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Three-dimensional computer-aided analysis of the intraganglionic topography of primary muscle afferent neurons in the rat.

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THE ANATOMICAL RECORD 227:405-417 (1990)
Three-Dimensional Computer-Aided Analysis of
the lntraganglionic Topography of Primary Muscle
Afferent Neurons in the Rat
JEAN-MARIE PEYRONNARD, JEAN-PIERRE MESSIER, MARTIN DUBREUIL,
LOUISE CHARRON, AND FRANCE LEBEL
Centre de Recherche en Sciences Neurologiques and Hbpital Hbtel-Dieu, Universite de
Montreal, Montrt?al, Quebec, Canada
ABSTRACT
A microcomputer system was used to reconstruct, in the L5 dorsal
root ganglion (DRG) of the rat, the three-dimensional arrangement of primary
neurons which had been labelled by application of horseradish peroxidase (HRP)
and fluoro-gold (FG) to various muscle nerves of the leg. Analysis of the data and
animation of the reconstructed images with commercially available software were
instrumental in identifying the preferential intraganglionic locations of the neurons innervating muscles such as the soleus (SOL), the gastrocnemius lateralis
(GL), and medialis (GM), or parts of the GM. These locations appeared to be somewhat related to the position of the muscles in the posterior compartment of the leg.
Additionally, the study provided quantitative estimates of muscle afferent neuronal populations, allowed a comparison of the labelling performances of HRP and
FG, and finally indicated that few DRG neurons project to two different muscles.
The somatotopical organization of many neural sys- Computerized reconstruction and manipulation in 3-D
tems is illustrated by the clustering of spinal motoneu- of the image data was undertaken to establish the rerons into cell columns (Burke et al., 1977; Hollyday, spective locations of the neuronal aggregates under
1980; Janjua and Leong, 1984; Landmesser, 1978b; study.
McHanwell and Biscoe, 1981; Nicolopoulos-Stournaras
MATERIALS AND METHODS
and Iles, 1983; Peyronnard et al., 1986; Romanes, 1964)
which may be further subdivided into compartment nuSurgical Procedures and Application of Cell Tracers
clei innervating specific parts of a muscle (Weeks and
Eighteen adult female Sprague-Dawley rats were
English, 1985,1987).In the sensory system, cytological anesthetized intraperitoneally with sodium pentobarand electrophysiological evidence points to a topo- bital. The nerve trunks derived from the posterior tibgraphical localization of the central projections of cu- ial nerve and supplying the soleus (SOL), the gastroctaneous and visceral afferents in the dorsal roots nemius lateralis (GL), and the gastrocnemius medialis
(Brown and Fuchs, 1975; Heaney e t al., 1984; Koerber (GM) muscles were isolated in the right hindlimb, as
and Brown, 1982; Koerber and Mendell, 1988; Light were the two terminal branches of the nerve to the GM
and Durkovic, 1984; Wilson et al., 1986), and of their which penetrate the superior (GMS) and inferior (GMI)
cell bodies in the spinal and trigeminal ganglia (Arl- parts of the muscle (Fig. 1).The proximal stumps of the
hal, 1968; Capra and Wax, 1989; Corner et al., 1978; transected nerves were immersed for 2 hours in a
Jacquin et al., 1983; Kausz and Rethelyi, 1985; Lende sealed capillary tube filled either with a 20% solution
and Poulos, 1970; Marfut, 1981; Oyagi e t al., 1989; Shi- of HRP (type VI) (Sigma Chemical. Co., St. Louis, MO)
genaga e t al., 1989). Primary muscle afferent neurons or a 3% solution of FG (Fluoro-chromes, Engelwood,
differ in this respect, a s they lack a n orderly intragan- CO) in 0.9% saline. During this time, diffusion of the
glionic distribution according to studies using either markers was prevented by ligating all neighboring
the retrograde chromatolytic reaction after peripheral nerves and by wrapping the operation site with a plasnerve injury (Norcio and De Santis, 1976) or cell trac- tic sheet. In order to assess their labelling capacity,
ing techniques (Kausz and Rethelyi, 1985; McLachlan HRP and FG were alternatively applied in some aniand Janig, 1983). We ourselves expressed this view in mals to the SOL, GM, and GL; but they were also used
a report (Peyronnard et al., 1986) on the neuronal in- simultaneously to map, in the same animal, the muscle
nervation of rat hindlimb muscles. However, further afferent neurons supplying either the SOL and GM
examination of the material led u s to believe t h a t a
definite conclusion required three-dimensional (3-D)
analysis of the location of these neurons in dorsal root
ganglia (DRG). For this purpose, the present study utiReceived August 16, 1989; accepted October 26, 1989.
lized horseradish peroxidase (HRP) and f luoro-gold
Address reprint requests to Dr. J.M. Peyronnard, Centre de Re(FG) to mark, in a given animal, the afferent neurons cherche
en Sciences Neurologiques, Faculte de Medecine, Universite
supplying one or two different muscle nerves, or alter- de Montreal, Case Postale 6208, Succ. A, Montreal, Quebec, Canada
natively, the different branches of a muscle nerve. H3C 3T8.
0 1990 WILEY-LISS, INC.
406
J.-M. PEYRON NARD ET AL
PL
GL
SOL
Fig. 1. Axillary view of the rat hindlimb showing, in black, the
posterior tibia1 nerve supply t o the gastrocnemius lateralis (GL), soleus (SOL), plantaris (PL),and gastrocnemius medialis muscles (GM),
including the two terminal nerve branches t o the GM.
(SOL-GM), the GM and GL (GM-GL),or the upper and
lower parts of the GM (GMS-GMI).
Seventy-two hours later, the animals were killed by
transcardiac perfusion of 1% paraformaldehyde and
1.25% glutaraldehyde in phosphate buffer (pH 7.4).
The right L4, L5, and L6 DRG were dissected, postfixed
for 30 min in the same perfusate, and immersed for 3
hours in phosphate buffer containing 10% sucrose (pH
7.4).
Tissue Processing and Analysis
Before freezing in liquid nitrogen, the DRG were positioned to bring the ventral roots to the bottom, with
the dorsal roots surrounded by the bulk of the ganglionic cells lying above (see Figs. 2 , 3 ) .Serial sections, 20
pm thick, were cut longitudinally with a cryostat
(-20°C) microtome and processed for HRP histochemistry with tetramethylbenzidine (Mesulam, 1978). In
the case of double-labelled ganglia, the fluorescent
cells were detected first, with a epifluorescenceequipped Zeiss photomicroscope. These cells were numbered and their diameter was computed (Macintosh
Plus) after projection onto a digitizing tablet. The same
procedure was then applied to HRP-stained cells, using
bright-field illumination.
All data were transferred onto photographic prints of
the sections enlarged to a final magnification of x 65.
After superimposition and marking with three reference points, these prints were used for a 3-D reconstruction of individual cells. In the case of neurons appearing on several adjacent sections, only the level
containing the largest diameter was retained. Subsequently, the position of the reference points and the
labelled cells, as well a s the contours of the ganglia,
ventral and dorsal roots were digitized on a graphic
tablet with a 3-D reconstruction software package
(MacReco 4.0, E. Otten and G.W. Dreschel, Groningen,
The Netherlands). The data were processed on a Macintosh I1 microcomputer equipped with a 5-megabyte
random access memory, a 40-megabyte hard disk, and
a 13-inch color monitor (Apple). To prevent crowding of
the reconstructed figures, DRG contours were digitized
a t 4 different levels only (out of approximately 50 sections for a L5 ganglion). The ventral and dorsal root
contours were also drawn at every third to fifth section,
and no attempt was made to follow the intraganglionic
pathway of the dorsal root fibers. To circumvent the
limited capacity of the program with respect to figure
animation, we employed one of its options to define the
X, Y, Z coordinates of all selected points. After calibration measurements, the coordinate documents together
with the information on cell diameters were turned
over to a spreadsheet (Excel 2.0, Microsoft Corp., Seattle, WA) and transferred to graphic data analysis software (MacSpin 2.0, D2 Software Inc., Austin, TX). The
MacSpin program, which we used to reconstruct roots
and DRG contours, makes i t possible to draw lines between sequentially connected events. Furthermore, it
offers the distinct advantage of allowing a 360"rotation
of any 3-D structure in six different directions, while
its multiple-window display options facilitates comparison between reconstructed DRG. One of its functions,
identified as the centroid connection operation, was
found to be most helpful for the visual enhancement of
the topographical location and spatial extent of neurons innervating the same muscle target. In this mode
of operation, the computer defines the center or centroid point of a neuronal cluster by averaging the X, Y,
and Z coordinates of all the cells belonging to this cluster. It then selects the neuron closest to the centroid
point and connects this centroid neuron linearly to every other related cell. We also used this function for a
quantitative localization of SOL, GM, and GL neurons,
as vai-iations in the DRG morphology from one animal
to the other made it extremely difficult to use reference
anatomical landmarks, such as contours of the ganglia
or roots. For this purpose, the centroid neuron of each
SOL-GM or GM-GL neuronal pool was determined and
served as a reference for positioning the X, Y, Z axes
within the ganglia. When viewed along the root or X
axis, the Z and Y coordinates delineated four quadrants: dorsal lateral, dorsal medial, ventral lateral, and
ventral medial. Within the pools (SOL-GM or GM-GL),
the percentages of SOL, GM, or GL neurons projecting
in each of these quadrants were determined, and the
values obtained for all animals were averaged. The
same procedure was followed after rotation in a lateral
view along the Y axis. In this position, the Z coordinate
NEURONAL ORGANIZATION IN DORSAL ROOT GANGLIA
407
X
VENTRAL ROOT
DORSAL ROOT
LAMINA VERTEBRAE
A
Fig. 2. A. Oblique orientation of a L5 DRG in vivo. B Position given to all ganglia prior to processing
and after rotation along the ventral root (X) axis.
the combined use of FG and HRP made i t possible to
establish that far more DRG neurons to the GM were
supplied by the inferior than the superior nerve branch
(Table 3). In one animal (GMS-GMI,), a relatively high
RESULTS
number of double-labelled neurons were found, possiSegmental Location and Numerical Estimates of DRG
bly representing contamination artefacts due to the
Muscle Afferent Neurons
close proximity of the nerve structures exposed to the
After application of the cell tracers to muscle nerves, markers. In all other instances, the percentage of douneurons containing fluorescent FG granules or the ble-labelled DRG cells was rather low, varying beHRP reaction product (Fig. 4A,B) were found in the tween 1 and 8.9 for a mean of 3.9. Finally, for all muslumbar spinal ganglia. As shown in Table 1, with a cles under study, DRG cell diameters ranged from 10 to
single exception (animal SOL,), the vast majority (96% 65 pm.
on average) of SOL neurons labelled with HRP or FG
were located in the L5 DRG, the remainder being found
lntraganglionic Location of Muscle Cell Afferent Neurons
a t the L4 level. A similar proportion (95%)of afferent
Since most SOL, GM, and GL neurons are located in
neurons innervating the GM nerve or its two distal
branches (Tables 2, 3) was present in the L5 spinal the L5 DRG, only results pertinent to these ganglia
ganglia, the rest being scattered in the L6 and occa- will be presented. We have already mentioned that,
sionally the L4 DRG. GL neurons were more equally prior to sectioning, the DRG were tilted approximately
distributed between the L4 and L5 DRG but prevailed 60" laterally along the roots axis from the oblique poin the latter (Table 4). Quantitative assessment of sition they occupy in vivo (Fig. 2A). In this process, the
whole muscle DRG cell populations with one or the bulk of the ganglia normally oriented towards the spiother tracer revealed little interindividual differences. nal canal was moved to a n upward and dorsal position,
However, the number of HRP-labelled cells of the SOL while the motor roots were internally displaced in a
and GM, averaging 103 and 136, respectively, was completely ventral location (Fig. 2B). By so doing, relower than the mean FG estimates: 144 and 162. Being constructions could proceed from DRG cut at approxiof the same magnitude in single- and double-labelled mately the same angle along the rostrocaudal axis,
animals, the difference indicated that FG had not been starting from what could be defined as the top of the
affected by treatment needed to reveal the HRP depos- ganglia and moving down towards the ventral roots
its. Despite their unequal performance a s cell markers, (Fig. 3A). The fact that contours of the ganglia, ventral,
served a s a dividing line between rostra1 and caudal
sectors. Selected views of the ganglia were printed on
a n Apple Laser Writer I1 NT and edited for illustration.
A
EXTERNAL VIEW
A
/
Y
Rostra1
Caudal
Fig. 3. Diagrammatic reconstruction of a L5 DRG depicting A) the
relationship between ganglionic cells (dotted areas), the ventral (VR)
and dorsal (DR) roots, as well as the plans of histological sections; and
B) the pattern of distribution of the dorsal root fibers as they appear
at two different cross-sectional levels (a,b)of the DRG. At level a,
dorsal root fibers exist as multiple bundles of axons (*) separated by
islands of DRG cells.
and dorsal roots had been digitized at selected intervals
only, for the sake of clarity and without trying to reproduce the complex intraganglionic organization of
the dorsal root fibers (Fig. 3B), did not interfere with
analysis of the relationships between these anatomical
elements and the position of the labelled cells.
From the three orthogonal views of a dually labelled
L5 DRG shown in Figure 5A-C, one can appreciate
without additional treatment of the data that, despite
an important spatial overlap, SOL and GM neurons
tend to be confined to separate ganglionic areas. The
centroid connection operation (Fig. 6A-C) illustrates
more vividly that GM cells are mostly located in the
dorsal and internal parts of the DRG, being closer to
409
NEURONAL ORGANIZATION IN DORSAL ROOT GANGLIA
Fig. 4. Photomicrographs of a section of a dually labelled L5 DRG (animal SOL-GM,). In A, epifluorescent illumination shows GM neurons filled with FG granules, while in B, brightfield illumination
reveals a SOL neuron containing HRP deposits. Bar: 20 Fm.
TABLE 1. Number of dorsal root ganglion cells labelled after exposure of the soleus nerve to horseradish
peroxidase or Fluoro-Gold
Animal
SOL-GM,
4
SOL,
SOL,
SOL-GM,
41
1
SOL,
SOL-GM,
10
-
Y)
=
HRP
L5
L4
97
(2)l
-
-
56
97
(4)
108
-
Total
L4
FG
L5
Total
101
-
-
-
97
98
6
-
149
-
155
118
-
3
130
(3)
-
-
133
No. of double-labelled cells.
TABLE 2. Number of dorsal root ganglion cells labelled after exposure of the gastrocnemius medialis nerve to
horseradish peroxidase or Fluoro-Gold
HRP
FG
Animal
SOL-GM,
L4
L5
L6
Total
L4
L5
L6
Total
-
-
-
-
4
9
183
SOL-GM,
-
-
-
-
-
170
(2)l
147
17
164
(4)
-
-
2
156
-
-
-
139
12
151
GM,
GM-GL,
-
135
-
-
135
-
3
SOL-GM,
-
-
137
-
GM-GL,
-
137
(3)
-
-
-
-
’(
151
(6)
1 = No. of double-labelled cells.
the spinal nerve than most of the SOL neurons which
occupy a more rostral, external and ventral position.
Drawing envelopes of the roots and ganglion (Fig. 7)
allowed a better appreciation of the relationships between these neuronal clusters and the anatomical elements of the DRG. Quantitative analysis performed in
three animals revealed a fair constancy in the location
of SOL and GM neurons within SOL-GM pools. As evidenced by Figure 8A, a n average of 83.8%of the SOL
neurons projected into the ventral and lateral parts of
these pools, while 88.2% of the GM neurons were found
in the dorsal and medial sectors. This clustering was
also apparent in the lateral view, since only 38.7% of
the SOL neurons were caudally situated as opposed to
64.7%of the GM cells. It was interesting t h a t the intraganglionic topography of GL neurons in relation to
GM cells (Fig. 9) was almost identical to t h a t described
for the SOL, these two muscles also sharing a similar
410
J.-M. PEYRONNARD ET AL.
Z
A
.
0
OO
0.
0 .
L,
Y
-X
0.
0
0
........ ........
...............
0
0
.....,..,...L................ n,, .....
...................
..... .............................~,,,,.....,,..
....:..,..... .....
S
.,..,;
0
,n........
I......nW.....,,,ll......
M......
1.11.111.1~
Z
B
4
Y
Ax
-------
0
00
Xo
0
0
. ..................................
Oo
0 O0
0 QQ--*---
,11111:
11111.11.1"
1111111111111111
::
Q
C
'rx
Y
I
200 pm
Fig. 5. A-C: Lateral, rostro-caudal, and dorsal views of a L5 DRG
(animal SOL-GM,) after exposure of the GM and SOL nerves to FG
and HRP, respectively. These drawings prepared from computer print
outs indicate the position of the GM- (solid dots) and SOL- (empty
dots) labelled cells and the outlines of the ganglion (G) and roots (VR
and DR). The few double-labelled cells are not shown. Despite overlapping, it can be appreciated that the GM and SOL neurons are
located in different ganglionic areas.
411
NEURONAL ORGANIZATION IN DORSAL ROOT GANGLIA
.
A
B
Y
C
7
tI
Z
I
X
-
200 pin
Fig 6 A-C: Reproduction of Figure 5 By drawing the centroid connections, the spatial segregation of
the GM and SOL neuronal aggregates IS vlsually enhanced
412
J.-M. PEYRONNARD ET AL
TABLE 3. Number of dorsal root ganglion cells labelled after exposure to horseradish peroxidase or
Fluoro-Gold of the terminal nerve branches to the gastrocnemius medialis muscle
L4
Animal
GMS-GMI,
-
GMS-GMI,
GMS-GMI,
'(
Superior nerve branch: HRP
L5
Lfi
67
(26)'
-
-
Total
67
L4
1
-
-
-
-
41
(6)
2
43
-
Inferior nerve branch: FG
L5
Lfi
164
(26)
121
139
5
(6)
Total
165
121
144
1 = No. of double-labelled cells.
TABLE 4. Number of dorsal root ganglion cells labelled after exposure of the gastrocnemius lateralis nerve to
horseradish peroxidase or Fluoro-Gold
HRP
Animal
GM-GL,
L4
37
GM-GL,
-
GM-GL,
40
'( ) =
L5
58
(GI1
FG
L6
L*
L,
Lf3
Total
-
Total
95
-
-
-
-
-
-
-
33
-
108
-
9
(2)
-
146
68
(3)
104
(11)
-
-
No. of double-labelled cells.
INTERNAL VIEW
Rostra1
200 pm
Caudal
Fig. 7. Same DRG a s in Figures 5 and 6 after illustration in 3-D through artistic drawing of the root
and ganglion contours. To give a sense of depth, the size of the spheres representing GM (black) and SOL
(white) neurons varies, depending on their proximity to the viewer.
location in the rat leg, lateral to the GM (Fig. 1).The
data in Figure 8B averaged from three different GMGL pools of neurons confirmed that 93.2% of the GL
neurons were, like SOL cells, laterally and ventrally
located, in contrast with the dorsal and medial location
of 90.1% of the GM neurons. As we mentioned earlier
for SOL neurons, GL and GM neurons were also
present in opposite proportions in the rostra1 and caudal sectors of the GM-GL neuronal pool. While use of
the centroid point and figure animation helped to de-
fine a clear spatial segregation of neuronal innervation
of the lateral and medial muscles in the posterior compartment of the leg, interindividual differences were
met in the position of cellular aggregates. As a result,
there were instances where demarcation between
groups of neurons was apparent in certain views (Fig.
10A,C) but not in others (Fig. 10B). Studies dealing
with nerve branches to the GM suggest a preferential
location of neurons in the dorsal aspect of the L5 ganglion innervating the upper part of the muscle, the
NEURONAL ORGANIZATION IN DORSAL ROOT GANGLIA
413
chicken (Hollyday, 1980; Landmesser, 1978131, mouse
(McHanwell and Biscoe, 1981), rat (NicolopoulosStournaras and Iles, 1983; Peyronnard et al., 19861, cat
(Romanes, 1964) and monkey (Janjua and Leong,
1984). Extant evidence suggests that primary sensory
neurons innervating cutaneous and visceral structures
or their centripetal processes have an orderly distribution. For instance, in lumbo-sacral DRG of the cat, a
link has been found between the position of a neuron in
LATERAL
MEDIAL
the ganglion or that of its peripheral receptive field and
the location of its central projection either in the dorsal
GM neurons
root or in the spinal dorsal horn (Brown and Fuchs,
1975; Burton and McFarlane, 1973; Koerber and
Brown, 1982; Koerber and Mendell, 1988; Light and
Durkovic, 1984; Wilson et al., 1986). Along the same
lines, electrophysiological studies have suggested a somatotopical organization of the saphenous nerve input
to the spinal cord in the cat and rabbit (Heaney et al.,
1984), and the clustering of cutaneous spinal ganglion
cells in the frog (Corner et al., 1978). Finally, microelectrode recordings (Arlhal, 1968) and cell tracing
techniques (Kausz and Rethelyi, 1985) have helped to
B
GL neurons
Z
demonstrate, in the S2 ganglion of the cat, a topographical arrangement of the neuronal somata supplying the
perineum and the urinary bladder. Similar observations have been made in the trigeminal ganglion of rats
and cats through the use of HRP or its wheat germ
agglutinin conjugate (Capra and Wax, 1989; Jacquin et
al., 1983; Marfut, 1981; Oyagi et al., 1989; Shigenaga
et al., 1989). In contrast, muscle afferent neurons have
been shown to be erratically dispersed throughout the
GM neurons
spinal ganglia, even in studies employing HRP (Kausz
z
and Rethelyi, 1985; McLachlan and Janig, 1983; Peyronnard et al., 1986) as a more appropriate method for
precise cell identification than the retrograde chromatolytic reaction (Norcio and De Santis, 1976). However,
these negative findings were derived solely from the
inspection of serial DRG sections, and not from a 3-D
reconstruction of the ganglia, as is the case in this report.
Fig. 8. Compartmental distribution of SOL, GM, and GL cells within
Such a reconstruction faced challenges which could
SOL-GM (A) or GM-GL (B) pools, as seen in two orthogonal views. not be met with conventional drawing techniques. InThe X, Y and Z axes, aligned on the centroid neuron (c) of these pools,
divide the neuronal populations into defined compartments. Values deed, only a computerized system could be expected 1)
are averaged percentages of neurons projecting in a specific compart- to position in 3-D up to several hundred muscle afferment in three different animals.
ent cells in relation to structural landmarks, such as
the outlines of the roots and the ganglion itself; and 2)
to rotate the reconstructed image in order to apprecineuronal supply to the other parts being somewhat in ate, from different perspectives, the relationships bea more ventral position. Finally, it is noteworthy that tween these anatomical elements or to position ganglia
no clustering by neuronal size was detected and that from different animals along the same angle for the
the few double-labelled cells appeared to be erratically purpose of comparison.
Computer-assisted image reconstruction is no longer
distributed.
the exclusive domain of mainframe systems of the kind
DISCUSSION
recently used to assemble a rat brain in 3-D (Toga et
The image-processing system used in this study was al., 1989). In recent years, numerous systems based on
instrumental in revealing that L5 DRG neurons, which microcomputer technology have been commercially desupply specific leg muscles of the rat, have a distinct veloped (Huijsmans et al., 1986; Jarvis, 1988). Howintraganglionic topography linked to some extent to ever, most remain expensive, while some have a limited capacity to manipulate the image or do not offer
the anatomical position of muscles in the leg.
Until now, muscle afferent nerve cells seemed rather the possibility of working with multiple screen winunique because of their lack of somatotopical organi- dows. We assembled our own system around a mediumzation common to most other primary neuromuscular priced Mac11 microcomputer equipped with a graphic
neurons. Indeed, the columnar arrangement of mo- tablet. The input data were photographic prints of DRG
toneurons in the ventral horn of the spinal gray matter sections which had to be first superposed to eliminate
is a feature observed in many animals, including the redundant neuronal cross-sections, a process which, for
A
Z
w
SOL neurons
Z
A
B
*-v,
Z
Y
P
A
X
Z
200 Lrn
Fig. 9. A-C: Three orthogonal views of the L5 DRG of animal GM-GL,, showing that most HRPlabelled GL neurons (empty dots), like SOL nerve cells (see Fig. 51, are located in the ventral, lateral, and
rostra1 part of the ganglion, while a majority of FG-labelled GM neurons (solid dots) are in a more dorsal,
internal, and caudal position.
A
B
Y-
i
X
C
Y
U
200 pm
Fig. 10. In this dually-labelled animal (SOL-GM,), the spatial segregation of the SOL (empty dots) and
GM (solid dots) neurons is mostly apparent in the lateral and dorsal views of the L5 DRG (A,C) and is
difficult to appreciate in the rostrocaudal projection (B).
416
J.-M. PEYRONNARD ET AI,
the time being, can hardly be done with scanned or compatibility with peroxidase cytochemistry (Cabrera
video-captured images. This procedure allowed the in- e t al., 1988; Pieribone and Aston-Jones, 1988; Schmued
clusion of reference marks on the photographs, a step and Fallon, 1986), it also led to neuronal estimates
critical to the realignment of the sections during recon- which, on average, were 26% higher than those obstruction. No attempt was otherwise made to correct tained with HRP (Cabrera et al., 1988). There was no
the potential distortions resulting from slicing and definite evidence that this difference could be artifactual, resulting, for instance, from the accidental labelmounting the tissue (Huijsmans et al., 1986).
The software package (see Materials and Methods) ling of adjacent nerve filaments through diffusion of
used to digitize the input images on a graphic tablet led the tracer. In fact, only one animal gave reason to susto a volumetric representation of the DRG which re- pect that such a phenomenon could have taken place,
mained nonetheless unsatisfactory due to the limited judging from the high number of dually labelled cells
capacity of the program for image animation. This de- after exposure of the superior and inferior branches of
ficiency was resolved by exporting the data to another the GM to HRP and FG, respectively. Such double laprogram allowing simultaneous rotation of several 3-D belling was otherwise infrequent and implicated on avDRG images in a n almost unlimited number of direc- erage no more than 3.9% of the neurons. This would
tions. However, a special graphic design was needed to confirm that despite the well-documented peripheral
unravel the intricate arrangement of muscle afferent dichotomy of DRG cells (Coggeshall, 1985, 1986; Honerve cells. Indeed, the preferential grouping of neu- heisel and Mense, 1987; Langford and Coggeshall,
rons innervating specific leg muscles of the rat in cer- 1981; Pierau et al., 1982; Taylor and Pierau, 19821, few
tain areas of the L5 DRG became obvious once the cen- afferent neurons send axons to separate nerves in the
troid point and its connections had been defined for rat (Devor e t al., 1984).
each neuronal aggregate. In so doing, we not only obACKNOWLEDGMENTS
served a fair constancy in the location of nerve cell
clusters but also a similarity in the intraganglionic
The authors are indebted to the Apple Company of
topography of GL and SOL neurons in relation to GM Canada for its technical support (of M.D.) and to C.
innervation. If a link exists between this observation Gauthier and G. Filosi for their artwork. Gratitude is
and the shared location, lateral to the GM, of the GL extended to 0. Da Silva for editing this manuscript.
and SOL in the posterior compartment of the leg, it
could suggest that, during development, muscle afferLITERATURE CITED
ent neurons, like sensory (Honig, 1982; Scott, 1982) Arlhal, A. 1968 Mise en evidence d’une somatotopie a u niveau du
and motor cells (Lances-Jones and Landmesser, 1981;
ganglion spinal chez le chat. C.R. SOC.Biol. (Paris), 162:1979Landmesser and Morris, 1975; Landmesser, 1978a;
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Tosney and Landmesser, 19851, respond to limb- Baron, R., W. Janig, and W. Kollmann 1988 Sympathetic and afferent
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