Conceived and designed the experiments: JL YL. Performed the experiments: YD. Analyzed the data: YD FZ. Contributed reagents/materials/analysis tools: JL. Wrote the paper: YD JL YL.
The authors have declared that no competing interests exist.
It is well known that the brainstem premotor neurons of the facial nucleus and hypoglossal nucleus coordinate orofacial nociceptive reflex (ONR) responses. However, whether the brainstem PNs receive the nociceptive projection directly from the caudal spinal trigeminal nucleus is still kept unclear. Our present study focuses on the distribution of premotor neurons in the ONR pathways of rats and the collateral projection of the premotor neurons which are involved in the brainstem local pathways of the orofacial nociceptive reflexes of rat. Retrograde tracer Fluoro-gold (FG) or FG/tetramethylrhodamine-dextran amine (TMR-DA) were injected into the VII or/and XII, and anterograde tracer biotinylated dextran amine (BDA) was injected into the caudal spinal trigeminal nucleus (Vc). The tracing studies indicated that FG-labeled neurons receiving BDA-labeled fibers from the Vc were mainly distributed bilaterally in the parvicellular reticular formation (PCRt), dorsal and ventral medullary reticular formation (MdD, MdV), supratrigeminal nucleus (Vsup) and parabrachial nucleus (PBN) with an ipsilateral dominance. Some FG/TMR-DA double-labeled premotor neurons, which were observed bilaterally in the PCRt, MdD, dorsal part of the MdV, peri-motor nucleus regions, contacted with BDA-labeled axonal terminals and expressed
The trigeminal motor nucleus (V), facial nucleus (VII) and hypoglossal nucleus (XII) mainly administrate the orofacial muscle, such as jaw, lips, tongue, palate, pharynx, esophagus, larynx, diaphragm and other respiratory muscles and are involved in the orofacial coordinated activities. Electrophysiological and morphological studies demonstrated that numerous premotor neurons were distributed principally in the pontomedullary reticular formation (RF), raphe nucleus, trigeminal sensory complex, parabrachial region between the cerebral motor cortex and the brainstem orofacial motonucleiand precisely coordinated for executing complex muscular functions
The caudal spinal trigeminal nucleus (Vc) is involved in conveying nociceptive input from the skin of the face, lips, tooth pulp, oral and nasal cavities, cornea, and so on. Previous studies identified that the Vc was not only the region where the trigeminal primary afferents terminated but also the potential structure underlying the local pathways of the ONR
In addition, most orofacial motor nuclei, such as the VII and XII, are largely implicated in synchronizing various oral movements and performing ONR. By using fluorescent retrograde double-labeling method, the previous morphological studies showed that the distribution of the premotor neurons related to the VII or XII was overlapped
Our present studies aimed at (1) detecting whether the projection fibers from Vc might make synaptic contact to the premotor neurons projecting to VII or XII by using a double-labeled technique of biotinylated dextran amine (BDA) anterograde tracing combined with Fluoro-gold (FG), tetramethylrhodamine-dextran amine (TMR-DA) or wheat germ agglutinated horseradish peroxidase (WGA-HRP) retrograde transport; (2) examining whether the premotor neurons sending collaterals to the VII and XII simultaneously receive afferents from the Vc; (3) further identifying the nociceptive premotor neurons in the brainstem NR pathways in which the immunoreactivity for Fos was induced by subcutaneous injection of formalin into the upper and lower lips.
A total of 35 adult male rats (Sprague-Dawley; China SH, Xi'an, People's Republic of China) weighing 250–300 g were housed in a standard laboratory condition (artificial light cycle 12 h on/12 h off) with food and water
All of rats were divided into four groups (
n | purpose | tracer injection sites | formalin stimulation (n/group) | |
Group 1 | 10 | light microscope | FG injected into the VII and BDA into the Vc | 4 |
Group 2 | 10 | light microscope | FG injected into the XII and BDA into the Vc | 4 |
Group 3 | 10 | light microscope | FG injected into the VII, TMR-DA into the XII and BDA into the Vc | 4 |
Group 4 | 5 | electron microscope | WGA-HRP injected into the VII (2 animals) or the XII (3 animals) and BDA into the Vc | no |
BDA was used as an anterograde tracer to label axonal terminal of the Vc projecting neurons for epifluorescence, confocal laser scanning microscopic and electron microscopic studies. The animals were anesthetized by sodium pentobarbital (40 mg/kg, i.p.) until no limb-withdrawal reflex was elicited by pinching the hind paw. They were placed on a stereotaxic frame, and the cisternal cavity of the caudal medulla oblongata was surgically exposed. An approximate volume of 0.2 µl of a 10% solution of BDA (10,000 MW, D1956, Molecular probes, Eugene, OR, USA) dissolved in 0.05 M phosphate-buffered saline (PBS, pH 7.4) was injected into the Vc by pressure through a glass micropipette (internal tip diameter = 15–25 µm) which was attached to a 1-µl Hamilton microsyringe.
For light microscopic study in the first two groups, 4% of FG (80014, Biotium, Hayward, CA, USA) dissolved in 0.05 M PBS was iontophoresed into the VII or XII, respectively, on the side ipsilateral to the side of the BDA injection into the Vc with 7 µA positive current pulses (7 s on/7 s off) for 15 minutes after BDA injection. In the third group, 4% of FG was ipsilaterally iontophoresed into the VII after BDA injection, and then an approximate volume of 0.05–0.1 µl of a 10% TMR-DA (D3308, 3000 MW, anionic, lysine fixable; Molecular Probes, Eugene, OR) dissolved in 0.1 M citrate-NaOH (pH 3.0)
After the FG or/and TMR-DA injection, the rats were allowed to survive for 7 days and then they were killed by transcardial perfusion. Two hours before the perfusion, the 12 rats (4/group) in the first three groups were anesthetized with ethyl ether. Then a volume of 0.2 ml of 1.5% formalin dissolved in physiological saline was injected subcutaneously into the upper and lower lips ipsilateral to the surgery using a 25-gauge needle. The other rats were used as normal control (n = 6, 2/group) or experimental control (n = 12, 4/group). The experimental control received a single injection of 0.2 ml of physiological saline instead of the formalin solution.
After surviving for 7 days, the rats in the first three groups were deeply anesthetized by sodium pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 100 ml of a solution consisting of 0.9% saline in 0.05 M phosphate buffer (PB, pH 7.4), followed by a volume of 500 ml of 0.1 M PB containing 4% paraformaldehyde and 75% (v/v)-saturated picric acid. The brains were removed and placed in 0.1 M PB containing 30% (w/v) sucrose at 4°C for 24 h. Subsequently, the brainstems were serially cut into transverse sections 30 µm thick on a freezing microtome. The sections were divided into five series and collected into 0.05 M PBS (pH 7.4).
The first series of every group was for observation of the injection site of the VII or/and XII, the distribution of retrograde labeled neurons projecting to the VII or XII (Group 1 and 2), and the retrograde double-labeled neurons projecting to both the VII and XII (Group 3) by an epifluorescence microscope (BX-60; Olympus, Tokyo, Japan).
The second series of every group was processed for visualization of the injection site of Vc and the distribution of BDA-labeled fibers in the brainstem. The sections were incubated in 0.5% Triton X-100 in 0.05 M PBS (pH 7.6) overnight prior to incubation in the fluorescent isothiocyanate (FITC) -labeled avidin D (1∶200, A-2001, Vector Laboratories, Burlingame, CA, USA) at room temperature for 2 h. After the incubation, all the sections were rinsed in 0.05 M PBS, mounted onto gelatin-coated glass slides, air-dried, cover-slipped by a mixture of 50% (v/v) glycerin and 2.5% (w/v) triethylene diamine (anti-fading agent) in 0.05 M PBS. Then the injection sites and distribution of BDA-labeled fibers were observed by the epifluorescence microscope.
The third series of Groups 1 and Group 2 was processed for double-labeling study of FG-labeled neurons and BDA-labeled fibers and terminals. The sections were incubated at room temperature first with rabbit anti-FG IgG (1∶5000, AB153, Chemicon, Temecula, CA, USA) overnight and then with a mixture of 10 µg/ml FITC-labeled donkey anti-rabbit IgG antibody (AP182F, Chemicon), and 10 µg/ml Texas Red (TR)-labeled avidin D (A-2006, Vector Laboratories) for 4 h. The incubation medium used for the primary antibodies was 0.05 M PBS (pH 7.4) containing 2% normal donkey serum (NDS), 0.5% Triton X-100, 0.05% sodium azide (NaN3) and 0.25% λ-carrageen (NDS-PBS). The incubation medium for the secondary antibodies was 0.05 M PBS (pH 7.4) containing 0.5% Triton X-100.
The third series of Group 3 was used to observe the triple-labeling in FG, TMR and BDA. Briefly, the sections were incubated at room temperature first with rabbit anti-FG IgG and guinea pig (Gp) anti-TMR IgG (1∶2000)
The fourth series of Group 1 and Group 2 was used to identify the contact between the BDA-labeled fibers and retrograde double-labeled neurons with FG and Fos. Briefly, the sections were incubated firstly with a mixture of rabbit anti-FG IgG, mouse anti-Fos IgG (1∶1000, SC-413, Santa Cruz, CA, USA) at room temperature for 24 h and at 4°C for 24 h, and then with a mixture of FITC-labeled donkey anti-rabbit IgG antibody, Cy5-labeled goat anti-mouse IgG (1∶200, AP127S, Chemicon) and Rh-labeled avidin D for 4 hours. The incubation mediums were similar to those in the third series. The fourth series of Group 3 was used for the immunofluorescence histochemical study of the coexistence among the FG, TMR retrograde doubled-labeled neurons and Fos. The process was similar to that in the third series of Group 1 and 3 except for substitution of TMR-immunohistochemistry for BDA-immunohistochemistry. For controls, some sections were also processed as above but with omission of the first anti-Fos IgG, which resulted in no staining for the Fos protein.
The sections in the fifth series of every group were mounted onto gelatin-coated glass slides and then stained with 1% cresyl violet. Large projection drawings of these cresyl-violet-stained sections were then prepared, and the location of FG or/and TMR-DA-labeled neuronal cell bodies were plotted on the projection drawings with and aid of a camera lucida attachment. Subsequently, the data were reconstructed onto projection drawings of sets of serial sections.
The rats in Group 4, which survived seven days after the injection of BDA, were allowed to survive for additional 48 h following WGA-HRP injection. These rats were deeply reanesthetized by injection of an overdose of sodium pentobarbital (100 mg/kg, i.p.), and then perfused transcardially with 100 ml of 0.025 M PBS(pH 7.4), followed by 500 ml of a fixative consisting of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PB (pH 7.4). The brains were removed and stored in 0.1 M PB (pH 7.4) containing of 4% paraformaldehyde at 4°C for 2–4 h.
Serial sections of the pons and medulla were cut transversely on a vibratome (Microslicer DTM-1000; Dosaka EM, Kyoto, Japan) at 50 µm thickness. Tissue sections were processed for the histochemical demonstration of WGA-HRP by using tetramethylbenzidine-sodium tungstate (TMB-ST) method
The BDA-labeled fibers and FG/TMR-labeled neurons were scrutinized and counted under the epifluorescence microscope (BX-60, Japan) with appropriate filters for TMR (excitation 540–552 nm; emission≥575 nm), FG (excitation 350–395 nm; emission 430 nm) and FITC or DTAF (excitation 450–490 nm; emission 515–565 nm). Digital images of fluorescent labeling were taken by a DP-70 CCD camera and software (Olympus Optical Co., Inc., LTD, Japan) connected to a computer for permanent documentation. Two separate images from the same field were captured with two different filters corresponding to the specific combination of the tracers.
The immunofluorescence histochemical results were observed by a confocal laser-scanning microscope (CLSM, FV1000, Olympus, Tokyo, Japan) by using laser beams of 490, 590 and 640 nm with the appropriated emission filter for FITC (520 nm), Rh (615 nm) and Cy5 (705 nm). Then the digital images were arranged and modified (15–20% contrast enhancement) in software Tiff files. The ultrastructures were observed by an electron microscope (CM100; Philips, Eindhoven, The Netherlands).
The cases in which injection sites were centered in the VII (
Fluorescent photomicrographs of a section through the brainstem, showing the sites of FG centered on the facial nucleus (VII) (a), hypoglossal nucleus (XII) (b) and BDA on the caudal spinal trigeminal nucleus (Vc)(c). Scale bars = 250 µm.
In all successful cases of the first two groups, most FG retrogradely labeled neuronal cell bodies were medium (15–34 µm), triangular and multipolar in shape. There were also a few large (≥34 µm) multipolar, small (≤15 µm) fusiform or circular neuronal cell bodies by chance.
In the case of the VII injection, the retrogradely labeled neurons were observed throughout the brainstem with a level of caudal segment prevalence, the distribution pattern of which was generally consistent with that described in our previous study
In the case of the XII injection, most FG-labeled neuronal cell bodies were observed bilaterally in the pontomedullary RF with a slight dominance on the side ipsilateral to the injection site. The pattern of distribution of these labeled neuronal cell bodies in the pontomedullary RF was the same as that after FG injection into the VII. Moreover, a few retrogradely FG-labeled neuronal cell bodies were observed in the superficial layer of the Vc ipsilateral with the injection site.
After FG and TMR-DA were injected into both the VII and XII at the same side, respectively, FG/TMR-DA double-labeled neuronal cell bodies were observed bilaterally in the brainstem with an ipsilateral dominance (
Projection drawing of transverse section through the brainstem of R23, showing the distribution pattern of the retrograde double-labeled neuronal cell bodies with FG injection into the VII and TMR-DA into the ipsilateral XII. The injection sites are blackened. Neuronal cell bodies single-labeled with FG, TMR-DA, or those dually labeled with FG/TMR-DA are represented by filled circles, open circles or filled triangles. The filled circles and open circles are plotted in one-to-three fashion, whereas the filled triangles are one-to-one fashion. Scale Bar = 1 mm.
a–b′, Fluorescent photomicrographs showing FG (a and b) and TMR-DA (a′ and b′) retrograde-labeled neurons in the brainstem reticular formation after FG injection into the VII and TMR-DA ipsilaterally into the XII. Double-arrowheads indicate FG/TMR-DA double-labeled neurons, arrowheads and arrows indicate FG or TMR-DA single-labeled neurons, respectively. a and b were taken from the parvicellular reticular formation and the dorsal medullary reticular formation, respectively. The fields of a–b corresponds with that of a′–b′. Scale bar = 35 µm.
rat number | pontomedullary RF | PBN | regions around the Vmo | ||||||
FG | TMR | FG/TMR (%) | FG | TMR | FG/TMR(%) | FG | TMR | FG/TMR (%) | |
R23 | 394 | 283 | 46(6.4) | 49 | 23 | 3(4.0) | 67 | 53 | 5(4.0) |
R24 | 365 | 257 | 39(5.9) | 43 | 19 | 3(4.6) | 63 | 41 | 4(3.7) |
R26 | 325 | 253 | 33(5.4) | 43 | 18 | 2(3.2) | 58 | 44 | 4(3.8) |
R27 | 386 | 288 | 47(6.5) | 51 | 27 | 4(4.9) | 69 | 47 | 6(4.9) |
R29 | 369 | 264 | 41(6.1) | 47 | 24 | 4(5.3) | 65 | 44 | 4(3.5) |
Total | 1839 | 1345 | 206(6.1) | 233 | 111 | 16(4.4) | 322 | 229 | 23(4.0) |
In each rat, the numbers of FG-, TMR-single-labeled and FG/TMR-double-labeled neurons were counted in the pontomedullary reticular formation (RF), parabrachial nucleus (PBN) and the regions around the Vmo on both sides. And then, the ratio of FG/TMR-double-labeled neurons to total retrograde-labeled neurons (FG+TMR+FG/TMR) was calculated in each region. Cell counts were obtained from every fifth section of a series of serial sections of 30 µm thickness.
Double labeling method of BDA anterograde tracing combined with FG retrograde transport examined if the fibers and terminals of the trigeminal nociceptive afferents contacted the premotor neurons which send their axons to the VII or XII by CLSM. After BDA injected into the Vc (
Digital images showing the close appositions (arrows) between axonal terminals of the Vc neurons and FG retrograde-labeled dendrites (a, d) or neuronal cell bodies (a, b and c) of the premotor neurons in the brainstem reticular formation after BDA injection into the Vc and FG into the VII (a, b) or XII (c, d). a and c were taken from the parvicellular reticular formation, b from the supratrigeminal nucleus, d from the dorsal medullary reticular formation. Scale bars = 20 µm in a, b and d, 18 µm in c.
To examine whether the premotor neurons receiving trigeminal nociceptive afferents sent their bifurcating axon simultaneously to the VII and XII, the triple-labeling method of BDA anterograde tracing combined with FG and TMR-DA retrograde transport was applied. After FG and TMR-DA were injected into the VII and XII, respectively, and BDA was injected into the Vc, the appositions between BDA-labeled terminals and FG/TMR double-labeled premotor neurons were observed in pontomedullary RF, mainly including the ventral part of Vmo, PCRt, MdD and MdV, in which the highest density of the double-labeled premotor neurons was distributed(
a, b and c, digital images showing the close appositions (arrows) between axonal terminals of the Vc neurons and FG/TMR retrograde-double-labeled neuronal cell bodies (a, b) or dendrite (c) after FG injection into the VII, TMR-DA into the XII and BDA into ipsilateral Vc. Immunoreactivities for FG, Fos and BDA are visualized with FITC (green), Cy5 (blue), and Rh (red), respectively. d, BDA-labeled axonal fibers and terminals making close apposition to dendrite or neuronal cell bodies of FG-labeled premotor neurons expressing Fos-like immunoreactivities. e, FG/TMR/Fos triple-labeled neurons after FG injection into the VII, TMR-DA into the XII combined with the formalin ipsilaterally subcutaneous stimulation. a, b, d and e are taken from the ventral medullary reticular formation, and c is from the parvicellular reticular formation. Scale bars = 10 µm in a, c and d; 15 µm in b and 5 µm in e.
To further identify whether the FG or FG/TMR double-labeled premotor neurons were located in the ONR pathway, Fos expression (blue color) was observed at the fourth section in each series of serial sections of Group 1, 2 and 3 in which FG or FG/TMR-DA were injected into the VII or/and XII and BDA into the Vc (
Group | Animal number | MdV | PCRt | ||
Fos | FG+Fos (%) | Fos | FG+Fos (%) | ||
1 | No. 4 | 94 | 9(9.6) | 158 | 12(7.6) |
No. 9 | 87 | 5(5.7) | 181 | 9(5.0) | |
2 | No. 13 | 123 | 10(8.1) | 104 | 7(6.7) |
No. 15 | 148 | 9(6.1) | 99 | 4(4.0) |
In each rat, the total number of Fos positive neurons were counted in the MdV and PCRt. Of these Fos-labeled neurons, the numbers of FG retrogradely labeled ones (FG+Fos) were also counted. The ratio of FG+Fos to total Fos (%) was calculated in each region. Cell counts were obtained from every fifth section of a series of serial sections of 30 µm thickness.
However, there was no FG/Fos double-labeled neurons observed in the normal and control groups without injection with formalin.
Synaptic connections of projection fibers from Vc with the premotor neurons which send their axons to the VII or XII were examined by electron microscopy with a double-labeled method after injection of BDA into the Vc, WGA-HRP into the VII or XII. After WGA-HRP was injected into the VII or XII, the distribution pattern of retrogradely WGA-HRP-labeled neurons was similar to that FG injected into the VII or XII.
WGA-HRP-labeled premotor neurons were detected by the presence of highly electron-dense clumps of crystalline, sometimes amorphous punctual structure in the cytoplasm of the dendrites and somata (
WGA-HRP was injected into the VII of the rat and BDA into its ipsilateral Vc. Note: BDA-labeled terminals were identified by fine granular and homogeneously distributed electron dense reaction product with the dense distribution on the membrane of the synaptic vesicles or outer membrane of the mitochondria,and most synaptic vesicles are clear spherical in their terminal. HRP-labeled postsynaptic components exhibited highly electron dense, clumps of crystalline or amorphous material (arrows) in the cytoplasm and dendritic profiles. BDA-labeled S-type terminals (T) containing sphere vesicles make asymmetric synapses with dendrites (D) of HRP-labeled premotor neurons (a). BDA-labeled terminal forms asymmetric synapses with two HRP-labeled dendrites(D1, D2)and one negative spine (S) (b). BDA-labeled terminal makes asymmetric synapse with neuronal cell body of HRP-labeled premotor neurons (Soma) (c). One BDA-labeled terminal (T1) and one BDA-negative terminal (T2) synapse simultaneously with dendrite of HRP-labeled premotor neurons, and empty-arrowhead indicates the dense-core vesicle in the negative terminal (d). The arrowheads indicate postsynaptic specializations; N means nucleus of the cell body. a–d are taken from gigantocellular reticular formation, the dorsal medullary reticular formation, parvicellular reticular formation and supratrigeminal nucleus, respectively. Scale bars = 0.3 µm in a; 0.45 in b and d and 0.5 µm in c.
WGA-HRP was injected into the XII of the rat and BDA into its ipsilateral Vc. BDA-labeled S-type terminals (T) containing spherical vesicles make asymmetric synapses with dendrites (D) of HRP-labeled premotor neurons (a). Two BDA-labeled terminals (T1, T2) make asymmetric synapses simultaneously with dendrite of HRP-labeled premotor neuron (b). A BDA-labeled terminal makes asymmetric synapses simultaneously with one HRP-labeled spine (S2), one negative dendrite (D) and one negative spine (S1) (c). One negative terminal makes asymmetric synapse with HRP-labeled dendrite (d). The arrowheads indicate postsynaptic specializations. a and b are taken from the dorsal medullary reticular formation, c and d from the parvicellular reticular formation. Scale bars = 0.5 µm in a; 0.35 µm in b and c and 0.45 µm in d.
The synaptic contact of BDA-labeled boutons with dendrites or somata of WGA-HRP-labeled neurons were observed, and these synapses were mainly distributed in PCRt, MdD, MdV, Vsup and PBN. The synapses were counted in the PCRt of five sections. Quantificative analysis showed 73% of BDA-labeled boutons (88 among 120) made synapses with dendrites of WGA-HRP-labeled premotor neurons, while 27% (32/120) formed axo-somatic synapses. The vast majority of synapses were asymmetric in which the presynaptic elements contained small, clear and spherical synaptic vesicles, and the postsynaptic membrane was overtly thickened and specialized (
In addition, there were also some synapses between BDA-labeled boutons and dendrites or somata without HRP product, or between negative boutons and HRP-labeled dendrites or somata of premotor neurons. Some negative axonal terminals included dense-core particles (
In our present study, the direct connection of projection fibers from Vc with the premotor neurons which sent their axons to the VII or XII, and both to the VII and XII by axon collaterals in the brainstem was examined using tracer tracing method under both light and electron microscopic levels. In addition, we detected that some promoter neurons in which Fos protein-like immunoreactivity was induced by subcutaneous injection of formalin into the lips apposed to the Vc projecting fibers. These results indicate that those premotor neurons which sent their axons to the VII or/and XII are involved in local orofacial reflex circuits.
The premotor neurons projecting to VII or XII had the same distribution pattern and were mainly located in the brainstem RF, including PCRt, MdD. The previous morphological experiments had shown that the last-order premotor neurons projecting to the VII or XII were mainly distributed in the PCRt and MdD
To confirm whether these premotor neurons receive afferent projecting from the Vc, we observed the close appositions between the BDA-labeled fibers and FG-labeled premotor neurons by CLSM as well as the synaptic contact between these two components by electron microscope.
The Vc has traditionally been considered as an essential brainstem relay for the orofacial peripheral nociceptive information transmission. The previous studies also demonstrated that the Vc may be a critical element in the neural pathways underlying ONR
A proto-oncogene
The synaptological characteristics of projecting from the Vc to premotor neurons were examined by electron microscopy. Our present ultrastructural observations demonstrated that the axonal boutons from the Vc were S-type containing small, clear and spherical synaptic vesicles, and predominantly formed asymmetric synapses with the dendrites of the premotor neurons projecting to the VII or XII. This result suggested that the innervations of the Vc neurons to the premotor neurons was excitatory, which is consistent with our previous study
The orofacial nociceptive afferents converge onto the neurons of the Vc. The Vc projecting (glutamatergic excitatory axons) act on the motoneurons of the VII or XII via inhibitory premotor neurons (N1, N2 and N3) in the brainstem, and then the tonic orofacial nociceptive reflexes are decreased. Real lines represent excitatory, and the dotted lines are inhibitory. ? represents other inhibitory neurotransmitters.
There are also some BDA-negative axonal terminals making asymmetric synapses on the dendrites of premotor neurons contain dense-core vesicles, which suggests these boutons are likely to contain substance P or enkephalin. In terms of neurochemical properties of projections from the premotor neurons to cranial motoneurons, previous studies indicated that some neuropeptides like substance P and enkephalins existed in the projecting terminals
Oral motor behaviors including feeding, mastication, swallowing, vocalization and respiration are complex movements. The precise coordination of different orofacial muscles and motor nuclei is necessary for executing the complex function. For example, during chewing, the masser begins rhythm activities concomitantly with the movement of tongue
Morphological studies have revealed that the distribution of the premotor neurons of Vmo, VII and XII is overlapping in the brainstem
The doubly labeled premotor neurons were mainly distributed in the brainstem, especially in the RF and probably subserved the coordination of orofacial movements required for the complex oral motor behaviors. Furthermore, physiological studies showed that stimulating various trigeminal nerve branches could elicit reflex responsiveness of different groups of brainstem motoneurons
After FG injection into the VII, TMR into XII and BDA into Vc, some FG/TMR double-labeled premotor neurons, receiving projections from Vc and sending their axonal collaterals simultaneously to the VII and XII, were mainly observed in the pontomedullary RF, such as the ventral part of Vmo, PCRt, MdD and MdV. Dauvergne
According to our experiments, not so many double-labeled premotor neurons closely appose with Vc fibers, which may be related with the limitation of the tracer-tracing method we used. The retrograde tracer FG and WGA-HRP cannot be trans-synaptically transported, so the bi-synapse pathways can only be traced
In conclusion, our study has corroborated the distribution of the premotor neurons which are involved in the brainstem local circuits of the nociceptive reflexes and has demonstrated that the orofacial nociceptive afferents axon terminals synapse upon the premotor neurons of the VII and XII under the electron microscope. Moreover, we provide some morphological evidence for existence of the premotor neurons projecting simultaneously to both the VII and XII by the axonal collaterals. These premotor neurons may play an important role in harmonizing different orofacial muscles movements and accomplishing orofacial complex nociceptive reflexes.
The authors thanks Dr. Wu Sheng-xi, Dr. Wang Wen and Dr. Zhou Yu-mei for their valuable comments on the first version of the manuscript.