentally in the size distributions of their DRG neurons. While there are exceptions (40), it is widely assumed that the vast majority of the perikarya of the slowly conducting, unmyelinated, primary afferent fibers belong to the small “B’‘-type DRG neurons that have perikaryal dimensions smaller than 29-30 pm (22,27,39-41, 63,64). Cutaneous nerves in the rat have large numbers of unmyelinated axons most of which presumably derive from small DRG neurons (5,9). Unless small axons
Segmental
Area
Sizes Area 667 1113 1021 1257 1091 672 794 797 765 819
-+ + + k f + k + f. f
Diameter 488 455 390 497 489 415 444 427 485 607
900 + 470
28.8 40.1 38.0 42.0 39.4 31.7 34.3 33.0 32.0 33.0
+ 11 + 8 + 7 rt 9 + 9 AZ 10 + 10 f 9 + 10 + 12
35.2 f 10
RAT
DRG
TABLE Total Distribution Nerve
L4
0 (0) 0 (0) 0 (0) 48 (1)
65 (2) 10 (1)
Actual Count % of total
123 1.2%
4 75 103 3234 2133 848
L5 (4) (5) (45) (68) (79) (96)
6397 61.8%
tions in the size representations of cell bodies and numbers of DRG neurons. These potential sources of error, not listed in the order of importance, are: (i) spurious labeling due to uncontrolled spread of the tracer substances; (ii) errors in reconstruction and data processing; (iii) incomplete labeling of a population of neurons whose axons were exposed to the tracer substances; (iv) inaccuracies in the split-cell error correction, (v) duplicate counting of DRG neurons with bifurcating axons that project into separate branches of the sciatic, and (vi) natural variability in numbers of neurons contribut-
------FEPOlEAL TIBIAL
-.-.-.-.-.-.-. Ala ‘1ER”ES
MC A,40
7
of Sciatic Afferent Neurons According to Segmental Level and Tributary L3
Med. gastroc. Sural LG and soleus Tibia1 Peroneal Lateral sural
97
NEURONS
LiGSOL
P‘ERVES
LATERAL SURAL AND SURN NERb!S
FIG. 7. Normalized histograms of DRG neuron populations of cutaneous, muscle, and mixed nerves. The diameter profiles of six pooled cutaneous nerves, three each from Tables 1 and 2 for the sural and lateral sural nerves. These nerves contain few (0%) muscle afferents. The histogram profile for typical mixed nerves, representing pooled cases of three peroneal and three tibia1 nerves, has a larger mean diameter; roughly 20% of its afferents innervate muscle. In the case of muscle afferents, three cases each from Tables 5 and 6, all of which innervate muscle; the mean size is the largest. The proportion of small neurons (t29 mm in diameter) in muscle nerves is greatly reduced in comparison to mixed and pure cutaneous nerves.
103 1564 126 1466 501 28
L6 (92) (93) (55) (31) (19) (3)
3788 36.6%
4
(4)
36 (2) 0.5 0 0 0
(tl) (0) (0) (0)
41 0.4%
Branch Total neurons (% of total) 111 1675 230 4748 2699 886
(1.1) (16.2) (2.2) (45.9) (26.1) (8.5)
10,349 100%
ing to a given tributary branch due to developmental or aging factors. (i) Unintentional or spurious labeling of neurons, other than those whose cut axons were exposed to the tracer solutions, could produce excess numbers of labeled profiles leading to an overestimate in the corrected numbers of neurons. The wax reservoir technique of labeling cut axons was developed specifically to render insignificant the incidence of such labeling errors (71,72). It is difficult to know with certainty from DRG material alone whether spurious labeling is present or not. Fortunately, with the exception of the lateral sural nerve, all branches of the sciatic used in this study also contained axons of motoneurons. If weakly labeled motoneurons had occurred in inappropriate ventral horn locations for the nerve labeled, spread of the labeling solution was assumed to have occurred. Such preparations were discarded. By thorough washing of the nerve ends and surgical wounds at the time of closure, and strict adherence to labeling criteria of motoneurons, spurious labeling of motoneurons was usually absent, or so limited as to be insignificant (71). Under these conditions it was assumed that spurious labeling among DRG neurons would be no greater than that observed for motoneurons. As a source of error in the present study, spurious labeling can be excluded. (ii) Errors in counting due to tissue reconstruction and data processing are inevitable when dealing with large quantities of numerical data as in the present study. The most common source of error in this category was the oversight of some labeled profiles in the tissue slices and subsequent failure to plot them on paper with the aid of the drawing tube. Such an error would lead to underestimation of neuron counts. This was circumvented by using a standard spot-check in about 25% of the sections to confirm that all labeled profiles in the sections were correctly transferred to paper in both numbers and sizes. Furthermore, every section for each DRG was double-checked to verify that it had a corresponding drawing and that the data for
98
SWETT
each had been appropriately entered into a spreadsheet file. Errors were caught and corrected in the raw data of several preparations in the early stages of the study but they seldom exceeded l-Z% of the profiles. The accuracy limit in drawing of the profiles too large or too small was probably in the range of +1.5-2 pm; distortions produced by this type of error are unknown but probably self-canceling for practical purposes. Error check protocols were also embedded in the spreadsheets to trap entry errors which could be flagged for editing before fragment correction procedures were applied for estimating true neuron counts. Error checks and corrections consumed about 15% of the total effort required to complete each reconstruction. Errors of omission eventually became rare or absent. No reconstruction was attempted of any nerve preparation unless all sections of every ganglion, especially DRGs L4 and L5, were captured and properly processed. Sometimes, if only one section was known to be missing from an L3 or L6 DRG, reconstructions were carried out as the loss of a section in these ganglia would not have had any impact on the total profile count. Thus, while the process of drawing, measuring, and inputting labeled profiles all introduced unavoidable errors and distortions, these proved to be negligible and can be discounted as having any significant impact on the results. (iii) Incomplete labeling of a neuron population is probably the most serious potential source of error in the study. This error is difficult to identify, circumvent, correct, or quantify. Even though nerve labeling procedures were geared to produce maximal retrograde filling and density of reaction products, the smallest DRG neurons associated with unmyelinated axons did not seem to be labeled as reliably as the large DRG neurons. Because motoneuron counts labeled with the same technique reached “ceiling” levels with small standard deviations (71), it was assumed that all myelinated DRG axons would transport and label their respective perikarya with comparable efficiency. As Sittiracha and McLachlan (59) pointed out, small unmyelinated axons may be unable to take up and transport enough tracer substance to be detected in some cases. A hint of this, they pointed out, may be gauged from cases in which lower mean cell counts are coupled with larger mean perikaryal diameters. There are a few HRP-labeled cases in Tables l-4 that show this type of pattern. They were admitted into the database because they could not be excluded by the criteria for labeling quality. It cannot be excluded that these cases reflect accurately the true compositions of these nerves, but such variability as seen in these tables must be attributable to other factors as well. One such factor, for example, may be attributed to the use of HRP or WGA-HRP alone as the labeling agent. It was reported earlier that HRP alone does not label unmyelinated axons as well as myelinated axons. Conversely, WGA-HRP appeared to label unmyelin-
7” I51
AL. a-
ated axons better than myelinated ones (66). If a mixture of these tracers is not used there is a risk that some neurons may escape labeling. For example, 2 HRP-labeled MG nerve cases (Table 5) showed fewer than 100 neurons and they also showed some of the largest mean perikaryal sizes. This suggests that a few small DRG neurons may have escaped labeling. The same phenomenon appears in the data for sural nerve case R80. In five peroneal cases (12, W3, Wll, W22, X2) low cell counts were again coupled with normal or larger than normal mean perikaryal ~mensions suggesting an underrepresentation of its small DRG neuron components. In many preparations there was an impression that a natural “ceiling” had been reached and that labeling of the available population had been essentially complete. These have large counts and medium to smaller mean cell sizes. If the few low-count, large-sized cases were excluded from Tables l-4, it is evident that the mean cell counts in each nerve would be increased by roughly 3-5%. There is another type of potential error in labeling that could also lead to an underestimate in neuron counts. If postlabeling survival times are not properly controlled, some elements will fail to be labeled. When postlabeling survival times are less than 24 h, large neurons may be incompletely labeled. Conversely, small neurons may be underrepresented if postlabeling survival times extend beyond 72-96 h (l&62). Such differential labeling has been claimed for motoneurons as well (47). This source of error does not apply to the present study. All experiments involved labeling for 4872 h when cell counts appeared to be maximal and stable. (iv) Another potential source of unavoidable error lies in the use of a mathematical “fragment correction” factor to cancel out the split-cell error. It is nearly impossible to arrive at any accurate estimate of DRG neuron counts without the use of one, but, as correctly pointed out by Schmalbruch (58), the use or misuse of such a tool can introduce errors of enormous proportions. It is probably one of the major causes underlying the lack of agreement in the literature over the number of DRG neurons or axons related to a given nerve. It became essential for the present study to develop a mathematical correction factor with a prouen margin of accuracy. A parallel series of experiments were undertaken to perfect an iterative mathematical technique for correction of split-cell error originally developed by Hendry (24) and further improved by others (55, 60). The procedure used in the present study, described in detail elsewhere (74), can reconstruct computer simulated sectioned populations of spherical “cells,,’ similar in numbers and size to those seen in Fig. 4, with 0% error in number and size. When applied to real motoneuron populations, whose perikaryal tend to be elliptical in shape, the result was a mean underestimate of true neu-
99
RAT DRG NEURONS ron numbers by 1.4 f 3.5%. When the total observed count in Table 7 is adjusted for this mean correction error, the total DRG neuron count is close to 10,500. Mean perikaryal sizes were reduced by about 2-3% due to the slight tendency of the correction procedure to underestimate the numbers of large cells and overestimate the numbers of small ones. (v) Another potential source of error lies in the knowledge that individual DRG neurons may have more than one axon projecting distally (28,37,43). It has been claimed that these dichotomizing axons project in large numbers into different nerve branches to innervate even widely separated peripheral structures (4, 52-54, 73). If such branching of DRG axons existed to any significant extent among the tributary branches of the sciatic nerve, it would produce an overestimate in DRG neuron numbers in the present work due to the nervelabeling strategy used. To control for this possibility double-labeling experiments were performed on pairs of sciatic nerve branches with a sensitive HRP and Fluoro-Gold combination (71). Although still in progress, these studies show that double-labeled neurons exist but they are so few in number that the potential error introduced by this would be tl% and of no significance to this study. This observation supports the conclusion of others; dichotomizing primary afferent axons exist but so few of them occupy adjacent branches of the sciatic that they are a doubtful source of error in the present study (5,13,X, 16). How can this be reconciled with reports that many primary afferent axons dichotomize (37) and that many of them are unmyelinated (26)? The most logical explanations are that they (i) do not extend into the periphery far enough to be labeled (see (26)) or (ii) remain in such close proximity to each other that they normally occupy the same labeled tributary branch. Axonal branching can be safely discounted as a significant source of error in the present study. (vi) Variability in the DRG counts for individual branches of the sciatic can be attributed in part to factors related to development and aging. It has been claimed that DRG neurons of the rat do not remain stable in numbers or perikaryal dimensions during aging; they reportedly increase in number and decrease in size with increasing age (14,15,23). If the animals in the present study had widely differing ages, the variability seen in the DRG counts could be partly attributed to such an age-related factor. This cannot apply to the present work. The mean DRG count for the sciatic nerve young to middle-aged rats in the present study was significantly higher than the count reported for old rats (14, 15). This observation, coupled with data from the cat (3), suggests that neurogenesis does not occur among DRG neurons as a normal feature of aging as has been claimed by others (14,15). Over 90% of the experiments were carried out on young, sexually mature female rats weighing 120-240 g. Less than 6 months dif-
ference, in the extreme, separated them in age. Age-related neurogenesis of DRG neurons would not have had any effect on the total counts of neurons. In any event, there is no evidence that such neurons could extend their axons far enough into the periphery to be labeled (14). Only a few animals in the study were male and 6 months older than the oldest female. These cases did not differ noticeably from the others so there was nothing to suggest that the variability seen could be attributed to sex or age. Variability in DRG counts for sciatic tributary branches could be partly related to factors governing pathfinding of growth cones of DRG axons which determine the boundaries andinnervation densities ofperiphera1 receptive fields in early development. Motoneurons and DRG neurons may differ in this respect. The mean variability of rat motoneurons is about +6% (71), nearly a quarter of that for DRG neurons (22%) with a range of 14% for the MG nerve to 33% for the lateral sural (Tables l-6). While muscle innervation is by nature compartmentalized, the components destined to innervate a cutaneous receptive field probably vary more widely from one animal to another due to factors that determine the branching patterns of nerve trunks during development and the number of neurons that succumb to normal, massive cell death near the time of birth (11, 21). The lateral sural nerve has the curious property of nearly doubling the number of DRG neurons from one animal to another. The small version contains a mean of about 630 DRG neurons while the larger one contains about 1140 DRG neurons, an apparent incremental difference of nearly 500 neurons (Table 2). The total surface area of their respective cutaneous receptive fields was not measured but would presumably differ in size accordingly. From its population profile (Fig. 2), the vast majority of its neurons are small and presumably supply unmyelinated axons. Assuming that homologous skin fields have a fairly constant innervation density from one animal to the next, the small lateral sural, in comparison with the large, must have relinquished 500 DRG neurons to one or more of the nerves supplying adjacent skin areas such as the saphenous or sural(72). Although this point was not discussed by Sittiracha and McLachlan (59), examination of their data in Table 1 for the rat MG and sural nerves shows comparable degrees of variability of DRG counts found in the present study. In view of this, it is interesting that axonal counts of the lateral sural by Schmalbruch (56) do not show the degree of variability reported here. Sciatic Nerve DRG Neurons-Accuracy
in Numbers
The results of the present study reveal that the normal rat sciatic nerve contains approximately 10,500 DRG neurons, the total value of Table 7 being increased
100
SWETT
by 1.4% to offset the known error of the fragment correction procedure. Assuming that this is an underestimate by 35% due to incomplete labeling in some cases, the true, mean count should be somewhere in the range of 10,500 and 11,000. This number is greater than that reported by investigators who used thinner sections, nucleolar counts, section sampling, and correction procedures of unproven accuracy to obtain their results (6, 14, 15). This number, however, is noticeably smaller than what can be calculated from the results of others who counted DRG neurons retrogradely labeled from tributary branches of the sciatic in a manner similar to the one reported here. Baron et al. (5) counted a total of 9900 DRG neurons for the tibial, sural, and muscle nerves, a number that is 30% greater than the sum of the same nerves listed in Table 7. Assuming that they would have reported a comparable difference for the peroneal and lateral sural branches, their sciatic nerve would presumably be supplied by a mean number of about 14,500 DRG neurons. The disparity between this number and the 10,500 figure reported in this work can be mostly explained by the different mathematical correction methods used in the two studies to compensate for split-cell error. Baron et al. (5) counted every labeled profile in every section, as done in this study, but their counts were made from sections 30-brn thick and reduced by 30% to correct for split-cell error. This resulted in an inflated cell count by at least 20% in their work because the mean diameter of rat sciatic DRG neurons is close to 30 pm (Tables l-4), the same as their section thickness. Nearly every cell on the average would have been cut in two to produce two labeled profiles; all neurons larger than 34 pm, a significant proportion of the total, would have produced three profiles. Their raw profile counts should have been reduced by at least 50% instead of 30%. If the mean, raw profile counts of six tibia1 nerve cases (containing more than 6000 profiles) from Table 1 of Baron et al. (5) are corrected according to the procedures used in the present study, i.e., by 50%, their count for tibia1 DRG neurons would be 3950, or 1 standard deviation below the results reported in Table 4. The latter difference could also be partly explained by the fact that Baron et al. (5) labeled the tibia1 1 cm above the ankle instead of near the popliteal fossa as done in this study. There would be more afferent neurons present at the more proximal labeling site. The data from Baron et al. (5) are compatible with our results when raw profiles are “fragment corrected” (74). Using the same rationale and correcting the mean count of the four sural casesfrom Table 1 of Baron et al. (5) by 50%, the count would be 2220 in contrast to our estimate of 1675 neurons (Table l), a difference of about 2 standard deviations. However, a better match for our sural count was found with a similar calculation
l!J'I‘AL. -- ‘-
from the data of Sittiracha and McLachlan (59) in which they must have had a raw profile count of about 3500. When corrected by 50% according to our requirements, their data would produce a count of about 1750 DRG, a value close to the mean total shown in Table 1. An almost perfect match is also found in recalculating the four largest muscle nerve cases in Table 1 of Baron et al. (5). Because of the larger mean size of these neurons, their raw profile counts must be reduced by at least 55%. The result is an recalculated estimate of 342 DRG neurons, a value nearly identical to the sum of the MG and LG-Sol nerves in Tables 5 and 6. These examples collectively suggest that the completeness of retrograde labeling of DRG neurons in the present study was comparable to that of others (5, 59) and that the estimates for neuron counts differ because of the alternate strategies used for correcting split-cell error (5,59). Given the evidence that the corrected data in Tables l-6 show relatively stable “ceiling” counts, the data from these other investigators would seem to reinforce our contention that the data summarized in Table 7 is very close to the true population count. There is some additional, indirect evidence from axonal counts that the data in Tables l-7 may also accurately reflect the sciatic DRG neuron population. Take, for example, the common peroneal nerve. The axon counts for this nerve from Jenq et al. (32) and Schmalbruch (56) are similar with a mean of about 1930 myelinated axons and 3900 unmyelinated ones. From the latter it is necessary to subtract 1100 unmyelinated postganglionic sympathetic axons (56) to yield a total of 2800 unmyelinated sensory (afferent) axons. According to our most recent data, there are about 630 motoneurons in the nerve (68,69,71). This corresponds well with the number of myelinated motor axons in the nerve (56). When the motoneuron axons are subtracted from the myelinated group the net result is a total count of 1300 myelinated afferent fibers. The total number of afferent fibers in the peroneal nerve must then be about 4100 axons. If DRG neurons on the average provide 2.3 axons to peripheral nerve (36, 37) then the total DRG count for the peroneal nerve would be close to 1800, significantly below the total estimated in Table 3. However, if only those DRG neurons giving rise to unmyelinated fibers give rise to 2.3 axons, then a total of 2700 DRG neurons should contribute to the common peroneal nerve. Interestingly, this is the same count derived from reconstruction of labeled DRG neurons listed in Table 3. The results of the above comparisons are obviously highly speculative and perhaps merely coincidental. It is instructive then to examine the sural nerve. It contains about 70 motoneurons (51,59,71), almost exactly 1000 myelinated axons, and 3800 unmyelinated axons (30, 32, 49, 51, 56). One study calculated that 28% of all axons in the sural were postganglionic sympathetic neu-
RAT
DRG
rons (56). If this number is correct, the total number of afferent axons, 930 myelinated plus 2460 unmyelinated, would equal 3390. If it is again assumed that all DRG neurons give rise to 2.3 axons, a total of 1470 DRG neurons would be estimated for the sural; if such branching is primarily a property of small DRG neurons then the total sural DRG population would be closer to 2000. These estimates are 1 standard deviation above and below the mean number of sural DRG neurons shown in Table 1. A third example is the MG nerve which has a mean of about 660 axons (30, 32, 51). Muscle nerves are rich in unmyelinated axons (61) and there is evidence that about 50% of all axons in muscle nerves may originate from postganglionic sympathetic neurons, 55% in cat (42), and 44% in rat (5). This leaves about 330 somatic motor and sensory axons in the MG nerve. If one assumes that myelinated axons in muscle nerves remain unbranched until they are intramuscular (7), 140 myelinated motoneuron axons (71) may be subtracted to obtain 190 sensory axons of all types. If rat muscle nerve is similar to cat muscle nerve (7), about three quarters of these (140) will be myelinated afferent axons (30,32, 51). Assuming that the remaining 50 are branched unmyelinated afferent axons, a grand total of about 160 DRG neurons should be associated with the MG nerve. Although the match in Table 7 is not as good as in the preceding two examples, the example nevertheless augments the evidence presented earlier that the numbers of DRG neurons in pure muscle nerves are few and that the numbers of small DRG neurons are very few (Tables 5 and 6; Figs. 5-7). The tibia1 nerve is reported to have about 13,000 axons (32, 56). About 1000 of them are myelinated axons of somatic motoneurons (56, 71). Of the remaining 12,000 axons, 27% of which are presumably from autonomic (sympathetic) postganglionic neurons (56). The total number of sensory axons would therefore equal 8800. Nothing is known about branching of tibia1 DRG neurons, but if it is assumed that there are roughly at least 1000 muscle afferents that do not branch and the remainder give rise to about two axons each, the tibia1 nerve should contain about 4900 DRG neurons, a value remarkably close to that found through retrograde labeling. The comparisons above, while speculative, nevertheless emphasize that the numbers of DRG neurons reported in Table 7 cannot be far from the true numbers of DRG neurons that contribute to the rat sciatic nerve.
able to contribute to the project thanks to the help of the Natural Sciences and Engineering Council of Canada. This work was made possible by a research grant from the National Institutes of Health (NS-25707).
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