Download Ventral Medial Nucleus Neurons Send Thalamocortical Afferents

Cerebral Cortex January 2015;25:221–235
doi:10.1093/cercor/bht216
Advance Access publication August 22, 2013
Ventral Medial Nucleus Neurons Send Thalamocortical Afferents More Widely and More
Preferentially to Layer 1 than Neurons of the Ventral Anterior–Ventral Lateral Nuclear
Complex in the Rat
Eriko Kuramoto1, Sachi Ohno1,2, Takahiro Furuta1, Tomo Unzai1, Yasuhiro R. Tanaka1, Hiroyuki Hioki1 and Takeshi Kaneko1
1
Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606–8501, Japan and
Department of Dental Anesthesiology, Graduate School of Medicine and Dentistry, Kagoshima University, Kagoshima 890-8544,
Japan
2
Address correspondence to: Prof. Takeshi Kaneko, Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University,
Kyoto 606-8501, Japan. Email: [email protected]
Not only inhibitory afferent-dominant zone (IZ) of the ventral
anterior–ventral lateral thalamic complex (VA-VL) but also the ventral
medial nucleus (VM) is known to receive strong inputs from the
basal ganglia and send axons to motor areas. We previously reported
differences in axonal arborization between IZ neurons and the other
VA-VL neurons in rats by single-neuron tracing with viral vectors. In
the present study, the axonal arborization of single VM neurons was
visualized by the same method, and compared with that of IZ
neurons. VM neurons formed fewer axon collaterals in the striatum,
but sent axon fibers more widely and more preferentially (79% of
fibers) to layer 1 of cortical areas than IZ neurons. Furthermore, the
VM seemed to contain at least 2 types of neurons; a major population of VM neurons sent axon fibers principally to motor-associated
areas as VA-VL neurons did, and the other population projected
mainly to orbital or cingulate areas. Although both VM and IZ
neurons receive strong basal ganglia inputs, these results suggest
that VM neurons, at a single neuron level, innervate the apical dendrites of cortical pyramidal neurons more intensely and more widely
than IZ neurons.
Keywords: layer 1, motor thalamic neurons, sindbis viral vector, singleneuron tracing, ventral medial thalamic nucleus
Introduction
The ventral medial thalamic nucleus (VM) receives afferents
from the basal ganglia (Carter and Fibiger 1978; Hendry et al.
1979; Di Chiara et al. 1979; Herkenham 1979; Deniau and Chevalier 1992; Sakai et al. 1998; Kha et al. 2001; Sakai and Bruce
2004; Kuramoto et al. 2011) and relays basal ganglia information
to the cerebral cortex including motor areas. Thus, the VM is at
least partly associated with the motor function as well as with
other cortical functions. In addition to the VM, the ventral
anterior and ventral lateral nuclear complex (VA-VL), the motor
thalamic nuclei, accepts abundant afferents not only from the
cerebellum but from the basal ganglia (for review, see Groenewegen and Witter 2004; Jones 2007). Those basal ganglia afferents to the VM and VA-VL are emitted in rats by the
entopeduncular nucleus (Ep; internal segment of the globus pallidus) and substantia nigra pars reticulata (SNr), and have been
suggested to be GABAergic by several lines of evidence. The destruction of the Ep and/or SNr has been reported to cause the
reduction of GABA content, glutamic acid decarboxylase (GAD)
activity, and GAD immunoreactivity in the motor thalamic
nuclei and VM (Di Chiara et al. 1979; Kilpatrick et al. 1980;
Penney and Young 1981; Kuramoto et al. 2011); nigrothalamic
transmission is blocked by GABA antagonist (MacLeod et al.
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1980); and GABA immunoreactivity has frequently been observed in the axon terminals derived from the Ep/SNr (Ilinsky
et al. 1997; Bodor et al. 2008). These inhibitory afferents principally enter the VM and inhibitory input-dominant zone (IZ) of
the VA-VL (Kuramoto et al. 2009, 2011), the latter being located
in the rostroventral portion of the VA-VL. On the other hand, the
caudodorsal portion of the VA-VL receives glutamatergic excitatory afferents mainly from the deep cerebellar nuclei (Schwarz
and Schmitz 1997; Kuramoto et al. 2011), and has been named
excitatory subcortical afferent-dominant zone (EZ) because of
the abundance of axon terminals immunoreactive for vesicular
glutamate transporter 2 (VGluT2) (Kuramoto et al. 2009).
It has been proposed that thalamic projection neurons are
classified into core-type and matrix-type neurons mainly on the
basis of projection targets in the cerebral cortical layers (for
review, see Jones 1998, 2001). Core-type neurons are located in
the specific relay nuclei and send axon fibers mainly to the
middle layers of the cerebral cortex, that is, L4 and the deep part
of L3, whereas matrix-type neurons are distributed throughout
the thalamic nuclei and project preferentially to L1 of the widespread cortical areas. Although the core- or matrix-type neurons
are also characterized by parvalbumin or calbindin immunoreactivity, respectively, in cat and monkey thalamic nuclei (Jones
1998, 2001), this chemical differentiation only partially works in
rodent thalamic nuclei. In the rat thalamus, no projection
neurons are immunopositive for parvalbumin, but many neurons
are positive for calbindin (Rubio-Garrido et al. 2007; Kuramoto
et al. 2009, 2011; Ohno et al. 2012). Neurons in the VM are well
known to project mainly to L1 of widespread cortical areas including motor-associated areas in the rat (Krettek and Price 1977;
Herkenham 1979, 1980; Arbuthnott et al. 1990; Mitchell and
Cauller 2001; Rubio-Garrido et al. 2009) and cat brains (Glenn
et al. 1982), although a similar projection of VM neurons has not
been reported in the monkey brain (Jones 2007).
We recently developed a replication-deficient Sindbis viral
vector which was designed to express palmitoylation siteattached green fluorescent protein (palGFP) (Furuta et al.
2001), and used the vector for anterograde tracing of neurons
(Nakamura et al. 2002, 2004, 2005; Ito et al. 2007; Furuta et al.
2008). The vector worked as a highly sensitive anterograde
tracer, because infected neurons produced an enormous
amount of palGFP under the strong subgenomic promoter of
the virus, and the palGFP was targeted to plasma membrane including axonal membrane by the addition of 2 long fatty acyl residues, palmitoyl residues, to the protein (Moriyoshi et al.
1996). We first applied this vector to single neuron labeling of
mesencephalic dopamine neurons, and were surprised to
observe far denser and more widespread axon fibers in the striatum than expected (Matsuda et al. 2009). We then started to
apply this single neuron-labeling technique to visualization of
thalamic projection neurons including motor and sensory thalamic nuclei (Kuramoto et al. 2009; Ohno et al. 2012). In the
VA-VL, IZ neurons were classified as matrix-type neurons from
the observation that more than half of their projecting axon
fibers were distributed in L1 of the cerebral cortex (Kuramoto
et al. 2009). VM neurons had similar input–output organization
to those of IZ neurons; both the neuron groups received inhibitory afferents from the basal ganglia and sent efferents preferentially to L1 of the cerebral cortex including motor-associated
areas. However, we noticed some differences between the 2
groups of matrix-type neurons during a series of single neuronlabeling studies in the motor thalamic nuclei and their surrounding nuclei. In the present study, we first tried to reveal the quantitative properties of axonal arborization of single VM neurons
and then compare them with those of single IZ neurons.
Materials and Methods
Animals
Adult male Sprague-Dawley rats (Shizuoka Laboratory Animal Center,
Shizuoka, Japan), weighing 300–400 g, were used in the present study.
The experiments were approved by the Committees for Animal Care
(Med Kyo 12013, 12014) and for Recombinant DNA Study (120093) in
Kyoto University. All efforts were made to minimize the suffering and
number of animals used in the present study.
Injection of Sindbis Viral Vectors and Fixation
Seventy rats were anesthetized by intraperitoneal injection of chloral
hydrate (30 mg/ 100 g body weight). For single neuron labeling, a
mixture of palGFP Sindbis viral vectors (1–2 × 102 infectious units (IU);
Furuta et al. 2001) and Sindbis viral vectors expressing palmitoylation
site-attached monomeric red fluorescent protein ( pal-mRFP) (1–2 × 102
IU; Nishino et al. 2008) in 0.3 µl of 5 mM sodium phosphate ( pH
7.4)-buffered 0.9% saline (PBS) containing 0.5% bovine serum albumin
(BSA) was injected into the VM (2.3–3.0 mm posterior to the bregma,
1.2–1.6 mm lateral to the midline, and 6.3–6.7 mm deep from the brain
surface) by pressure through a glass micropipette attached to Picospritzer II (General Valve Corporation, East Hanover, NJ, USA). The
virus-injected rats survived for 51–54 h after the injection.
The 70 virus-injected rats were anesthetized again with chloral
hydrate (70 mg/100 g), and perfused transcardially with 200 mL of PBS,
followed by 200 mL of 3% formaldehyde, 75% saturated picric acid, and
0.1 M Na2HPO4 (adjusted with NaOH to pH 7.0). The brains were then
removed and postfixed for 4 h at room temperature with the same fixative. After cryoprotection with 30% sucrose in PBS, the brains were cut
into 40-µm-thick parasagittal or 50-µm-thick frontal sections on a freezing microtome, and the sections were collected serially in PBS.
Characterization of palGFP- or pal-mRFP-Expressing
Thalamic Neurons
The sections including the injection site were observed under epifluorescent microscope Axiophot (Zeiss, Oberkochen, Germany) to find
thalamic neurons infected with the viruses. All the following incubations were performed at room temperature and followed by a rinse
with PBS containing 0.3% Triton X-100 (PBS-X). The sections containing palGFP- or pal-mRFP-positive thalamic neurons were incubated
overnight with 2 µg/mL mouse monoclonal IgG2a to recombinant GAD
of 67 kDa (GAD67; MAB5406, EMD Millipore, Temecula, CA, USA) in
PBS-X containing 0.12% lambda-carrageenan, 0.02% sodium azide,
and 1% donkey serum (PBS-XCD), and then for 4 h with 1 µg/mL AlexaFluor 647-conjugated anti-[mouse IgG] goat antibody (A21236; Life
Technologies, Gaithersburg, MD, USA) in PBS-XCD. Under an LSM5
PASCAL confocal laser-scanning microscope (Zeiss), the location of
222 Single Thalamocortical Axon Fibers of Ventral Medial Nucleus
•
Kuramoto et al.
palGFP- or pal-mRFP-labeled neurons was examined in reference to
GAD67 immunoreactivity as described in the Results section. Further,
the sections containing palGFP- or pal-mRFP-labeled neurons were incubated 2 h with 10 µg/mL of propidium iodide or 1/200-diluted NeuroTrace 500/525 green fluorescent Nissl stain (N-21480; Life
Technologies), respectively, in PBS-X, and the location of the labeled
neurons was re-examined in reference to Nissl-like staining.
Immunoperoxidase Staining for GFP or mRFP
GFP or mRFP immunoreactivity was visualized by combining the avidinbiotinylated peroxidase complex (ABC) method with the biotinylated
tyramine (BT)-glucose oxidase (GO) amplification (Furuta et al. 2009;
Kuramoto et al. 2009). All the serial sections containing one palGFP- or
pal-mRFP-labeled VM neuron in a hemisphere were incubated overnight
with 0.5 µg/mL affinity-purified rabbit antibody to GFP (Tamamaki et al.
2000; Nakamura et al. 2008) or 0.5 µg/mL affinity-purified rabbit antibody to mRFP (Hioki et al. 2010), respectively, in PBS-XCD. After a rinse
with PBS-X, the sections were incubated for 2 hrs with 10 µg/mL biotinylated anti-[rabbit IgG] goat antibody (BA-1000; Vector, Burlingame,
CA, USA) and then for 1 hr with ABC (1:100; Elite variety, Vector) in
PBS-X. After a rinse in 0.1 M sodium phosphate buffer (PB; pH 7.4), the
sections were incubated for 30 min in the BT-GO reaction mixture containing 1.25 µM BT, 3 µg/mL of glucose oxidase (16831-14; nacalai
tesque, Kyoto, Japan; 259 U/mg), 2 mg/mL of β-D-glucose and 1% BSA
in 0.1 M PB (pH 7.4), followed by a wash with PBS. Subsequently, the
sections were again incubated for 1 h with ABC in PBS-X, and the
bound peroxidase was finally developed brown by reaction for 30–60
min with 0.02% diaminobenzidine-4HCl (DAB), and 0.0001% H2O2 in
50 mM Tris–HCl, pH 7.6. All the above incubations and reactions were
performed at room temperature. All the stained sections were serially
mounted onto gelatinized glass slides, dried up, dehydrated in an
ethanol series, cleared in xylene, and finally coverslipped. After reconstruction of palGFP- or pal-mRFP-labeled neurons, the sections were
counterstained for Nissl with 0.2% Cresyl violet to determine cytoarchitecture of the cerebral cortex. The cytoarchitectonic areas of the cerebral
cortex were mainly determined after Nissl staining according to the atlas
of Paxinos and Watson (2007), and, specifically in case of orbital areas,
to Van De Werd and Uylings (2008). Cortical lamination was delineated
according to Swanson (2004) and Zilles (1985) with an appropriate reference to Vogt et al. (2004) for cingulate areas.
Reconstruction and Morphological Analysis of Single
VM Neurons
The cell body and dendrites of stained VM neurons were reconstructed
onto a sagittal plane under a microscope attached with camera lucida
apparatus or with Neurolucida 10 (MicroBrightField, Williston, VT,
USA). The reconstructed figures were captured and digitized by a conventional scanner. The axonal arborization was reconstructed as described previously (Ohno et al. 2012). Briefly, a whole parasagittal or
frontal section was automatically captured into a large color image
with a spatial resolution of 1.038 µm/pixel using digital slide scanner
TOCO (CLARO, Aomori, Japan), and at most 174 parasagittal or 270
frontal images (file size = 50–150 MB/image) were obtained from a
hemisphere. On the images, we traced and digitized the axon fibers
with a pen tablet (Bamboo Tablet; Wacom, Saitama, Japan) and software CANVAS X (ACD Systems International, Inc., Victoria, Canada).
The axon fibers were thereby reconstructed 2-dimensionally to a collection of many short Bézier curves section by section onto a sagittal
plane, and the digitized fibers from all the sections were superimposed
in the computer. The length and color code of Bézier curves in a
Canvas file was automatically determined curve by curve and written
out to an Excel file by using an Applescript macro, which was programmed by Y.R.T.
For 3D reconstruction, we needed to change the Bézier curves of
axon fibers into a TIFF image temporarily, and to re-trace the axon
fibers on the TIFF image in software Neurolucida, because the Neurolucida did not have a software module to directly transform the Bézier
curve information to the Neurolucida file format (DAT file). More
specifically, the Bézier curves were exported section by section to a
black-and-white TIFF file (file size of 15–20 MB/section) with the
maximum resolution of software CANVAS X, and were re-traced using
submodule AutoNeuron (settings: bright field, max process diameter =
3 µm, and seed detection sensitivity = 100). Black-and-white images
were used because of the file size limitation (≤20 MB/image) in the
Neurolucida, and the maximum resolution was required to re-trace the
image of Bézier curves smoothly. All the re-traced lines were arranged
3-dimensionally in the Neurolucida, and the 3D image was rotated,
when necessary, and visualized with enhancement of depth perception
using the Depth Cueing option in submodule Explorer. The cerebral
cortex was outlined in the Neurolucida at every 200 µm distance by
tracing the contour on black-and-white TIFF images (1.42 µm/pixel)
made from original photo-images (1.038 µm/pixel) of every fifth serial
sections.
The fine morphological indices, such as intervaricosity interval,
were measured with an ×100 objective lens (PlanApo100; NA = 1.4;
Nikon, Tokyo, Japan). For statistical analysis, such as Bonferroni post
hoc multiple comparison test following 2-way analysis of variance
(ANOVA), Mann–Whitney’s U test and Student’s t-test, softwares GraphPad Prism 4 (Graphpad Software, Inc., San Diego, CA, USA) and Excel
(Microsoft, Redmond, WA, USA) were used.
proximal sites of their axons were well labeled without any
pathological signs, we suspected that the infection of these
neurons was delayed for substantial hours after the injection,
and the remaining survival time was not enough for sufficient
production or transport of the reporter protein. Excluding
these 5 neurons from 19 neurons, we reconstructed the axonal
arborization of the remaining 14 VM neurons as far as possible.
The axons of the 14 neurons were clearly stained up to the
exact end of fine axon fibers without any fading away, and the
ends always formed small terminal varicosities. This suggests
that the axonal arborization of the 14 neurons was mostly, if
not completely, reconstructed. The 14 neurons were numbered
according to the location of their cell bodies in the VM from
the lateral to the medial portion (Fig. 2).
Results
Selection of palGFP- or pal-mRFP-Labeled Neurons
for Single-Cell Tracing
Because almost all thalamic neurons projected to ipsilateral
hemispheres (Donoghue and Parham 1983), a mixture of
palGFP- and pal-mRFP-expressing Sindbis viral vectors was injected at an adequate dilution into both hemispheres of 70 rat
brains (140 hemispheres) as reported previously (Ohno et al.
2012). The survival time was as short as possible, that is, 51–54
h, to avoid possible effects of viral infection on the axonal morphology of the infected neurons. Fifty-five hemispheres containing only one palGFP- or pal-mRFP-labeled neuron were selected
under an epifluorescent microscope and further processed. We
previously found that immunoreactivity for GAD67 was clearly
more intense in the VM and the IZ of the VA-VL than in the EZ,
and that these intensely GAD67-immunoreactive region mainly
received GABAergic inputs from the basal ganglia (Kuramoto
et al. 2011). In addition, GAD67 immunoreactivity was useful
for demarcating the border between the VM/IZ and submedius
thalamic nucleus, because the submedius nucleus shows clearly
weaker immunoreactivity than the VM and IZ (Kuramoto et al.
2011). Thus, immunofluorescence staining for GAD67 immunoreactivity revealed that 27 of the 55 labeled neurons were
located in the VM or IZ showing intense immunoreactivity
(Fig. 1A′,C′). Subsequently, the fluorescent Nissl-like staining
showed that the 27 neurons in the intensely GAD67-immunoreactive region were further differentiated into 19 VM and 8 IZ
neurons by the cytoarchitecture (Fig. 1A,C) in reference to the
atlas of Paxinos and Watson (2007).
The sections of the 19 hemispheres containing singlelabeled VM neurons were then processed for immunoperoxidase staining for GFP or mRFP by the ABC method with the
BT-GO amplification (Fig. 1B′,D′). Thus, the axonal arborizations of 10 palGFP-labeled and 9 pal-mRFP-labeled VM
neurons were visualized. Of the 19 VM neurons, 10 neurons
were revealed to mainly send axons to the primary (M1) and
secondary motor areas (M2), whereas 9 neurons did not
chiefly project to the motor areas, but to the orbital and/or cingulate areas. Furthermore, when reconstructing the axonal arborization, we noticed that the axon labeling of 5 VM neurons
faded away at the distal sites of their arborization. Because the
Figure 1. Identification of VM neurons infected with palGFP- or pal-mRFP-expressing
Sindbis viral vectors. The parasagittal sections which contained palGFP- or
pal-mRFP-labeled neuronal cell bodies (arrowheads in A–D) were immunostained for
GAD67 (A′,C′). Under the confocal laser-scanning or fluorescent microscope, GFP was
detected in 488 nm or 450–490 nm excitation and 505–530 nm emission condition,
mRFP was in 543 nm or 540–552 nm excitation and 590–625 or 575–625 nm
emission condition, and GAD67 immunoreactivity (AlexaFluor647) was in 633 nm
excitation and ≥650 nm emission condition. The sections were further stained with
propidium iodide (PI; A, B, excitation 540–552 nm, emission 590–625 nm) or
NeuroTrace 500/525 green (C, D, excitation 450–490 nm, emission 505–530 nm).
After the identification, the labeled neurons were visualized by immunoperoxidase
staining with the anti-GFP (B′) or anti-mRFP antibody (D′). AD, anterodorsal thalamic
nucleus; AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; CL,
central lateral thalamic nucleus; LD, laterodorsal thalamic nucleus; LHb, lateral
habenula; LP, lateral posterior thalamic nucleus; MD, mediodosal thalamic nucleus; Pc,
paracentral thalamic nucleus; Pf, parafascicular thalamic nucleus; Rt, thalamic reticular
nucleus; VPpc, parvocellular part of the ventral posterior thalamic nuclei. Scale bar in C′
applies to A, A′, C, C′; that in D′ applies to B, B′, D, D′.
Cerebral Cortex January 2015, V 25 N 1 223
Axonal Arborization of VM Neurons
All the 14 VM neurons reconstructed in the present study extensively projected axon fibers to the cerebral cortex (Fig. 3A,B),
but no neurons possessed axon collaterals inside of the dorsal
thalamus. When the axons exited from the thalamus, VM
neurons always emitted a few axon collaterals to the thalamic reticular nucleus (Fig. 3C). In the course to the cerebral cortex, 12
of the 14 VM neurons sent axon collaterals to the neostriatum
(Fig. 3D,E), although neurons 10 and 13 mainly projected to the
orbital and cingulate areas (Table 1) without any axon collaterals emitted to the neostriatum. Thus, the main targets of VM
neurons were the cerebral cortex, neostriatum, and thalamic reticular nucleus with exceptions that some neurons formed a tiny
axon collateral in the zona incerta (Fig. 4A), claustrum (Fig. 5A),
or external segment of the globus pallidus (Fig. 6D, Supplementary Figs 1A and 2A). Every axon fiber in the cerebral cortex,
striatum, and thalamic reticular nucleus was clearly labeled, and
its axon varicosities were easily detectable (Fig. 3B,E) by the
combination of the ABC method with the BT-GO amplification.
The target cortical areas of single VM neurons were listed in
Table 1, and their axonal arborization was almost completely reconstructed as shown in Figures 4–7 and Supplementary
Figures 1 and 2. Although the axon fibers of each VM neuron
were widely distributed to many cortical fields in the present reconstruction, 8 VM neurons sent axon fibers mainly to the M1,
M2, forelimb (FL), and/or hindlimb areas (HL), and 6 neurons
projected chiefly to the orbital and/or cingulate areas (Table 1).
The M1 and M2 corresponded to the lateral and medial
agranular fields of Donoghue and Wise (1982), whereas the HL
and FL were granular fields, which were often included in the
primary somatosensory area. However, because the HL and a
medial part of the FL were reported to share the electrophysiological and morphological characteristics of the M1 (Hall and
Lindholm 1974; Donoghue et al. 1979; Donoghue and Wise
1982), the 8 VM neurons (neurons 1, 3–7, 9, and 14) were considered to innervate motor-associated areas principally, and
were tentatively named “motor-preferring VM neurons.”
In addition, the VM contained the second population of
neurons (6/14 = 43 or 9/19 = 47%), named “nonmotor-preferring
VM neurons” here, which did not seem to principally innervate
for the motor-associated areas but mainly headed for the orbital
and/or cingulate areas (neurons 2, 8, 10–13). However, the latter
population of nonmotor-preferring VM neurons did not appear
to be a single entity, because the population contained the
neurons without projection to the orbital area (neuron 2) and
without projection to the cingulate area (neuron 12).
Orbital areas were divided into the medial, ventral, dorsolateral, lateral, and ventrolateral orbital areas in the present study
according to a recent cytoarchitectonic and chemoarchitectonic work of Van De Werd and Uylings (2008). These orbital
subareas were reported to be identified in frontal sections by
the cytoarchitecture with Nissl staining. Because of the difficulty in delineating these subareas on parasagittal planes, a
complete series of parasagittal Nissl-stained sections of a rat
brain was made, and the orbital subareas in parasagittal planes
were determined in reference to another complete series of
Figure 2. The somal location of VM neurons reconstructed in the present study. VM neurons were projected onto the nearest parasagittal plane of Nissl-stained sections, and
serially numbered from the lateral to medial portions of the VM. The broken lines indicate the border of the VM, which was determined in the Nissl-stained sections with the aid of
GAD67 immunoreactivity in the adjacent sections. APt, anterior pretectal nucleus; ml, medial lemniscus; mt, mammillothalamic tract; Po, posterior thalamic nuclei; Sm, submedius
thalamic nucleus; VPM, ventral posteromedial thalamic nucleus; ZI, zona incerta. For the other abbreviations, see the legends of Figure 1. Scale bar in D applies to A–D.
224 Single Thalamocortical Axon Fibers of Ventral Medial Nucleus
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Kuramoto et al.
Figure 3. Axon fibers and varicosities of VM neurons. VM neurons projected axon fibers to the superficial layer of the M1 (A, B), thalamic reticular nucleus (Rt; C) and neostriatum
(D, E). Arrowheads in B and E indicate axon varicosities which were located at a focus plane of the microscope. To determine cortical areas and layers, we counterstained sections
for Nissl with Cresyl violet after the reconstruction. The counterstaining was not clear in the figure because the Cresyl violet color was photographically suppressed with a
450-nm-centered band path filter.
frontal Nissl-stained sections (Supplementary Fig. 3). On the
other hand, cingulate areas were the same as Cg1 and Cg2 of
Paxinos and Watson (2007), which mostly corresponded to the
dorsal and ventral subdivisions of the anterior cingulate area
(Krettek and Price 1977) or to areas 24b and 24a (Vogt and
Peters 1981). When VM neurons projected axons to the cingulate areas, the axons were mainly distributed in the Cg1 or
dorsal subdivision of the anterior cingulate area.
As the lateral portion of the VM was reported to receive
nociceptive information from the brainstem reticular formation
(Villanueva et al. 1996, 1998), the location of cell bodies might
be functionally segregated within the VM according to their
roles or projection targets. Thus, the somal location of motorpreferring VM neurons was statistically compared with that of
nonmotor-preferring neurons. As shown in Figure 2, where
the circled and boxed numbers indicated 8 motor-preferring
and 6 nonmotor-preferring VM neurons, respectively, the
nonmotor-preferring neurons were seemingly located more
medially than the motor-preferring ones. However, no statistically significant difference in the mediolateral or rostrocaudal
location was noticed between the 2 groups (P = 0.18 or
P = 0.28, respectively, by the 2-tailed unpaired U test).
Although the main targets of motor-preferring VM neurons
were M1, M2, FL, and HL (motor-associated areas), they sent
axon fibers to many other cortical areas. For example, the fine
varicose axon fibers of VM neuron 3 were distributed in very
wide cortical fields over M2, M1, HL, primary somatosensory, cingulate, medial parietal association, prelimbic, secondary visual,
frontal association, FL, lateral parietal association, primary visual,
and retrosplenial dysgranular areas (Fig. 4A,B; Table 1). The cortical fields covered by this single neuron were spread more than
7 mm rostrocaudally and more than 3 mm mediolaterally
(Fig. 4C). A similarly widespread distribution of axon fibers was
observed in all the motor-preferring VM neurons (Figs. 5, 7A–F,
Supplementary Fig. 1). Most of the neurons sent axon fibers to
cortical regions spreading more than 5 mm rostrocaudally. Even
when the list of cortical projection targets was limited to the
Table 1
Target cortical areas of reconstructed VM and IZ neurons
Neurons
VM neuron
1
2
3
4
5
6
7
8
9
10
11
12
13
14
IZ neuronc
1
2
3
4
5
Target areas listed in a descending order of the total axonal length
FL a, M1, S1b, M2, HL, LPtA a
Cg, V1, V2, Au, M2, PtP, IL, MPtA, M1, LPtA, S1, PL, HL
M2, M1, HL, S1, Cg, MPtA, PL, V2, FrA, FL, LPtA, V1, RSD
M1, FL, LO, FrA, S1, VLO, DLO, DI, M2, AI
M1, S1, S2, M2, VLO, FL, LPtA, FrA, LO, Cg, PtP, DLO, VO, MO, AI
M2, M1, S2, VLO, LO, FrA, Cg, FL, S1, LPtA, MO, DLO, VO
M2, Cg, M1, FrA, VLO, MO, Ent, Ect, LO, VO, PL, MPtA, AI, RSD
VO, MO, Cg, VLO, LO, FrA, PL, Fr3, DLO, M2, AI
M1, M2, Cg, FL, HL, VLO, FrA, PL, S1, MO, DLO
MO, VO, PL, M2, VLO, Cg, FrA, LO, AI, DLO, IL, M1
Cg, VO, PL, M2, MO, FrA, VLO, Fr3
VLO, LO, M1, AI, VO, M2, MO, FrA, DLO, DI, S1
VO, LO, PL, MO, Cg, VLO, M2, M1, DLO, FrA, AI
M2, M1, LO, VLO, S1, LPtA, FrA, PL, V1, MPtA, AI, HL, DLO, VO, V2, PtP, FL
M1, M2, HL, Cg, V1, RSD, PL
FL, M1, S1, FrA, HL, M2, Fr3, LO, DLO
FL, M1, S1, HL
M1, M2, FL, FrA, S1, DLO, LO, AI, VLO, Fr3
M2, Cg, M1, HL, S1, MPtA, FL, PL, FrA, V2, RSD
Note: AI, agranular insular area; Au, auditory area; Cg, cingulate area; DI, dysgranular insular area;
DLO, dorsolateral orbital area; Fr3, frontal cortex, area 3; Ect, ectorhinal area; Ent, entorhinal area;
FrA, frontal association area; IL, infralimbic area; LO, lateral orbital area; LPtA, lateral parietal
association area; MO, medial orbital area; MPtA, medial parietal association area; PL, prelimbic
area; PtP, parietal cortex, posterior area; RSD, retrosplenial dysgranular area; S2, secondary
somatosensory area; V1, primary visual area; V2, secondary visual area; VLO, ventrolateral orbital
area; VO, ventral orbital area. For the other abbreviations, see text.
a
Underline, boldface, and italic fonts indicate the target area containing varicose axon fibers of
≥60, ≥30, and <5 mm, respectively. The axon length of VM neurons in each cortical area is listed
in Supplementary Table 1.
b
FL and HL are excluded from the primary somatosensory area (S1).
c
The data of IZ neurons were obtained from the IZ neurons published in Kuramoto et al. (2009).
areas receiving axon fibers of more than 30 mm (underlined
and/or bold letters in Table 1), a large variety of cortical areas including the primary somatosensory, secondary somatosensory,
cingulate, orbital, and parietal association areas received an
axonal projection from the motor-preferring VM neurons.
Cerebral Cortex January 2015, V 25 N 1 225
Figure 4. Reconstructed axon fibers of VM neuron 3. The main axon of neuron 3 sent a few axon collaterals to the thalamic reticular nucleus when it exited from the thalamus, and
then provided several collaterals with the striatum during the course to the cerebral cortex (A). After entering the subcortical white matter or cerebral cortex, the main axon formed
branches and the branches were distributed to widespread cortical field including the motor-associated areas and their surrounding areas. The colors of axon fibers indicate fine
varicose axon fibers in different cortical layers (B). The varicose axon fibers were preferentially distributed in L1 of widespread cortical areas. In (C), the cortical distribution of
varicose axon fibers is shown in a 3D manner. Gray figures in C are the outlines of cerebral cortex of the rat. St, striatum; ZI, zona incerta. For the other abbreviations, see text and
the legends of Table 1 and Figure 1.
In Figures 6, 7G–J, and Supplementary Figure 2, the axonal arborization of 6 nonmotor-preferring VM neurons is
shown in detail. The axonal arborization of these nonmotorpreferring VM neurons (except neuron 2) was concentrated in
226 Single Thalamocortical Axon Fibers of Ventral Medial Nucleus
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Kuramoto et al.
a smaller cortical field than that of the 8 motor-preferring
VM neurons. Of the 6 neurons, neurons 8, 10, and 13 sent
varicose axon fibers totaling more than 30 mm in length
to both the orbital and cingulate areas (Table 1). Neuron 12
Figure 5. Axonal arborization of 4 VM neurons projecting principally to the motor-associated areas. The axon fibers were widely distributed in the motor-associated areas (A, E, I,
K) and especially in their L1 (red colors in B–D, F–H, J, L, M). Compared with neurons 5, 6, and 14, neuron 1 sent many axon collaterals to the striatum (I). Cl, claustrum; St,
striatum; for the other abbreviations, see text and the legends of Table 1 and Figure 1.
projected mainly to the orbital areas and M1 (Supplementary
Fig. 2C,D), whereas neurons 2 and 11 chiefly projected to the
cingulate area (Table 1). Neuron 2 further projected to the
visual, auditory, and secondary motor areas widely (Fig. 6G–J).
Thus, the present results clearly show that the individual
VM neurons innervated widespread cortical areas as the
Cerebral Cortex January 2015, V 25 N 1 227
Figure 6. Axonal arborization of 3 VM neurons projecting mainly to the orbital and/or cingulate areas. Neurons 8 and 13 sent axon fibers mainly to L1 of the orbital areas (A–F),
where the spread of axon fibers appeared smaller than those of motor preferring VM neurons in Figures 4 and 5. Of the 14 reconstructed VM neurons, the most abundant striatal
axon collaterals were observed in neuron 8 (A). In contrast, no striatal axon collaterals were provided by neuron 13 (D). The axon fibers of neuron 2 were widely spread (G) and
97.0% of varicose axon fibers were distributed in L1 (H–J). Dense distribution of axon fibers in the visual areas was another characteristic of this neuron. GPe, external segment of
the globus pallidus; St, striatum; for the other abbreviations, see text and the legends of Table 1 and Figure 1.
228 Single Thalamocortical Axon Fibers of Ventral Medial Nucleus
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Figure 7. Examples of single VM neurons reconstructed from the frontal sections. Neuron 7 projected mainly to the motor areas (A–F), whereas neuron 10 projected chiefly to the
orbital areas (G–J). Both the neurons sent axon fibers preferentially to L1 of the cortical areas. St, striatum; for the other abbreviations, see text and the legends of Table 1 and Figure 1.
nucleus as a whole is well known to innervate wide areas
(Herkenham 1979; Glenn et al. 1982; Arbuthnott et al. 1990;
Desbois and Villanueva 2001). However, each VM neuron did
not send their axons throughout the cerebral cortex, but preferred the motor-associated, orbital, and/or cingulate areas to
the other areas.
Cerebral Cortex January 2015, V 25 N 1 229
Quantitative Data and Laminar Distribution of Varicose
Axon Fibers of VM Neurons
In Table 2, the length of varicose axon fibers and estimated
number of axon varicosities of each VM neuron were described. The axon varicosity was defined by the localized swelling of axon fibers with ≥1.5-fold larger diameters than the
thickness of intervaricosity axon fibers (Fig. 3B). Intervaricosity interval was measured by random sampling, and the mean
interval was calculated area by area and layer by layer (Supplementary Table 2). The number of axon varicosities was estimated by dividing the axon length by the mean intervaricosity
interval. The total length of varicose axon fibers ranged from
200 to 579 mm with the mean ± SD = 351 ± 107 mm, and the
estimated total number of axon varicosities was from 34 360
to 101 480 with 60 264 ± 18 997, showing a large variation
neuron by neuron. When compared with VA-VL neurons
(Kuramoto et al. 2009), the total axon length of VM neurons
was longer than IZ (206 ± 48 mm; P = 0.011 by unpaired
2-tailed t-test) or EZ neurons (234 ± 92 mm; P = 0.046).
The varicose axon fibers emitted from the EZ and IZ of the
VA-VL were targeted at middle cortical layers and L1, respectively (Kuramoto et al. 2009). In contrast, all the VM neurons reconstructed in the present study preferred the superficial part
of L1 to the other layers. Furthermore, all but one of the VM
neurons sent more abundant varicose axon fibers to L1 than IZ
neurons. In Figures 4B, 5–7, and Supplementary Figures 1 and
2, fine varicose axon fibers were labeled with different colors.
As indicated by red color in these figures, most VM neurons
sent a high percentage of varicose axon fibers to L1; the percentage of L1 axon length in total cortical axon length was
78.6 ± 14.4% in mean ± SD (Table 2), being significantly larger
(P = 0.002 by the 2-tailed unpaired t-test) than that of IZ
neurons (53.5 ± 7.3%; Kuramoto et al. 2009). All the VM
neurons except neuron 12 sent more than 70% of varicose
axon fibers and varicosities to L1, whereas IZ neurons projected at most 66% of varicose axon fibers to L1. This finding
suggests that VM neurons constitute a different population
from IZ neurons, although both the VM and IZ neurons receive
strong inhibitory inputs from the basal ganglia. VM neuron 12
was located in the center of the VM (Fig. 2C) and showed no
clear difference in the dendritic arborization from the other
VM neurons (Fig. 8A), but the neuron was different in cortical
axonal arborization from the surrounding VM neurons,
sending only 40% of axon fibers to L1 of the orbital areas (Supplementary Fig. 2C,D).
As reported previously (Herkenham 1979; Arbuthnott et al.
1990), the axon fibers of the VM neurons were sometimes ramified moderately in L2/3 (neurons 1, 5 and 6 in Fig. 5; neuron
9 in Supplementary Fig. 1; neuron 12 in Supplementary Fig. 2;
neuron 13 in Fig. 6). Varicose axon fibers of the VM neurons
were distributed in L2/3 and L5, ranging 2.7–128.9 mm and
4.1–71.2 mm, respectively, although the length of axon fibers
in these layers was much shorter than that in L1 (Supplementary Table 3).
In Table 3, the morphological parameters of axons were
compared between motor-preferring and nonmotor-preferring
VM neurons, the latter of which projected mainly to the orbital
and/or cingulate areas. Neither the total length of varicose
axon fibers in the cortex or striatum nor the relative axon
length in L1 differed significantly between the 2 populations.
Quantitative Comparison of Axon Fibers of
Motor-Preferring VM Neurons with Those of IZ Neurons
in Motor-Associated Areas and Striatum
Because both VM and IZ neurons preferred L1 of motorassociated areas (M1, M2, FL, and HL) as their cortical target,
we compared the morphological data of axon fibers between
VM and IZ neurons, using the recalculated data of the 5 IZ
neurons reported before (Kuramoto et al. 2009). In addition, to
compare functionally related neurons, we selected the 8 VM
neurons projecting mainly to the motor-associated areas, and
collated them with the IZ neurons (Table 4). In the striatum,
the mean axon length of the 8 motor-preferring VM neurons
was less than half of the length of IZ neurons, being significantly shorter than that of IZ neurons (P < 0.05 by the 2-tailed
unpaired t-test; Table 4), although the difference of striatal collaterals was not significant between all the 14 VM neurons
(27.5 ± 31.1 mm) and the 5 IZ neurons (P = 0.056). On the
other hand, the varicose axon fibers of the motor-preferring
VM neurons in the motor-associated areas were longer than
Table 2
Fine varicose axon fibers of VM neurons in the cerebral cortex
VM neuron
Total axon length in cortex (mm)a
Estimated number of varicosities in cortexb
Relative axon length in L1 (%)
Estimated number of varicosities in L1b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Mean ± SD
378.9
579.4
428.6
265.7
478.6
438.3
364.2
200.3
234.3
362.9
221.6
364.1
311.0
283.8
350.8 ± 107.4
65 772
101 480
75 631
44 602
82 605
75 655
61 737
34 360
40 158
62 815
37 897
58 037
52 291
50 663
60 264 ± 18 997
69.8
97.0
78.0
78.6
75.2
71.6
76.9
95.1
71.6
92.7
89.8
40.3
76.0
88.4
78.6 ± 14.4
47 618 (72.4%)
98 872 (97.4%)
61 332 (81.1%)
36 579 (82.0%)
64 407 (78.0%)
56 648 (74.9%)
49 781 (80.6%)
32 814 (95.5%)
30 223 (75.3%)
58 729 (93.5%)
34 445 (90.9%)
24 975 (43.0%)
40 858 (78.1%)
45 630 (90.1%)
48 779 ± 18 948 (80.9 ± 13.6%)
Note: aThe length of axon fibers was estimated by multiplying the length of varicose axons projected onto parasagittal planes by π/2. The length in each layer was presented in Supplementary Table 2. The
measured axons did not contain thick straight axon fibers which might be myelinated portions of thalamocortical axons and make no synaptic contacts.
b
The estimated number of varicosities in each cortical layer of each area was calculated by dividing the axon length by the mean intervaricosity interval (Supplementary Table 3). The mean intervaricosity
interval was measured with 30 nearby varicosity pairs on the axon fibers which ran parallel to the section surface.
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Table 3
Comparison of varicose axon fibers between motor-preferring and nonmotor-preferring VM neurons
Number of neurons
Total axon length in striatum (mm)
Total axon length in cortex (mm)
Axon length in L1 (mm)
Relative axon length in L1 (%)
Motor-preferring
Nonmotor-preferring
P valuea
VM neurons
VM neurons
U test
t-test
8
24.3 ± 24.6
359.1 ± 89.3
272.6 ± 64.1
76.3 ± 5.9
6
31.8 ± 40.3
339.9 ± 136.2
278.4 ± 153.0
81.8 ± 21.7
0.950
0.414
0.573
0.142
0.672
0.755
0.923
0.500
Note: aCalculated by the 2-tailed unpaired U- or t-test.
Table 4
Comparison of fine varicose axon fibers between motor-preferring VM and IZ neurons
VM neuron
1
3
4
5
6
7
9
14
Mean ± SD
IZ neuronb
1
2
3
4
5
Mean ± SD
P valuec
Total axon length in
the striatum (mm)a
Total axon length in
motor-associated areas
(mm)a
Relative axon length in L1 of
motor-associated areas (%)
32.6
11.4
37.2
4.1
6.4
15.3
77.5
10.1
24.3 ± 24.6
355.9
251.8
202.2
269.2
315.1
253.2
187.8
170.4
250.7 ± 63.6
68.8
77.5
83.1
73.2
71.9
73.6
67.6
89.9
75.7 ± 7.6
47.1
97.8
67.9
43.6
39.4
59.2 ± 24.2
0.0295
152.9
151.2
171.1
269.5
117.4
172.4 ± 57.6
0.0473
44.0
50.0
53.1
47.3
75.7
54.0 ± 12.6
0.0024
Note: aThe length of axon fibers was estimated by multiplying the length of varicose axons
projected onto parasagittal plane by π/2. Thus, the measured axons did not contain thick straight
axon fibers which might be myelinated portions of thalamocortical axons and make no synaptic
contacts.
b
The data were measured and calculated from the samples in the previous report (Kuramoto et al.
2009) for comparison with the present data.
c
P value between motor-preferring VM and IZ neurons was calculated by the 2-tailed unpaired
t-test.
those of IZ neurons (P < 0.05). It was more impressive that the
relative axon length (76 ± 8%) of VM neurons in L1 of the
motor-associated areas was clearly greater than that of IZ
neurons (54 ± 13%; P < 0.005). This suggests that the VM
neurons are more specialized for activating the apical dendritic
tufts of pyramidal neurons in the motor-associated areas than
IZ neurons.
Cell Bodies and Dendrites of VM Neurons
The somal size of VM neurons was 230 ± 24 µm2 (mean ± SD;
Table 5), being similar to that of IZ or EZ neurons (Kuramoto
Figure 8. Dendrites and cell bodies of VM neurons. Extents of dendritic processes of
VM neurons (1–14; A) were elongated in rostrocaudal direction. Circled and boxed
numbers indicate motor-preferring and nonmotor-preferring VM neurons, respectively.
The dendrites of neurons 7 and 10 were traced in the Neurolucida 3-dimensionally,
rotated, and projected to a parasagittal plane. The Sholl analysis revealed that the
differences between the VM and IZ neurons were statistically significant at distances
of 140–220 µm from the cell body (B). In addition, the dendritic arborization was
significantly different between motor-preferring and nonmotor-preferring VM neurons
at distances of 40–120 µm away from the cell body (C). Circles and bars in B and C
indicate mean and SD, respectively, and asterisks point to the statistical significance
(*P < 0.05, **P < 0.01, ***P < 0.001 by Bonferroni multiple comparison test). The
data of IZ neurons were taken from Kuramoto et al. (2009).
Cerebral Cortex January 2015, V 25 N 1 231
Table 5
Cell body and dendrites of single-labeled VM and IZ neurons
Number of neurons
Somal area (μm2)
Dendritic spread
Rostrocaudal (μm)
Dorsoventral (μm)
Mediolateral (μm)
VM neurons
IZ neuronsa
P valueb
14
229.7 ± 24.2c
5
245.2 ± 24.3
0.2356
648.3 ± 164.2
330.4 ± 58.5
556.4 ± 119.4
339.3 ± 39.8
329.7 ± 47.1
432.0 ± 99.6
0.0008
0.9813
0.0534
Note: aThe somal area of IZ neurons was obtained from the previous report (Kuramoto et al. 2009),
and the dendritic spread was measured using the same samples.
b
P value was calculated by the 2-tailed unpaired t-test.
c
Mean ± SD.
et al. 2009). The region around each palGFP- or pal-mRFP-positive neuron was darkly immunostained (Fig. 1B′,D′) probably
because of extracellularly leaked palGFP or pal-mRFP,
suggesting an extremely strong expression of protein by the
subgenomic promoter of Sindbis viral vectors. However, neither
spread of the fluorescent proteins to nor uptake by adjacent
cells was found.
The reconstructed VM neurons were multipolar with many
dendrites (Fig. 8A) as reported previously in rat thalamus
(Sawyer et al. 1989, 1994; Yamamoto et al. 1991). When the reconstructed dendrites were projected to a parasagittal plane
(Fig. 8A), it was noticed that the dendritic arborization of VM
neurons was elongated rostrocaudally. When compared with 5
IZ neurons sampled in the previous report (Kuramoto et al.
2009), only the rostrocaudal extent of dendritic arborization
was significantly larger in VM neurons than in IZ neurons
(Table 5). This difference was also reflected in the Sholl analysis (Sholl 1953); the dendrites of VM neurons were significantly
longer than those of IZ neurons, although the branching of
both the VM and IZ neurons was in a similar range up to 100
µm apart from the cell body (Fig. 8B). The dendritic arborization of 6 nonmotor-preferring VM neurons was denser than
that of 8 motor-preferring neurons (Fig. 8A). This is confirmed
quantitatively by the Sholl analysis showing that the intersections of the nonmotor-preferring neurons were significantly
more numerous at 40–120 µm from the cell body than those of
the motor-preferring neurons (Fig. 8C).
Discussion
We here showed the morphological detail of axonal arborization of VM neurons at a single-neuron level using viral vectors.
The present results indicate that VM neurons are different
from IZ neurons of the VA-VL, although both the neuronal
groups receive strong GABAergic inputs from the basal ganglia
and mainly send axon fibers to L1 of the motor-associated
cortical areas. This is because 1) the relative distribution
of axon fibers in L1 was much higher in VM neurons than in
IZ neurons, 2) VM neurons sent fewer axon collaterals to the
striatum than IZ neurons, and 3) the dendritic arborization
of VM neurons was more extensive rostrocaudally than that
of IZ neurons. Furthermore, VM neurons were seemingly
divided into at least 2 types, “motor-preferring” and “nonmotorpreferring” neurons according to their cortical projection targets
(Fig. 9), although the nonmotor-preferring VM neurons might
be heterogenous.
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Figure 9. Summary diagram of axonal projections of VM neurons in comparison with
IZ and EZ neurons of the VA-VL. Although the 2 populations of VM neurons receive
basal ganglia inputs, one population sends axon fibers principally to motor-associated
areas and the other projects mainly to orbital and/or cingulate areas. Almost all the VM
neurons innervate L1 of the cerebral cortex more preferentially than IZ neurons. See
the text for more detail.
Cortical Projection of VM Neurons
The VM has been shown, in previous morphological studies
with anterograde tracers, to innervate the superficial part of L1
of widespread cortical areas, including the motor-associated,
orbital, cingulate, and visual areas, in the rat (Krettek and Price
1977; Herkenham 1979; Arbuthnott et al. 1990; Desbois and Villanueva 2001; Mitchell and Cauller 2001; Rubio-Garrido et al.
2009) and cat brains (Glenn et al. 1982). The projection of VM
neurons to L1 has been confirmed by the retrograde tracing technique with superficial application of horseradish peroxidase or
fluorescent tracers (Rieck and Carey 1985; Desbois and Villanueva 2001; Monconduit and Villanueva 2005; Rubio-Garrido et al.
2007, 2009). Furthermore, widespread L1 distribution of axon
fibers at a single-neuron level has been suggested by the double
retrograde tracer technique, which detects doubly retrogradelabeled neurons in the VM after the 2 tracers are separately
applied into the superficial portions of 2 distant cortical areas
(Monconduit and Villanueva 2005). The present study clearly
showed that all the VM neurons examined sent axon fibers to
widespread cortical areas even at a single-neuron level, and that
they highly preferred L1 as the target layer, irrespective of target
areas. Thus, the present results on VM neurons, which are in
good agreement of those previous findings, indicate that VM
neurons are classified as matrix-type neurons (Jones 1998,
2001). Recently, a further distinction has been made between
matrix-type neurons with widely ramified axons targeting multiple, distant cortical areas (multiareal subtype) and those with
an axon that arborizes within a single or few adjacent areas
(focal subtype) (Clascá et al. 2012). VM neurons are obviously
classified as the multiareal subtype, because all the examined
VM neurons projected to multiple distant cortical areas.
Furthermore, we noticed the presence of 2 comparable
populations of VM neurons in the present study (Fig. 9); a
major population of VM neurons sent axon fibers mainly to the
motor-associated cortical areas, and the other population
projected chiefly to the orbital and/or cingulate areas.
Although, from the present sampling of VM neurons, we did
not detect a clear spatial grouping of somal locations for the 2
populations within the VM, nonmotor-preferring VM neurons
had more abundant dendritic arborization than motorpreferring neurons (Fig. 8A,C). This suggests that the VM
contained 2 distinct groups of neurons that differ in dendritic
and axonal arborizations.
Functional Implication of VM Neurons in the Motor
Systems
As described in the Introduction section, IZ and EZ neurons in
the VA-VL receive strong inputs from the basal ganglia and cerebellum, respectively (Kuramoto et al. 2011), and project
mainly and widely to the motor-associated cortical areas
(Fig. 9). In addition, VM neurons have been reported to accept
strong basal ganglia inputs as IZ neurons (Di Chiara et al.
1979; Deniau and Chevalier 1992; Sakai et al. 1998; Bodor
et al. 2008; Kuramoto et al. 2011), and send axon fibers to L1
of widespread cortical areas including motor-associated areas
in the rat brain (Krettek and Price 1977; Herkenham 1979,
1980; Arbuthnott et al. 1990; Mitchell and Cauller 2001;
Rubio-Garrido et al. 2009). The present study further showed
that a major population of VM neurons sent axon fibers principally to the motor-associated areas. Thus, the motor-associated
areas receive 3 kinds of information: 1) cerebellar information
through the EZ to their middle cortical layers, 2) basal ganglia
information through the IZ mainly to L1, and 3) basal ganglia
information through the VM highly preferentially to L1. Taking
the shape of pyramidal neurons into consideration, the basal
dendrites of L2/3 and L5 pyramidal neurons receive cerebellar
information through the EZ and L4 spiny neurons, and the
apical dendritic tufts of those pyramidal neurons admit information from the basal ganglia through the IZ and VM. As the
axonal arborizations of single EZ, IZ, and VM neurons are
widely distributed in the motor-associated areas, these 3 kinds
of inputs are considered to converge on single pyramidal
neurons in the motor-associated areas. Thus, the cerebellar
and basal ganglia information would be integrated in those
pyramidal neurons and used for an appropriate motor control.
These findings hence raise a question of why the basal
ganglia information is transferred by the 2 routes through the
IZ and VM. One possible answer is that the 2 routes convey
different lines of information, which are produced in the basal
ganglia. Actually, the IZ or rostroventral portion of the VA-VL
receives afferents mainly from the ventral edge of the SNr,
whereas the VM accepts afferents chiefly from the central and
dorsal portion of the SNr in the rat brain (Deniau and Chevalier
1992; Nishimura et al. 1997; Sakai et al. 1998). Thus, the basal
ganglia information transferred by motor-preferring VM
neurons might be different from that conveyed by IZ neurons.
However, to our knowledge, no functional difference has been
reported between the 2 portions of the rat SNr, except for the
proposal that the ventral SNr–IZ pathway is concerned with
oculomotor function (Sakai et al. 1998).
The main target areas (receiving ≥30-mm-long varicose
axon fibers) of single IZ neurons included not only the motorassociated areas but also the cingulate area (Table 1).
However, when compared with the target areas of motorpreferring VM neurons, the target areas of IZ neurons were
less widespread, and motor-preferring VM neurons innervated,
in addition to the areas listed above, many cortical fields such
as medial orbital, ventrolateral orbital, cingulate, medial parietal association, and primary and secondary somatosensory
areas (Table 1). Considering this wide distribution of axon
fibers, we suppose that VM neurons are in association with
general arousal mechanism as suggested repeatedly in the
literature (Glenn et al. 1982; Arbuthnott et al. 1990; Sakai et al.
1998). The present study indicates that, even when a small
number of VM neurons are activated, many L2/3 and L5 pyramidal neurons in widespread cortical areas would be directly
activated through the apical dendritic tufts. Actually, a lesion
of the VM has been reported to result in a large reduction of
glucose utilization of widespread cortical areas (Girault et al.
1985). Thus, this system is efficient in recruiting many pyramidal neurons in wide cortical areas, and may thus be associated
with general arousal or attentional mechanism.
Function of VM Neurons in the Orbital
and Cingulate Areas
For more than 30 years, neurons in the rat VM are known to
project densely not only to the dorsolateral frontoparietal areas
but also to the prefrontal areas including the orbital and
anterior cingulate areas (Krettek and Price 1977; Herkenham
1979). In the present study, 6 of 14 VM neurons sent many
axon fibers principally to L1 of the orbital and/or cingulate
areas, but many fewer fibers to the medial prefrontal area composed of the infralimbic and prelimbic areas. This finding is
compatible with the previous findings that, when the injection
site of anterograde tracer was confined to the VM, the labeled
terminals were distributed densely in the dorsolateral frontoparietal, cingulate, or orbital areas, but less frequently in the
medial prefrontal area (Herkenham 1979; Arbuthnott et al.
1990). In contrast, when the injection site included the submedius nucleus, many terminals were found in L1 of the medial
prefrontal area (Krettek and Price 1977; Herkenham 1979; Arbuthnott et al. 1990). It is thus likely that, in the prefrontal
areas, VM neurons mainly innervate L1 of the orbital and cingulate areas, whereas the submedius nucleus primarily
supplies L1 of the medial prefrontal area with axon fibers.
Although all 6 nonmotor-preferring VM neurons also projected to the motor-associated areas, the present finding differentiates VM neurons from IZ neurons since the main target
fields of IZ neurons were almost constantly the motor-associated
areas (Kuramoto et al. 2009). This indicates much wider function of VM neurons beyond the motor control task in the motorassociated areas. Because the dorsal division of the anterior cingulate area is associated with visuomotor integration and nocifensive processing in rats (Vargo et al. 1988; Pastoriza et al.
1996; Yamamura et al. 1996; Donahue et al. 2001), and because
neurons in rat orbital areas are activated in association with the
odor discrimination task and in the odor-based conditioned
associative task (Schoenbaum and Eichenbaum 1995; Yonemori
et al. 2000), VM neurons are considered to contribute to these
higher-order functions.
The thalamocortical axon fibers of these VM neurons were
mostly distributed in L1 of those orbital and cingulate areas.
However, the thalamocortical axon terminals, which are specifically characterized by VGluT2 immunoreactivity in the cerebral
cortex (Fujiyama et al. 2001; Kaneko and Fujiyama 2002), are
densely distributed in middle cortical layers as well as in the
superficial part of L1. Thus, these areas may receive afferents at
their middle layers from thalamic nuclei other than the VM
(Fig. 9). The mediodorsal thalamic nucleus (MD) is generally
known to project to the “prefrontal areas,” which consists of 1)
infralimbic and prelimbic areas, 2) orbital areas, 3) anterior cingulate areas, and 4) agranular insular areas in rat brain (Krettek
and Price 1977; Divac et al. 1978; Vogt et al. 1981; Groenewegen
Cerebral Cortex January 2015, V 25 N 1 233
1988; Zeng and Stuesse 1991; Ray and Price 1992; Reep et al.
1996). The MD sends axon fibers to the middle layers as well as
to L1 of cingulate and orbital areas (Krettek and Price 1977;
Vogt et al. 1981; Groenewegen 1988), and thus their thalamocortical fibers may collaborate with the afferents from VM
neurons that mainly project to orbital and/or cingulate areas.
This relation of MD axons to VM axons in the cingulate and
orbital areas might be similar to that of EZ axons to IZ/VM
axons in the motor-associated areas. Thus, a further singleneuron-labeling study on the axon fibers of MD neurons is
necessary to understand the structural organization of VM
afferents to these higher-order prefrontal areas.
Supplementary Material
Supplementary Material can be found at http://www.cercor.oxfordjournals.org/.
Funding
This study was supported by Grants-in-Aid from the Ministry
of Education, Culture, Sports, Science, and Technology
(MEXT) for Young Scientists (23700413 and 25830034 to E.K.);
for Scientific Research (22300113 and 25250006 to T.K.,
24500409 to T.F., and 24500408 to H.H.); for Exploratory Research (23650175 to T.K.); for Research Activity Start-up
(24890179 to S.O.); and for Scientific Research on Innovative
Areas, “Mesoscopic Neurocircuitry” (23115101 to T.K.), “Brain
and Information Science on Material Perception” (23135519 to
T.F.), “Neuronal Diversity and Neocortical Organization”
(23123510 and 25123709 to H.H.) and “Foundation of Synapse
and Neurocircuit Pathology” (22110007 to H.H.).
Notes
Conflict of Interest: None declared.
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