Download Morphology of Thalamocortical Neurons Projecting

THE JOURNAL OF COMPARATIVE NEUROLOGY 361:1-24 (1995)
Morphology of Thalamocortical Neurons
Projecting to the Primary Somatosensory
Cortex and Their Relationship
to Spinothalamic Terminals
in the Squirrel Monkey
TING SHI AND A. VANIA APKARIAN
Department of Neurosurgery, Computational Neuroscience Program, State University
of New York Health Science Center, Syracuse, New York 13210
ABSTRACT
This study examined the morphology of thalamocortical neurons projecting to the primary
somatosensory cortex (SI; hand region of areas 3a, 3b, 1, and 2) and their relationship to the
spinothalamic (STT) terminals in the squirrel monkey. Retrogradely labeled thalamocortical
neurons were intracellularly filled with Lucifer yellow (LY), and the STT terminals were
anterogradely labeled with biotinylated dextran. Both filled neurons and labeled terminals were
differentially visualized in the same field by a dual immunocytochemical staining method.
SI-projecting neurons appeared at the light level to be in contact with STT terminal boutons in
the ventroposterior lateral (VPL), ventroposterior inferior (VPI), and centrolateral (CL) nuclei
and the posterior complex (PO).The analyses of the neuronal morphology revealed that somatic
and dendritic morphologies of SI-projecting neurons in these thalamic nuclei, as well as in the
anterior pulvinlar (Pulo), centromedial (CM), and ventrolateral (VL) nuclei, were generally
comparable with some exceptions: VL neurons had the largest soma sizes, the most primary
dendrites, and the longest total dendritic length among all neurons studied; VPI neurons had
the smallest soma sizes; VPL SI-projecting neurons were different from those in VPI in their
soma sizes, shape factors, and orientations; in VPL the cells projecting to the superficial layers
of SI were smaller than those projecting to the deeper layers, but in VPI the two groups of
neurons were similar in soma sizes. In general, the SI-projecting neurons in VPL, VPI, and CL
were similar in their dendritic morphologies and branching patterns, and varied from those in
P u b , PO, CM, and VL. D 1995 WiIey-Liss, Inc.
Indexing terms: intracellular injection, retrograde, anterograde, biotinylated dextran, fractal
dimension
The primary somatosensory cortex (SI) receives thalamocortical afferents from many thalamic nuclei. Its main
input is from the ventroposterior lateral (VPL) and ventroposterior medial nuclei (VPM: for review, see Jones, 1985).
A smaller number of cells projecting to SI are also found in
the ventroposterior inferior (VPI), centrolateral (CL), and
anterior pulvinar (Pulo) nuclei as well as in other thalamic
nuclei (Jones and Leavitt, 1974; Jones et al., 1979; Friedman and Jones, 1981; Pons and &as, 1985; Cusick and
Gould, 1990; Gingold et al., 1991). The neuronal morphologies in VPL or VPM (Yen and Jones, 1983; Yen et al., 1985;
Harris, 1986; Chiaia et al., 1991; Nomura et al., 1992;
Havton and Ohara, 1993, 1994; Ohara and Havton, 19941,
CL (Yamamoto et al., 1985a; Tombol et al., 19901, the
centromedial nucleus (CM, Hazlett et al., 1976; Yamamoto
o 1995 WILEY-LISS, INC.
et al., 1985b; Fenelon et al., 19941, and the ventrolateral
nucleus (VL, Yamamoto et al., 1984, 1985a; Kultas-Ilinsky
and Ilinsky, 1991) in the cat, rat, or monkey have been
studied by means of Golgi preparations or intracellular
injections of physiologically recorded neurons. Golgi preparations stain numerous neurons, but particular afferent
and efferent connections may not be determined with these
preparations alone, and although intracellular injections in
physiologically characterized neurons can produce detailed
morphological information in neurons with verified inputs
Accepted March 16,1995.
Address reprint requests to A. Vania Apkarian, SUNY Health Science
Center at Syracuse, Neurosurgery Research Laboratory, 3118 WSK Hall,
766 Irving Avenue, Syracuse, NY 13210.
T.SHI AND A.V. APKARIAN
2
and outputs, only a limited number of neurons can be
evaluated in each animal.
This study is aimed at examining the morphology of
SI-projecting neurons in different thalamic nuclei and the
relationship of these neurons to spinothalamic (STT) terminals in the squirrel monkey. In order to stain all the
dendrites of identified SI-projecting neurons and STT
terminals, anterograde and retrograde tracing techniques
were combined with intracellular Lucifer yellow (LY) injection in fixed tissue (Buhl and Lubke, 19881, and a dual
immunocytochemical staining method was developed. With
this approach, the complete dendritic and somatic morphologies of labeled thalamocortical neurons were evaluated, and
their proximity to anterogradely labeled STT terminals was
identified in the same tissue. The somata and dendrites of
the SI-projecting neurons from various thalamic nuclei
were quantitatively analyzed and compared between the
nuclei, and their relationships with the spinothalamic
(STT) terminals were studied.
From the earliest studies of Golgi impregnated cells (e.g.,
Cajal1911; Guillery, 1966; Tombol, 1967,19691,it has been
observed that dendritic shapes in any given nucleus (or
region) are highly variable. Cell morphology, size, and
packing density are the main parameters by which various
regions or nuclei have been delineated in the central
nervous system. However, the variability in dendritic shapes
is large enough that one can postulate that the morphology
of neurons projecting to a single target from multiple nuclei
may be more homogeneous than the general differences
observed between these nuclei. The latter would imply that
the innervating target would determine the detailed neuronal dendritic branching pattern. Alternatively, the neuronal morphology within multiple nuclei may be more homogeneous owing to a sharing of common afferent input.
These hypotheses were directly tested in this study, and
both were shown to be partially correct.
MATERIALS AND METHODS
Extracellular tracer injections
This study utilized five adult squirrel monkeys (500-700
kg, both sexes). Three of the monkeys were used in an
earlier study (Shi et al., 1993) to describe the locations of
the retrogradely labeled neurons and their overlap with
STT inputs. The housing, care, and surgical procedures
followed the institutional guidelines established by the
Committee for the Humane Use of Animals. One day prior
to surgery, the monkeys were treated with dexamethasone
(0.25 mgikg, i.m., twice daily) to prevent edema. On the day
of surgery, doses of dexamethasone and the antibiotic
Rocephin (75 mgikg, i.m.) were injected, and each animal
was initially anesthetized with Ketamine (30 mgikg, i.m.).
The surgical level of anesthesia in one animal was maintained with a Nembutal drip (0-10 mg/kg/h, i.v.) and
Ketamine (10 mgikgih, i.m.1, whereas the other animals
were maintained with 0.5-1.5% halothane or isoflurane
mixed with ?hO2and % NzO. The animals were intubated in
order to insure a patent airway. Intravenous fluid replacement with 5% dextrose lactated Ringer's solution was given
during the experiment. Body temperature, expired GOz
concentration, oxygen saturation (using a pulse oximeter),
and heart rate were monitored non-invasively and were
maintained within physiological limits.
The anesthetized animals were placed in a stereotactic
frame, and a craniotomy was performed under sterile
conditions. The body map in SI was physiologically determined and marked on an enlarged color photograph of the
cortical surface around the central sulcus. On one hemisphere, a retrograde fluorescent tracer, 2% Diamidino
Yellow (DY), was deposited on the surface of the SI hand
region (including areas 3a, 3b, 1,and 2) by placing a piece of
DY soaked filter paper on the cortex for 20-40 minutes (for
details see Shi et al., 1993; and Gingold et al., 1991). On the
other hemisphere, or in different animals (see Table l ) ,
green beads (GB), 2% fast blue (FBI, or 2% DY, was injected
into all cortical layers of the equivalent region of SI with a
Hamilton syringe. In four of these animals, the cervical
spinal cord was exposed through a laminetomy and 3.5-5 p1
of 2% wheat germ agglutinin-horseradish peroxidase (WGAHRP, Sigma) or 3.5-5 pl of 10% BD (biotin-dextran;
Vector) in normal saline was injected in the C5-Tl gray
matter with a glass pipette (for injection details, see Shi et
al., 1993). The spinal cord WGA-HRP injections were done
at the time of the cortical injections. The spinal cord BD
injections were made 4-6 weeks prior to the cortical
injections. Following surgery the animals were administered antibiotics for 3 days, and the animals that exhibited
pain symptoms during recovery were administered analgesics (Torbugesic 0.05 mgikg, i.m.1.
Tissue processing and intracellular injections
Following appropriate survival periods, the animals were
overdosed with Nembutal and perfused transcardially with
normal saline and 2.5% or 4% paraformaldehyde in 0.1 M
phosphate buffer (PB, pH 7.2). In addition, 0.025-0.05%
glutaraldehyde was added to the fixative for two of the
animals. Then, the brain was removed from the skull and
three to four series of coronal vibratome sections were
obtained and collected in a 0.1 M phosphate buffer (PB).
One series of the sections (150 pm in thickness) were stored
in 0.1 M PB at 4°C and used for intracellular filling within 2
weeks.
Retrogradely labeled thalamocortical cells were observed
under an epifluorescent microscope (excitation filters 395415 nm wavelength for DY and GB, 330-380 nm wavelength for FBI. Under direct visualization, retrogradely
labeled cells in each of the thalamic nuclei were picked for
intracellular injection of luicifer yellow. A glass pipette
pulled on a vertical pipette puller (David Kopf Instruments,
model 700C) and filled with a 4% aqueous solution of LY
(LY-CH,dilithium salt, 41H3620, Sigma) was mounted on a
micromanipulator attached to the microscope. A coronal
thalamic section was placed on a plain glass slide, covered
with a piece of filter paper in which a hole was punched, and
TABLE 1. Tracer Injections'
~
Side
SI injection
sites
Tracers
Left
Right
Hand, surface SI
Hand, all layer SI
DY
GB
2
Left
Right
Hand, surface SI
Hand, all laver SI
DY
GB
3
Left
Right
Hand, surface SI
DY
4
Left
Right
Left
Hand, all layer SI
FB
Hand. all laver SI
DY
Animal
no.
1
5
Spinal
iniection
C5-T1,3 5 pl
WGA-HRP
C5-Tl. 5
WGA-HRP
C5-T1, 3.5 61
BD
C5-T1,5 pl BD
'S1. somatosensory cortex; DY, diamidino yellow; GB, green heads; FB, fast blue;
WGA-HRP, wheat germ agglutinin-horse radish pexoxidos.
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
3
wetted with 0.1 M PB. Under visual control and a long
working-distance objective (50 x ), a retrogradely labeled
cell was intracellularly filled by delivering a negative current to the LY solution in the pipette through a piece of silver
wire (5-10 nA, 0.5-2 min, W-P Instruments Inc., see Maranto,
1982;Buhl and Lubke, 1988).When the dendrites of a cell were
completely filled, another cell was chosen and filled.
with the Tablecurve program (Jandel Scientific). With the
Tablecurve program, the distribution histograms of the
soma sizes (the soma size versus the number of neurons)
were fitted to normal distributions (one or two Gaussians).
Staining of LY-filled neurons and BD-labeled
STT terminals
The extent of the cortical and spinal cord injections in
three of the five animals is described in detail in an earlier
study (Shi et al., 1993). The cortical and spinal cord
injections in the other two animals were similar to the rest,
i.e., the cortical injections were concentrated in the hand
region of areas 3a, 3b, 1, and 2, whereas the spinal cord
injections were mainly located in the gray matter of the
cervical enlargement.
The number of retrogradely labeled cells from the
superficial SI were much fewer than those retrogradely
labeled from all layer-SI, but their distributions in the
thalamus were quite similar. Most retrogradely iabeied
thalamocortical neurons from the SI hand region were
primarily located in VPL, VPI, Pulo, and CL (for more
details, see Shi et al., 1993). A small number of labeled
neurons were seen in the posterior complex (PO), CM, and
VL. Nearly 300 neurons whose dendrites were completely,
or almost completely, filled were used in the dendritic
analysis, which were located in VPL (n = 761, VPI (n = 651,
Pulo (n = 611, PO (n = 30), CL (n = 411, CM (n = 121, and
VL (n = 12). Figure 1 shows samples of coronal sections
with LY-filled, immunocytochemically stained SI-projecting neurons in VPI.
LY-filled cells were either photoconverted (see Maranto,
1982; Buhl and Lubke, 1988) or immunocytochemically
reacted. Based on immunocytochemical methods to stain
LY-filled cells (Brandon, 1985; Brandon and Criswell, 1991)
and for BD (Brandt and Apkarian, 1992), a dual immunocytochemical staining method was developed. A section containing LY-filled cells was first placed in phosphate-buffered
saline containing 0.1% Triton-X 100 and 1% normal goat
serum (PBS-TX-NGS) for 20 mintues and then incubated
in PBS-TX-NGS containing the Fab fragment of anti-LY
antibody raised from rabbit (Fab-HCLY, 1:250, a gift from
Dr. C. Brandon) overnight at room temperature. After
washing the section twice in PBS-TX (0.5% Triton-X 100)
for 10 minutes, the section was immersed in avidin-biotin
complex (ABC-Kit, Vector) in PBS-TX solution for 2 hours
at 37°C. After two washes in PBS-TX for 10 minutes, the
section was reacted with 0.015% 3,5-diamino benzidine
(DAB)in Tris-buffer (pH 7.6) containing 0.4% nickel ammonium sulfate and 0.005% H202for 10 minutes. This procedure stained the BD labeled terminals in a blue-black color.
Following rinses in PBS, the tissue was incubated in
PBS-TX-NGS containing goat antiserum against FabHCLY (150, Cappel) for 2 hours, and then in PBS-TX-NGS
containing PAP (a complex of HRP and rabbit Fab fragment of antiserum to HRP, 1:100, Jackson Immunoresearch Laboratories, Inc.) for another 2 hours at 37°C.
After each antibody incubation and the PAP reaction, the
tissue was washed twice in PBS-TX-NGS for 10 minutes.
The section was then incubated in 0.05%DAB in Tris buffer
(pH 7.6) with 0.005% Hz02for 15 minutes at room temperature, rinsed in 0.1 M PB, and mounted out of 0.033M PB.
All of the LY-filled cells in a section were stained brown,
which was easily differentiated from the previously stained,
blue-black STT terminals (see Fig. 16).
RESULTS
Soma morphology of SI-projectingneurons
in different thalamic nuclei
In VPL, SI-projecting neurons usually had medium-sized
or larger somata with multipolar shapes and four to eight
primary dendrites. Samples of LY-filled,immunocytochemically stained SI-projecting neurons located in VPL are
shown in Figure 2. Most SI-projecting neurons in VPI were
medium-sized or small, and had four to eight primary
dendrites (see Fig. 3). The labeled SI-projecting neurons in
Pulo were medium-sized to large, multipolar, and usually
had five to nine primary dendrites with many branches (see
Fig. 4). labeled SI-projecting neurons in PO were mediumsized or large, multipolar or round, and had three to seven
primary dendrites (see Fig. 5 ) . The somata of SI-projecting
Data collection and analysis
neurons in CL were large or medium-sized, multipolar or
The locations of the LY-filled SI-projecting neurons and triangular-shaped, and had three to eight primary denlabeled STT terminals were determined relative to thalamic drites with branched dend itic trees (see Fig. 6). The
nuclear boundaries which were based on the background SI-projecting CM neurons usually had medium-sized, mulDAB staining and adjacent sections stained with cresyl tipolar somata and three to seven primary dendrites (Fig.
violet or cytochrome oxidase (for details regarding determi- 7). The SI-projecting VL neurons were large in their soma
nation of nuclear boundaries, see Gingold et al., 1991; Shi et sizes and had eight to 13primary dendrites (see Fig. 8).The
al., 1993; Apkarian and Shi 1994). Samples of these filled locations of the neurons shown in Figures 2-8 are illuscells and STT terminals were drawn with a camera lucida trated in a schematic diagram of coronal sections of the
and a computerized microscope system (Eutectic Electron- thalamus (Fig. 9).
Samples of stained SI-projecting neurons are shown in
ics, Raleigh, NC). The soma areas, soma shape factor, soma
widths and heights, and the approximate dendritic fields Figures 2-8. In general, the soma areas of the retrogradely
were measured by a computerized video system (Bioquant, labeled SI-projecting neurons varied from 78 pm2 to 752
R. M. Bioquantries). The shape factor = 4*a*areal km2 (mean 2 SD 325 ? 110, n = 262). Table 2 is a sumperimeter squared. The fractal dimension of the camera mary of the soma sizes and other measures of the SIlucida drawn neurons were measured using the box- projecting neurons in VPL, VPI, Pulo, PO, CL, CM, and VL.
counting method (Krauss et al., 1994). The total dendritic The soma areas of the SI-projecting neurons in VPL
length was measured with a computer program Mocha (351 & 135 pm2), Pulo (353 2 79 pm2),and CL (351 2 89
(Jandel Scientific). Statistical analyses were done with pm2) were slightly larger than those in PO (333 2 63 pm2),
Sigmastat (Jandel Scientific), and curve fitting was done CM (320 2 60 km2), and VPI (249 ? 91 pm2). The SI-
4
T. SHI AND A.V. APKARIAN
Fig. 1. A photomicrograph of Lucifer yellow (LY)-filled,immunocytochemically somatosensory cortex stained (SI) projecting neurons in
ventro posterior inferior nucleus (WI) region of the left thalamus.
Spinothalamic fibers coursing along the ventral border of VPI and
within WI and ventroposterior lateral nucleus (VPL) can also he ob-
served. The dashed line shows the border between VPI and dorsally
adjacent VPL and ventroposterior medial nucleus (VPM). Scale bar = 50
km. Insert is the plot of this thalamic section indicating the location of
the photomicrograph. Dorsal is up and lateral is left. The orientations
of all other figures are the same, except Figure 17C, D.
projecting neurons in VL had the largest soma areas
(410 ? 142 pm2) among all of the LY-filled SI-projecting
neurons in these thalamic nuclei. The comparison of the
soma sizes between the different thalamic nuclei resulted in
a significant difference (Kruskal-Wallis one-way ANOVA,
P c .0001, Fig. 10A). Post-hoc pairwise multiple comparison (Dunn’s method) shows that VPI soma sizes are
significantly smaller than VPL, Pulo, PO, CL, and VL
( P < ,051.All other pairs were not significantly different.
The shapes of these SI-projecting neurons were multipolar, triangular, round, and flat. The average shape factor
was highest for the VL cells (0.742 ? 0.051) and lowest for
VPI cells (0.567 0.123, Fig. IOB).There was a significant
difference in soma shape factor between the thalamic nuclei
(Kruskal-Wallis one-way ANOVA, P < .0002). Post-hoc
pairwise multiple comparison (Dunn’s method) shows that
VL shape factors are significantly larger than VPI, VPL,
Pulo, PO, and CL ( P < .05). All other pairs were not
*
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
E
Fig. 2. A-E: Camera lucida drawings of SI-projecting neurons in VPL. Scale bar = 30 pm. F: A
photomicrograph showing the smooth dendrites of SI-projectingneuron in W L . Scale bar = 10 km.
5
T.SHI AND A.V. APKARIAN
6
A
D
\-
Fig. 3. Camera lucida drawings (A,D) and computer plots (B,C) of SI-projectingneurons in VPI. Scale
bars = 30 km. E, F: Photomicrographs of typical dendritic branching patterns for WI SI-projecting
neurons. Scale bar = 10 km.
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
i
B
/
7
i
\
Fig. 4. A-C: Camera lucida drawings of SI-projecting neurons in anterior puloinlar (Pulo). Scale
bars = 30 pm. D,E: Typical dendrites of 51-projectingneurons in Pulo on which there are a few tiny spines.
Scale bar = 10 pm. (Figure 4 continued on next page.)
8
T. SHI AND A.V. APJSARIAN
Figure 4
(Continued from previous page.)
significantly different. The average ratio of the soma width
to the soma height in VPI was the largest (1.183 ? 0.409).
This ratio was smallest for SI-projecting neurons in CL
(0.894& 0.275, Fig. 1OC).However, these orientation differences were not statistically significant. These measures for
the SI-projecting neuronal somata indicate that the soma
area, shape factor, and orientation are usually similar
across the nuclei, with the exceptions of VL and W I .
Dendritic morphology of SI-projecting
neurons in different thalamic nuclei
In coronal sections, the VPL neurons had dendritic trees
with two to four order branches in dichotomous and
tuft-like patterns, that covered all possible directions from
the somata (Fig. 2). Generally, the dendritic sizes and
branching patterns of the neurons in VPL and VPI were
similar (Fig. 3), except for the VPI neurons located on or
near the ventrolateral border which rarely had dendrites
directed outward (Fig. 3C). Dendritic appendages, e.g.,
dendritic swellings, beads, or spines, were only occasionally
found on VPL and VPI dendrites (Figs. 2, 3). The primary
dendrites of Pulo neurons were usually short and richly
branching. Dendritic appendages were rarely seen on the
Pulo dendrites (Fig. 4).A few Pulo neurons had a small
number of primary dendrites with long dendritic branches
(Fig. 4D). The PO neurons usually had fewer primary
dendrites and branches, and their dendrites were basically
smooth (Fig. 5). The CL neurons branched in patterns
similar to W L and VPI neurons, and some had dendritic
swellings (Fig. 6 ) . The primary dendrites of CM neurons
seemed to be of two different types: sparsely branching and
densely branching. The densely branched dendrites often
had curves and spines (Fig. 7A,C,D) and the sparsely
branched ones were straight and smooth (Fig. 7B). Some
primary dendrites in the VL neurons branched near the
somata, whereas the others had no branches. The VL
dendrites were basically smooth (Fig. 8). In the majority of
the neurons the axons were not readily identifiable.
The numbers of primary dendrites of SI-projecting neurons were significantly different between the different
thalamic nuclei (Kruskal-Wallis ANOVA, P < .0001). The
VL neurons had the most primary dendrites (10.16 ? 1.751,
and the CM neurons had the least (4.483? 1.19, Fig. 11A).
Post-hoc pairwise multiple comparison (Dunn's method)
shows that the numbers of primary dendrites for VL and
Pulo were different from the neurons in the other nuclei
( P < .05). The total dendritic length for individual SIprojecting neurons were measured from two-dimensional
drawings. The dendritic lengths of SI-projecting neurons
were significantly different between the different thalamic
nuclei (Kruskal-Wallis ANOVA, P < .05). Post-hoc pairwise multiple comparison (Dunn's method) shows signifi-
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
9
C
Fig. 5. A-C: Camera lucida drawings of SI-projecting neurons in posterior complex (PO). Scale
bar = 30 Fm. D, E: Photomicrographs of SI-projecting neuron and dendrites in PO. Dendritic spines can be
seen in D. Structures out of focus are mainly spinothalamic fibers. Scale bar = 10 Fm.
cant differences between PO and VL, and PO and Pulo
( P i.05). The total dendritic lengths of the VL neurons
were the longest (4,684 ? 582 pm) among all-layer SIprojectingneurons, followed by Pulo neurons (3,699 2 1,865
pm), and were shortest for PO neurons (1,251 2 388 pm,
Fig. 11B). In addition, the fractal dimensions of the dendrites of individual neurons were measured, and the result
of such a measurement using box-counting method for a
VPI cell is shown in Figure 12A. On average, the fractal
dimensions in VL were the highest (1,410 2 0.050) among
all these nuclei, whereas those in PO were the smallest
(1,273 0.115, Fig. 12B and Table 2). The fractal dimensions of SI-projecting neurons were significantly different
between the different thalamic nuclei (Kruskal-Wallis
ANOVA, P < .05). The relative pattern of fractal dimension across the thalamic nuclei was very similar to the
pattern observed for the number of primary dendrites in
the same nuclei (see Fig. 12A,B).
*
T. SHI AND A.V. AF’KARIAN
10
A
\ -
Fig. 6 . Camera lucida drawings (A,D) and computer plots (C) of SI-projecting neurons in centrolateral
nucleus (CL). Scale bars = 30 pm. B: A photomicrograph of dendrites shown in A. The corresponding
dendritic branches are indicated in A and B with “v.” Scale bar = 10 am.
Comparisons of SI-projectingneurons
in VPL and VPI
The SI-projecting neurons in VPL and VPI were compared, since these two lateral thalamic nuclei were consid-
ered as important relay nuclei to SI (Gingold et al., 1991).
The cytoarchitecture of these two nuclei looked different in
Nissl-stained or cytochrome oxidase-stained sections (ref.
Fig. 1 in Apkarian and Shi, 1994). The present study
uncovered a number of morphological differences between
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
11
B
Fig. 7. A-C: Camera lucida drawings of SI-projecting neurons in centromedial nucleus (CM). Scale
bars = 30 Fm. D: A photomicrograph of dendrites from neuron in C, indxated with “v.” Dendritic spines
can be seen. The thick fiber on the upper right corner is a spinothalamic axon. Scale bar = 10 Fm.
the SI-projecting neurons in VPL and VPI. The soma sizes
or areas of SI-projecting neurons in VPL (351.5 t 135.2
pm2) were larger than those in VPI (249.5 t 90.8 pm2,
t-test P < .0001). The soma size distribution of the VPL
neurons could not be well fitted by a single Gaussian, but
was fitted by two Gaussians (r2= 0.9139, Fig. 13A). The
two-Gaussian fit remained the better fit for the cell size
histograms after changing the histogram bin size. The
sharper curve (mean = 335 pm2,SD = 28, amplitude = 26)
was 23.3% of the total area, and the second curve
(mean = 389 pm2, SD = 158, amplitude = 15) was 76.7%
of the total area. The soma size distribution of the VPI
SI-projecting neurons was also fitted to a two-Gaussian
distribution (r2= 0.7269, Fig. 13B). The two Gaussian
curves had very little overlap. The first curve (mean = 273
pm2, SD = 90, amplitude = 20) occupied 96.8% of the total
area, and the second curve (mean = 565 pm2, SD = 28,
amplitude = 2.1) occupied the rest of the total area. The
soma shape factors of the VPL neurons (0.607 2 0.107)
were slightly larger than those of the VPI neurons
T. SHI AND A.V. APKARIAN
12
B
Fig. 8. A, B: Camera luicda drawings of SI-projecting neurons in ventrolateral nucleus (VL). Scale
bar = 30 km. C: A photomicrograph of another VL neuron with smooth dendrites. Scale bar = 10 pm.
(0.567 t 0.123, t-test P < .05). The ratios of soma width to
soma height of the VPL neurons were smaller than those of
VPI (t-test P < . O l ) , implying that the VPI neuronal
somata are more horizontally oriented.
Although there was a significant difference in the average
soma sizes of all-layer %projecting neurons between VPL
and VPI, the soma sizes of the superficial SI-projecting
neurons in VPL and VPI were very similar to each other
(249.41 5 102.16 vs. 239.59 t 97.01, Fig. 14). The soma
sizes of the superficial SI-projecting neurons in VPL were
smaller than those of the all-layer SI-projecting neurons in
VPL (402.13 t 125.25, t-test P < .0001). The soma sizes of
the superficial SI-projecting neurons in VPI were not
different from those of all-layer SI-projecting neurons in
VPI (271.75 t 72.26, t-test P > .05). When the soma size
distributions of the superficial SI-projecting cells were
fitted with Gaussian curves, these soma sizes of the VPL
cells were adequately fitted with one Gaussian (r2= 0.975,
mean = 245 pm2,SD = 61, Fig. 13C). Those of the VPI cells
were still better fitted with two Gaussians (r2= 0.98, Fig.
13D). The sharper peak (mean = 205 p,m2, SD = 25, amplitude = 7.1) covered 32% of the total area, and the second
curve (mean = 245 pm2, SD = 82, amplitude = 4.6) covered the rest of the area. The relative contributions of each
curve for the superficial-SI projecting neurons seem different from that described above for all-layer SI-projecting
neurons in VPI.
Figure 14 shows other morphological properties of superficial SI and all-layer SI-projecting cells in VPL and VPI.
The ratio of the soma width to height of superficial SIprojecting neurons in VPI was higher than that in VPL
(1.228 2 0.44 vs. 1.010 & 0.32, t-test P < .05) and also
higher than that of the all-layer SI-projecting neurons in
VPI (1.080 & 0.300, t test P < ,051, which means that this
group of neurons was more horizontally oriented than the
others. The rest of the parameters measured showed that
MORPHOLOGY OF Sl-I'ROJECTING THALAMIC CEI,I,S
AP 4.6-5.0
13
5.3-5.6
6.2-7.4
Fig. 9. A schematic diagram of coronal thabdrnic sections a t difkrent anterior-posterior levels. 'I'hc
locations of the %projecting neurons in each of these thalamic nuclci, shown in Figures 2 -8, are shown
with t h e same alphabets a s used in Figures 2-8.
TAULE 2. Summary of Soma a n d I l e n d r i t i c M e a s u r e s i n Each Thalamic N ucleus'
VPL
VPI
Number of neurons
Soma area (krn2)
Soma shape factor
Ratio of soma x l y
Number of primary dendrites
Total dendritic length (pmV
76
351 i 135
0.607 0.107
1.013 2 0.266
6.21 i 1411
2,976 e 770
l+:ict,:d dirncnswri2
I . X l I0.49
n = 19
65
249 ? 9 1
0.567 ? 0.123
1.183 t 0.49
6.40 ? 1.60
2,864 t 1,093
n = 13
1.352 i 0.081
n = 11
*
=
m
Pulo
the dendrites of the superficial SI-projecting cells were
similar to those of all-layer SI-projecting cells in the two
nuclei.
Nine SI-projecting neurons in VPL (total of 41 dendritic
trees) and five SI-projecting neurons in VPI (total of 32
dendritic trees) were analyzed after tracing them with the
computerized neuron tracing system. The stem diameters
of the primary dendrities, the total dendritic length, the
total dendritic membrane areas, and the numbers of the
dendritic branch points for individual dendritic trees were
similar between these two nuclei. There were strong positive correlations between the primary dendritic diameter
and the total dendritic length (r = 0.584, P < .0001 in
VPL; r = 0.599, P < .001 in VPI), the total dendritic area
(r = 0.684, P < .0001 in VPL; r = 0.688, P < .0001 in
VPI), and the number of the branch points (r = 0.559,
P < .0001 in VPL; r = 0.654, P < ,0001 in VPI; see Fig.
15A-C). The slopes of diameter versus total length and
diameter versus total area were not significantly different
between VPL (370.6, 650.0) and VPI cells (272.7, 389.9,
Fig. 13A, B). The slopes for diameter versus branch points
for VPL (6.54) and VPI (3.27) were significantly different
PO
CL
CM
vr,
30
333 t 63
0.592 i 0.100
1.045 I(l.240
5.43 t 1.57
1,376 i 472
n = 3
1.273 i 0.115
n = 6
41
351 ? 89
0.572 -t 0.121
0 8514 I0 275
5.58 ? 1.51
2,350 i 757
n=9
1.352 t 0.027
n = 9
12
320 i 60
0.624 2 0.091
12
410 i 142
0.742 i 0.051
1 00.1 t 0 214
1 Ill7
4.48 i 1.19
3,127 i 1,126
n=4
1.283 ? 0.100
n=4
10.16 2 1.75
4,684 ? 582
n = 8
1.410 i 0.050
n = 8
6
0 20%
from each other (t-test, P < .0001). There were also strong
positive correlations between the numbers of the branch
points and the total dendritic length in these two nuclei
(r = 0.848, P < .0001 in VPL; r = 0.90, P < ,0001 in VPI;
see Fig. 15D). The slope between the numbers of the branch
points and the total dendritic length in VPL (45.7) was
significantly less than that in VPI (82.0, t-test, P < .0001).
Relationship between SI-projectingneurons
and spinothalamic terminals
The S'I'T terminals anterogradely labeled from the contralateral cervical enlargement were found in several thalamic
nuclei, including VPL, VPI, PO, CL, and sparsely in Pulo as
well as in the medial thalamic nuclei (see Gingold et al.,
1991; Shi et al., 1993). Since the labeled STT terminals in
the thalamus were a series of clusters, the approximate
locations of STT terminals were determined before intracellular injections by examining the stained, BD-labeled STT
terminals in the alternative serial sections. Figure 16A is a
color photograph of a Pulo SI-projecting cell (brown), and
Figure 16B shows a field in CL where STT terminals can be
T.SHI AND A.V. AF'KARIAN
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Fig. 11. A The numbers of the primary dendrites (A) and the total
dendritic (den.) lengths (B) measured from individual neurons for
SI-projecting neurons in the thalamic nuclei indicated.
T
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VPL VPIPulo PO CL C M VL
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Fig. 10. The soma areas of the SI-projectingneurons in each of the
thalamic nuclei indicated. B: The soma shape factors of the neurons. C :
The ratio of soma width (x)to soma height (y).
seen in blue-black, while the dendrites of two SI-projecting
cells are shown in brown. In these sections, many STT
terminal boutons were located between Nissl-stained somata, and only a few appeared to contact the somata,
implying that most STT terminals synapse on the dendrites
of thalamic neurons. A stereo-pair of color photos of a
SI-projecting cell in C1 with STT terminals abutting one of
its dendrites is shown in Figure 16C,D (three-dimensional
microscope, Edge Scientific Instrument Gorp., Santa
Monica, CAI. The same neuron and the STT terminals are
shown in more detail in drawing in Figure 17A. A total of
ten SI-projecting cells were observed in similar close proximity to, or seemingly directly contacting, STT terminals, of
which, two cells were in VPL, four were in VPI, two were in
CL, and two were in PO. Drawings of synapse-like STT
boutons abutting SI-projecting cells and other nearby STT
axons and terminals are shown for four neurons in Figure
17. All the synapse-like boutons were contacting the body of
the primary, secondary, and tertiary dendrites.
DISCUSSION
Intracellular labeling of the entire neuron
Neurons intracellularly filled with LY reveal neuronal
morphology at the same degree of detail as Golgi-impregnated material, as a result the method is widely used (Buhl
and Lubke, 1988; Buhl et al., 1989; Buhl, 1993). Many
laboratories have tried intracellular injection of HRP or
other tracers in thalamic neurons of live animals and have
filled a few labeled neurons in the cat (Yen and Jones, 1983;
Yamamoto et al., 1984; Yen et al., 1985; Nomura et al.,
1992), rat (Ohara and Havton, 19941, or monkey (Havton
and Ohara, 1993).Since the number of the neurons intracellularly injected in alive animals, especially in the monkey, is
MORPHOLOGY OF SI-PROJECTING THALAMIC CELIAS
A
h
104,
I
I
I
i
103
1
15
missed, but this should not significantly influence the
results of the present study.
Thalamocortical neurons
in the lateral thalamus
In the lateral thalamus, thalamocortical neurons have
been
studied in the cat (Yen and Jones, 1983; Yen et al.,
W
1985; Penny et al., 19831, rat (Peschanski et al., 1984;
U
Harris, 1986; Chiaia et al., 1991; Ohara and Havton, 1994),
a,
and monkey (Havton and Ohara, 1993, 1994). Yen and
1o2
Jones (1983; Yen et al., 1985) intracellularly injected HRP
v)
into physiologically identified neurons in the cat ventrobasal complex, and divided these neurons into three classes:
0
type I, type 11, and type 111. The type I and I1 neurons are
rn 10'
relay neurons, and the type I11 neurons are interneurons.
Many of our LY-filled neurons in VPL looked like their type
I neurons, with medial or large somata, large and smooth
primary dendrites radiating in all directions from the soma,
100
and tufts of multiple second-order dendrites. A small
1
10'
1o2
103
104 number of the LY-filled VPL thalamocortical neurons
resembled type I1 neurons, with smaller soma sizes, thinner
dichotomously branching primary dendrites, and with some
Size of boxes
dendritic appendages. The soma size distribution of the
VPL SI-projecting cells was better fitted to a two-Gaussian
1.5
curve, which implies two populations of SI-projecting neurons in VPL. The population with large mean and standard
n
T
W
deviation
is similar to cat type I neurons, and the popula[I: 1.4
tion with small mean and standard deviation is similar to
0
.cat type I1 neurons (compare Fig. 7 in Yen et al., 1985 to
Fig. 14A).
1.3
In general, the dendritic branching pattern of the VPL
SI-projecting neurons in the present study was similar to
5 1.2
the macaque VPL thalamocortical neurons intracellularly
recorded and filled with HRP by Havton and Ohara (19931,
a
.c.'
but the soma sizes and the total lengths of the dendrites of
1.1
our VPL neurons were smaller and shorter. The differences
L
should be due to the different species and also, perhaps, to
1
differences in methodology.
In the monkey, VPI neurons project to both SII (FriedVPL VPI Pulo PO CL CM VL
man and Murray, 1986; Stevens et al., 1993) and SI (Cusick
Fig. 12. A The fractal dimension measurement of a SI-projecting and Gould, 1990; Gingold et al., 1991; Shi et al., 1993).
Because little is known about the neuronal morphology in
neuron in VPI. B: The fractal dimensions of the SI-projectingneurons
in each of these thalamic nuclei.
VPI, we explored many SI-projecting neurons in this
nucleus. In comparison with VPL SI-projecting neurons,
the VPI SI-projecting neurons generally have smaller soma
sizes, more horizontally oriented somata, and similar denusually very low, intracellular tracer injection in the fixed dritic lengths and fractal dimensions. The soma size distritissue is much more convenient to systematically study a bution in both VPL and VPI neurons projecting to all layers
great number of neurons in a small number of animals. For of SI was well fitted to a two-Gaussian curve. These fits
example, as many as 40 neurons were filled in one fixed imply that the VPL and VPI neurons consist of two
section in the present study. Although LY-labeled cells can populations. Some VPI neurons had relatively more denbe directly observed with an epifluorescent microscope, dritic appendages than most VPL neurons. The dendriticpermanent staining is necessary for long-term or electron rich domain of VPI neurons implies that VPI neurons are
microscopic study. Both photoconversion and immunocyto- able to receive at least as much information as VPL neurons
chemical staining methods were used in the present study. and play an important role in the processing of somatosenThe LY photoconversion method only stained a couple of sory inputs. Recently, we studied the physiological features
LY-filled cells per section. In contrast, the immunocyto- of VPL and VPI neurons in the squirrel monkey (Apkarian
chemical method (Brandon, 1985; Brandon and Criswell, and Shi, 1994).Although the proportions of the nociceptive
1991) is better in that all LY-filled cells in a section can be neurons in the two nuclei were different, the general
stained at once. Only retrogradely labeled cells with somata physiologic features of VPI neurons were similar to those of
estimated to be in the middle of the thickness of the section VPL neurons, i.e., both of them could be classified into
were filled with LY. If such a cell was successfully injected, low-threshold, wide dynamic range, and high-threshold
most dendrites of the cell appeared to be filled and stained. types. The morphological similarities between filled VPL
The possibility cannot be excluded that a few of the and VPI neurons are consistent with the physiological
dendrites toward the top or bottom of the section were findings.
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Fig. 13. The curves of the soma size distributions of VPL (A,C) and W I neurons (B,D).A: All-layer
SI-projecting neurons in W L . B: All-layer SI-projecting neurons in VPI. C: Superficial SI-projecting
neurons in VPI. D: Superficial SI-projecting neurons in P I . The inserts show individual or multiple
Gaussian fits.
Within cat ventrobasal complex (Penny et al., 1983;
Rausell and Avendaiio, 1985) and monkey VPL (Rausell et
al., 1992), the neurons that project to the superficial layers
of SI usually have smaller soma sizes than the neurons that
project to the deeper layers. We retrogradely labeled thalamic neurons from the superficial layers or all layers of SI
and compared the superficial SI-projecting neurons with
all-layer SI-projecting neurons in VPL and VPI. The soma
sizes between these two groups of neurons are different in
VPL, but not in VPI. The superficial SI-projecting neurons
in VPL were similar in soma size to both superficial and
all-layer SI-projecting neurons in VPI. The soma size
distribution of the superficial SI-projecting neurons in VPL
was better fitted to a one-Gaussian curve, whereas the
superficial SI-projecting neurons in VPI was better fitted to
a two-Gaussian curve. It seems that only one population of
neurons in VPL, the group with smaller mean and standard
deviation in soma size, projects to the superficial layers of
SI. Conversely, the curve fits for VPI cells imply the
existence of three different populations of SI-projecting
neurons: one group that projects to superficial and deep
layers of SI, medium-sized with large variability; a second
group that projects mainly to superficial SI, small-sized
with small variability; and a third, largest-sized population
that only projects to the deeper layers.
In the present study and in previous studies (Gingold et
al., 1991; Shi et al., 19931, tracer injections in SI of the
squirrel monkey always resulted in some retrogradely
labeled neurons located in caudal VL. We cannot exclude
that the VL neurons were retrogradely labeled from the
adjacent primary motor cortex area 4, owing to tracer
spreading, since early studies (Friedman and Jones, 1981;
see reviews in Jones, 1985) show that the cells in the caudal
part of VL project to the primary motor cortex, but not to
SI. In the cat, Yamamoto et al. (1985a) intracellularly
recorded and stained neurons with HRP. A few of the
neurons were thalamocortical relay neurons and the rest
were thalamo-caudate relay, entopeduncular-responsive,
and cerebellar-responsive neurons. The soma sizes of the
VL neurons in the present study were smaller than those
described in the cat, whereas the dendritic fields were
similar. Some WGA-HRP retrogradely labeled VL neurons
in the macaque monkey were studied a t light and electron
microscopic levels (Kultas-Ilinsky, 1991). This retrograde
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
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Fig. 14. Comparisons between superficial SI-projecting neurons
and all layer SI-projecting neurons in VPL and W I . A Soma areas. B:
Soma shape factor. C : The ratio of the soma width (xJto soma height
(y). D: The numbers of primary (prim.) dendrites. E: The approximate
dendritic fields. F: The ratio of the dendritic (den.) field width (x)to the
dendritic field height (y). Asterisk indicates a significant difference (t
test,P < .05).
tracing technique only stains the somata and some primary
dendrites, as well as a few secondary dendrites. Compared
to the WGA-HRP-labeled VL thalamocortical neurons,
LY-filled neurons in the present study had more inflated
somata and more primary dendrites. The soma sizes, the
number of primary dendrites, total dendritic lengths, and
fractal dimensions of the VL neurons were the greatest
among all filled SI-projecting thalamic neurons in the
present study. Unlike other somatosensory thalamic nuclei,
VL does not receive direct inputs from the medial lemniscal
projection, nor from STT. The difference between the VL
neurons and the other SI-projecting neurons may reflect
their functional difference.
Thalamocortical neurons
in the posterior thalamus
The retrogradely labeled PO neurons from SI were only
located in the anterior portion of PO, the portion called
posterior nucleus. Only the neurons in the more posterior
portion of PO (the supergeniculate and limitan nuclei) were
studied in the cat by Winer and Morest (1983). The soma
sizes and shapes of our PO neurons were similar to the
somata of those principal cells in the suprageniculate and
limitans nuclei shown by Winer and Morest (1983), whereas
their dendritic domains were different. These morphological differences may be due to species differences and to the
T. SHI AND A.V. APKARIAN
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Fig. 15. The relationships between the primary (prim.) dendritic
(den.) diameter and the dendritic length (A), the dendritic areas (B),
and the number of the branching points (C) on individual dendritic
trees of SI-projectingneurons in VPL (triangles and solid line) and VF'I
(dots and dashed line). D: The relationships between the number of
branching points and the total dendritic length.
different subdivision where PO neurons were located. Alternatively, when compared with the SI-projecting neurons in
VPL and VPI, PO SI-projecting neurons were similar in
soma size and different in total dendritic length and
dendritic fractal dimension. The differences in the dendrites is probably due to the fact that some PO dendrites
had sparse branches.
Earlier studies indicate that Pulo neurons can be retrogradely labeled from area 2 of SI (Pons and Kaas, 1985).
Tracer injections in SI of squirrel monkeys result in a great
Fig. 16. A: Photomicrographs of a Lucifer yellow (LY)-filled and
immunocytochemically stained SI-projecting neuron in Pulo. B: The
dendrites of a LY-filled and immunocytochemically stained SIprojecting neurons (orange brown) and nearby BD-labeled spinothalamic (STT) terminals (blue black) in CL. In A and B, dorsal is left. C,
D: A pair of stereoscopic photomicrographs of a SI-projecting neurons
(orange brown) contacted by STT terminal boutons (blue black) in
centrolateral (CL).This pair of photomicrographswere taken through a
three-dimensional microscope (Edge Scientific Instrument Corp.). In C
and D, dorsal is up. Scale bars = 20 pm.
Figure 16
Figure 17 Aand B
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
21
Fig. 17. Drawings of SI-projecting neurons with contacting STT terminal boutons in W L (A),WI (B)
and CL ( C ) .Arrows point to the contacts between the dendrites and the boutons. The left neuron in C is the
same as in Figure 16C,D. Scale bars = 20 Fm.
number of retrogradely labeled cells in Pulo (Gingold et al.,
1991; Shi et al., 1993). In the present study, the LY-filled
neurons in Pulo had soma sizes similar to those in VPL and
PO. However, the Pulo neurons had more primary dendrites, longer total dendritic length, and slightly higher
dendritic fractal dimension than the SI-projecting neurons
in the other thalamic nuclei, except those in VL. The
pattern of the dendritic distribution of some Pulo neurons
is similar to VPL neurons, whereas the dendritic distribution of other Pulo neurons resembles VL SI-projecting
neurons. As found in most other thalamic nuclei, the Pulo
neurons also had few dendritic swellings. A comparison of
neuronal morphologies between Pulo, PO, VPL, VPI revealed that the SI-projecting neurons in Pulo were quite
different from those in the other thalamic nuclei in their
dendritic domain, suggesting that SI-projecting neurons in
Pulo may have a distinct physiologic function.
Thalamocortical neurons in thalamic
intralamina nuclei
Anatomic studies have shown that CL is a major intralaminar nucleus which projects to SI (Jones and Leavitt,
1974; Jones et al., 1979; Bentivoglio et al., 1983; Gingold et
al., 1991; Shi et al., 1993), whereas only a few CM neurons
can be retrogradely labeled from SI (Bentivoglio et al.,
1983; Gingold et al., 1991; Shi et al., 1993). CL and CM
neurons can respond to noxious and other sensory stimuli
(Dong et al., 1978; Dostrovsky and Guilbaud, 1990). Recently, Fenelon et al. (1994) have studied the neuronal
morphology in the central complex (CM and Pf in our
terminology) of both macaque monkey and human thalami
by means of the Golgi method. These researchers classified
the central complex into three subdivisions, and indicated
that the large, principal neurons in the different subdivisions were different in their dendritic domains. The dendritic branch pattern and the dendritic shapes of our CM
SI-projecting neurons are similar to those of the macaque
neurons located in the most lateral subdivision, pars paralateralis, and in the medial subdivision, pars media. To
date, the neuronal morphology of only a few CL neurons
have been were explored in the cat (Yamamoto et al., 1985a;
Tomb61 et al., 1990). We intracellularly labeled many
SI-projecting neurons in CL, and compared them with
other SI-projecting neurons. The measured parameters for
somata and dendrites of the CL neurons were not significantly different from those parameters measured for the
SI-projecting neurons in CM, VPL, VPI, and PO. However,
the CM dendrites had many dendritic spines, but the CL
dendrites did not, although both nuclei are in the intralaminar region. Surprisingly, the SI-projecting neurons in CL
were more similar to those in VPL than those in CM in their
soma sizes, dendritic branching patterns, and dendritic
appendages. Since both CL and VPL receive medial lemniscal and STT inputs, the similarities between the SIprojecting neurons in the two nuclei implies that similar
afferent inputs may somehow alter the dendritic morphology of their target neurons, and that the neurons in CL may
22
play an important role in processing somatosensory information.
Relationship between SI-projectingthalamic
neurons and STT terminals
For many years it has been indicated that the STT inputs
can be conveyed into SI (Jones, 1985; Kaas and Pons, 1988;
Gingold et al., 1991; Shi et al., 1993; Apkarian and Shi,
1994) since STT projects to the thalamus and the thalamic
neurons project to SI. Ralston and his coworkers (1985)
studied STT terminals anterogradely labeled with HRP
within VPL of the cat, rat, and monkey at the electron
microscopic level. They showed that the STT terminals
synapsed on the dendrites of VPL neurons that were
regarded as thalamocortical because these neurons were
not interneurons or GABAergic neurons (Ralston and
Ralston, 1992). In order to anatomically explore STT
connections with thalamocortical neurons whose cortical
projections are identified, multiple tracing techniques are
necessary. Buhl et al. (1989; Buhl, 1993) used an anterograde degeneration technique together with intracellular
injection of LY in retrogradely labeled cortical neurons.
Degeneration technique does not adequately trace the STT
terminals in the primate. Recently, a technique of anterograde tracing with Phaseolus uulgaris-leucoagglutinin
(PHA-L) and retrograde fluorescent tracing combined with
intracellular injection of LY was developed in the rat by
Wouterlood et al. (1990). PHA-L has been tried in our
laboratory for labeling monkey STT terminals, and few or
no terminals were well-labeled. Since BD can label both
large and small fibers or terminals in the monkey (Brandt
and Apkarian, 1992; Shi et al., 19931, we used BD to label
STT terminals and LY to further label SI-projecting cells in
the present study. We found that many STT boutons were
near the filled SI-projecting cells and their dendrites.
However, only a few STT boutons seemed to contact the
dendrites of the intracellularly labeled cells. This is the first
study to show STT terminals contacting the identified
SI-projecting thalamic neurons in the primate at the light
microscopic level. This finding is consistent with the electron microscopic study by Ralston et al. (1985) and further
supports the idea that STT inputs are able to be directly
relayed to SI not only through VPL but also through VPI,
CL, and PO.
Compared with earlier studies (Gingold et al., 1991; Shi
et al., 1993), the percentage (less than 5%) of the SIprojecting cells contacted by STT boutons among all LYfilled SI-projecting cells in the present study is much lower
than the percentage (about 24%) of the cells overlapping
STT terminals of all retrogradely labeled SI-projecting cells
in our earlier studies. The difference between the present
study and the earlier studies must be due to the completely
different methods. In Gingold et al. (19911, only the somata
or nuclei of the SI-projecting cells were retrogradely labeled
and the STT afferents were anterogradely labeled with
WGA-HRP. Unlike the BD technique which clearly labels
the profiles of terminal structures, in WGA-HRP material it
is difficult to distinguish the terminal boutons from the
axons or fibers. Furthermore, in the Gingold et al. study
(1991) the SI-projecting cells were counted as STT overlapping cells when the labeled STT afferents were located
within 100 pm from the neuronal somata. In the present
study, synaptic contacts are identified as only those structures where a bouton was attached to a labeled neuronal
dendrite without a visible gap between them (examined
T. SHI AND A.V. AF'KARIAN
under lOOx objective). Whether or not the contacts are
actual synapses can only be proven with electron microscopy. We have tried to further identify these contacts at the
electron microscopic level, but the issue preservation was
not adequate. Moreover, we could not see the BD-labeled
terminals in the same section during LY injections. Therefore, it was difficult to always fill the cells closest to the
BD-labeled terminals. We conclude that this is one of the
reasons why only a small number of cells were found
contacted by STT terminals.
The results show that the neurons in VPL, VPI, and PO
had few STT terminal-dendritic contacts, implying that the
STT input is highly divergent in these nuclei. The SIprojecting neurons in CL seemed to have more STT contacts than the VPL or VPI neurons, which suggests that CL
is an important nucleus in relaying STT inputs to SI.
CONCLUSIONS
In the present study, both the SI-projecting neurons in
VPL, VPI, Pulo, PO, CL, CM, and VL and the STT
terminals were well labeled. SI-projecting neurons in VPL,
VPI, CL, and PO were found to have contacts with STT
terminal boutons, indicating that the method used to stain
the entire identified thalamocortical neuron and the anterogradely labeled terminals is useful for studying the morphology of multisynaptic structures. The quantitative analyses
and comparisons of the neuronal somata and dendrites
between the various thalamic nuclei revealed that the
SI-projecting neurons in these different nuclei were rather
similar in the soma and dendritic morphology with some
exceptions: 1)VL neurons had the largest soma sizes, the
most spherical soma shapes, the most numbers of the
primary dendrites, and the greatest total dendritic length
among all of the studied neurons. 2) Pulo neurons also had
more primary dendrites and longer total dendritic lengths
than the neurons in VPL, VPI, CL, PO, and CM. 3) The
SI-projecting VPL and VPI neurons were different in their
soma sizes, shape factors, and orientations. The superficial
SI-projecting cells in VPL were smaller than the rest of
SI-projecting cells in VPL. However, the VPI cells projecting to superficial SI were not different from the VPI cells
projecting to all SI layers.
These results indicate that although there were some
differences in the morphology of SI-projecting cells in the
different thalamic nuclei, morphological similarities existed
among SI-projecting neurons. Actually, VPL, VPI, and CL
neurons projecting to SI were similar in dendritic morphology and branching pattern, and varied from PO, CM, and
VL neurons projecting to SI. The first group of nuclei share
common inputs (medial lemniscal and STT) and some
common cortical targets (SI and SII). The inputs to the
second group are from more diverse regions (e.g., inferior
colliculus, STT, dentate nucleus, globus pallidus), and their
outputs are also more divergent (e.g., SI, insular, auditory,
and motor cortices). Therefore, it is implied that both the
afferent inputs and efferent targets may, at least partially,
affect the determination of detailed dendritic branching
pattern of cortically projecting neurons. It should be emphasized, however, that this hypothesis remains to be rigorously tested by examining morphologic differences between
cells with different input and output connectivities, within
a given nucleus (or region).
MORPHOLOGY OF SI-PROJECTING THALAMIC CELLS
ACKNOWLEDGMENTS
We thank Dr. C. Brandon for providing antibodies, Dr.
D.R. Chialvo for help regarding fractal analysis, and Dr.
J.A. Robson for providing a computerized microscope. The
technical help of R.T. Stevens, H.M. Newman, and B.R.
Krauss is appreciated. This research was funded by the
Department of Neurosurgery at SUNY HSC.
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