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Introduction
The trigeminal sensory nuclear complex was originally
divided into 4 nuclei: principalis (Vp), oralis (Vo), interpolaris
(Vi), and caudalis (Vc) by
Olszewski[1]. Vp comprises a
ventral division and a dorsal division (Vpd), which are
cyto-architectonically different and have been proposed to serve
the different functions of relaying and processing of
sensory signals from the head, such as tactility and trigeminal
proprioception[2_4]. Such functions of the Vpd can be
supported by the findings that this structure receives direct
primary input from intraoral structures and possesses diverse
afferent and efferent fiber connections with other brain
areas[5_8].
It is well known that glutamate and gamma-aminobutyric
acid (GABA) are important excitatory and inhibitory
neutrotransmitters in the brain, respectively. Several lines
of evidence suggest that these transmitters are involved in
signal transmission or processing within the
Vpd[9_11]. The Vpd contains a high density of different-sized,
immunohisto-chemically heterogeneous neurons, many of which are likely
to contain glutamate[12], and constitute most of the Vpd
projection neurons[11]; therefore, these neurons play essential
roles in relaying mechanical sensory signals from the head.
In addition, the observation that myelinated trigeminal
primary afferent terminals are immunoreactive for glutamate
implies the glutamatergic nature of their associated input to
the Vpd[9]. However, in the central nervous system (CNS),
glutamate can also serve as the substrate for GABA
synthesis, and phosphate-activated glutaminase (PAG), a
marker for glutamatergic structures, was expressed only in
the glutamatergic cell bodies and
dendrites[13]. So because of the lack of suitable immunocytochemical markers for
glutamatergic axons, an understanding of the sensory
transmission through the glutamatergic primary afferents from
the orofacial area has been hampered. Recently, 3 vesicular
glutamate transporters, vesicular glutamate transporter
1 (VGluT1), VGluT2 and VGluT3, have been identified and
shown to be present selectively in axons belonging to largely
nonoverlapping populations of glutamatergic neurons
throughout the CNS[14_16]. Previous immunocytochemical
studies have provided evidence that VGluT1 is strongly
expressed in the myelinated afferent fibers of the Vpd,
whereas VGluT2 and VGluT3-IRs are expressed at very low
levels[17,18]. On the other hand, many axon terminals in the
Vpd were immunostained for GABA[9]. Based on the
aforementioned studies, we infer that VGluT1-LI axon terminals,
coming from trigeminal primary afferents, could activate Vpd
glutamatergic projection neurons which may be modulated
by GABA-LI axon terminals. Therefore, an attempt was made
to examine whether or not the VGluT1-LI axon terminals of
the Vpd come from the primary afferent fibers by performing
a trigeminal rhizotomy in rats, and examining the relationship
between the PAG-LI neurons and VGluT1/glutamate
decarboxylase (GAD; a marker for GABAergic neurons)-LI axon
terminals in the Vpd under the confocal laser-scanning
microscope (CLSM) and electron microscope (EM).
Materials and methods
All procedures for the experiments were approved by the
Animal Care and Use Committees at the Fourth Military
Medical University, Xi'an, China. A total of 10 adult
Sprague-Dawley male rats weighing 200_250 g were used. The rats
were anesthetized with chloral hydrate (70 mg/100 g body
weight) and perfused transcardially for light and electron
microscopic experiments[19].
Triple-immunofluorescence histochemistry for VGluT1,
GAD and PAG was performed. Briefly, the sections were
incubated at room temperature sequentially with: (1) a
mixture of 50 µg/mL mouse anti-PAG
IgM[20] and 0.8 µg/mL guinea pig anti-VGluT1
IgG[21] or mouse anti-GAD IgG (1:500; Chemicon, Temecula, CA, USA) overnight; (2) biotinylated
donkey anti-mouse IgM (1:100; Jackson ImmunoResearch,
West Grove, PA, USA) for 4 h; and (3) a mixture of 10%
(v/v) normal mouse serum, fluorescein isothiocyanate
(FITC)-labeled avidin-D (1:1000; Vector Labs, Burlingame, CA, USA)
and rhodamine-labeled goat anti-guinea pig IgG (1:100;
Chemicon, USA) and Cy5-labeled donkey anti-mouse IgG
(1:100; Chemicon, USA) for 3 h. The incubation medium in
steps 1 and 2 was prepared by using 0.05 mol/L
phosphate-buffered 0.9% saline (PBS) containing 0.5%
(v/v) Triton X-100, 0.25% (w/v) l-carrageenan, 0.05%
(w/v) NaN3 and 5% (v/v) normal donkey serum. The incubation medium in step 3
was prepared by using 0.05 mol/L PBS containing 0.3%
(v/v) Triton X-100. After the incubation, the sections were rinsed
in 0.05 mol/L PBS, mounted onto gelatin-coated glass slides,
and then observed under the CLSM (LSM 410; Zeiss,
Oberkochen,Germany) by using a laser beam of 488 nm, 543
nm and 633 nm with appropriate emission filters for FITC
(510_525 nm), rhodamine (590_610 nm) and Cy5 (670_810
nm). In the control experiments for the immunofluorescence
histochemistry, one of the primary antibodies was omitted
or replaced with normal IgG; no immunoreactivity for the
omitted or replaced antibody was found.
Electron microscopically, the immunogold-silver method
for VGluT1 or GAD, combined with the immunoperoxidase
method for PAG, was performed. Briefly, the sections were
incubated for 24 h at room temperature with a mixture of 0.8
µg/mL guinea pig anti-VGluT1 IgG or 1/500-diluted mouse
anti-GAD IgG (Chemicon, USA) and 75 µg/mL mouse
anti-PAG IgM, each diluted in 50 mmol/L Tris-buffered saline
(TBS; pH 7.4) containing 2% (v/v) normal goat serum
(TBS_NGS). Then the sections were washed in TBS and further
incubated overnight at room temperature with a mixture of
1/100-diluted biotinylated donkey anti-mouse IgM antibody
(Jackson ImmunoResearch, USA) and 1/100-diluted
anti-guinea pig IgG or anti-mouse IgG antibody conjugated to
1.4 nm gold particles (Nanoprobes; Stony Brook, NY, USA),
each diluted in TBS-NGS. Subsequently the sections were
processed for: (1) 1% postfixation with glutaraldehyde in 0.1
mol/L phosphate buffer PB for 10 min; (2) silver
enhancement with an HQ Silver Kit (Nanoprobes, Stony Brook, NY,
USA); (3) incubation with an ABC reagent (Vector Labs,
Burlingame, CA, USA) diluted at 1:50 in 50 mmol/L TBS for
3 h at room temperature; (4) visualization of
PAG-immunoreactivity by incubation with diaminobenzidine
tetrahydro-chloride and H2O2; (5) osmification with 1%
OsO4 in 0.1 mol/L PB for 1 h; (6) counterstaining with uranyl acetate; and (7)
flat-embedding in Durcupan (Fluka, Buchs, Switzerland)
after dehydration and mounting on silicon-coated glass
slides. Ultrathin sections were prepared and examined as
described[19].
Trigeminal rhizotomy was prepared as
described[22]. Briefly, after anesthesia, unilateral trigeminal rhizotomy was
performed on 3 rats. A 10-day postoperative period was
allowed for degeneration, and then the rats were fixed and
tissue was prepared for VGluT1 immunohistochemistry as
mentioned earlier. The sections containing the Vpd were
incubated sequentially at room temperature with: (1) 0.5
µg/mL guinea pig anti-VGluT1 IgG overnight; (2) 1/100-
diluted biotinylated anti-guinea pig IgG donkey antibody
(Jackson ImmunoResearch, USA) for 4 h; and (3)
1/50-diluted avidin-biotinylated peroxidase complex (Vector Labs,
USA) for 2 h. The incubation medium was the same as those
used in the immunofluorescence histochemistry.
Subsequently the sections were reacted with 0.02%
(w/v) 3,3'-diaminobenzidine tetrahydrochloride and 0.003%
H2O2 (v/v) for 10_30 min in 0.05 mol/L Tris-HCl buffer (pH 7.6).
Results
Under CLSM, the bodies of PAG-LI neurons were easily
identified with FITC fluorescence (green channel);
VGluT1-LI axon terminals with rhodamine fluorescence (red channel)
and GAD-LI axon terminals with Cy5 fluorescence (blue
channel). The PAG-IR was seen in small or medium-sized
cell bodies of the Vpd neurons. On the other hand,
VGluT1-IR was found mostly in the varicosities' characteristic of
axon terminals. We also found many GAD-LI axon
varicosities distributed in the Vpd. Both the VGluT1- and GAD-LI
axon terminals were frequently seen in close apposition to
PAG-LI cell bodies, and there were some VGluT1- and
GAD-LI axonal terminals which formed close appositions on the
same PAG-LI cell body (Figure 1).
Under the EM, some terminals were labeled with immunogold-silver grains indicating VGluT1-LI axon
terminals. Most of the VGluT1-LI terminals contained
abundant small clear round vesicles, and a number of VGluT1-LI
terminals made asymmetric synaptic contacts with
PAG-positive dendritic profiles (Figure 2A). No VGluT1-LI axon
terminals containing flattened vesicles and forming symmetrical
synapses were found. On the other hand, in the material
processed for dual immunohistochemical labeling of
GAD/PAG, most of the GAD-LI terminals made symmetric
synapses with PAG-LI dendritic profiles (Figure 2B).
It was examined whether or not VGluT1-IR is expressed
in primary afferent fibers. After unilateral trigeminal
rhizotomy, it was clearly shown that VGluT1-IR was less
intense in the Vpd (Figure 3A) on the side ipsilateral to the
trigeminal rhizotomy compared to the contralateral side
(Figure 3B, 3C). Thus, the trigeminal rhizotomy experiments
indicated that VGluT1 was expressed in primary afferent
fibers terminating within the Vpd.
Discussion
The results of present study show that PAG-LI neurons
receive orofacial sensory information transmitted from the
VGluT1-LI axon terminals, mainly arising from primary
afferent fibers of the trigeminal ganglion. It has been reported
that most of the neurons in the Vp projecting to the
ventro-posterior medial nucleus of the thalamus are
glutamatergic[9,11] and that glutamate-IR is present in some primary afferent
terminals and functions as an important excitatory
transmitter involved in the relay of sensory information to the
Vp[10]. Recently, Varoqui et al suggested that mechanoreception at
the Vp predominantly involved VGluT1 expressing axonal
terminals[17]. Combined with the previous studies, our
present results suggest that glutamatergic axon terminals
might act on the PAG-LI neurons in the Vpd and transmit the
orofacial region non-nociceptive sensory information to
them.
Bae et al[9] tested whether GABA could act on the
glutamatergic primary afferents through the axoaxonic
synapses to modulate the transmission of the orofacial sensory
information in the Vpd. In the present study, we observed
that the GAD-LI axon terminals formed symmetric synapses
with PAG-LI neurons and inferred that the GABA functioned
as an inhibitory transmitter acting on the neurons of the Vpd
in addition to the primary afferent fibers.
In summary, the present results, together with the
previously reported ones, provide strong anatomical evidence
that Vpd neurons receive trigeminal orofacial sensory
information from VGluT1 expressing glutamatergic terminals and
that GABAergic postsynaptic regulation might occur at the
level of the Vpd neurons.
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