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Keywords:

  • catecholamine;
  • dopamine-β-hydroxylase;
  • masticatory control;
  • motoneurons;
  • tyrosine hydroxylase

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The noradrenergic (NA) innervation in the trigeminal motor nucleus (Vmot) of postnatal and adult rats was examined by light and electron microscopic immunocytochemistry using antibodies against dopamine-β-hydroxylase or tyrosine hydroxylase. NA fibers were identified in the Vmot as early as the day of birth (postnatal day 0; P0). A continuous increase in the density of labeled fibers was observed during development up to P20, with a slight decrease at P30 and in the adult. Electron microscopic analysis of serial ultrathin sections revealed that, at P5, nearly half (46%) of the examined NA terminals made synaptic contact with other neuronal elements with membrane specializations. The percentage of examined NA varicosities engaged in synaptic contacts increased at P15 (74%), then decreased in the adult (64%). At all developmental ages, the majority of contacts made by these boutons were symmetrical, the postsynaptic elements being mainly dendrites and occasionally somata. Interestingly, some of the NA terminals made axo-axon contacts with other unidentified boutons. These results show that, although the density of NA fibers increases during postnatal development, functional NA boutons are present in the Vmot at early postnatal ages. Some of these fibers might exert their effects via nonsynaptic release of noradrenaline, the so-called volume transmission, but, in the main, they form conventional synaptic contacts with dendrites, somata, and other axonal terminals in the Vmot. These results are consistent with previous electrophysiological studies that propose an important role for the NA system in modulating mastication. Anat Rec, 290:96–107, 2007. © 2006 Wiley-Liss, Inc.

The trigeminal motor nucleus (Vmot) consists mainly of the cell bodies and dendrites of motoneurons, which project to the masticatory muscles of the jaw (Limwongase and DeSantis, 1977; Hamos and King, 1980). The trigeminal motoneurons receive excitatory monosynaptic inputs from group Ia primary afferents originating from jaw-closer muscle spindles (Appenteng, 1990; Luo and Li, 1991; Taylor et al., 1995). They also receive both excitatory and inhibitory inputs from interneurons, which are located lateral and caudal to the Vmot (Appenteng et al., 1989, 1990; Shigenaga et al., 2000; Yoshida et al., 2001) and are suggested to play an important role in the rhythmical patterns of jaw movement associated with mastication (Goldberg and Chandler, 1990; Donga and Lund, 1991; Min et al., 2003). The transmitter for the fast excitatory synaptic inputs onto motoneurons is glutamate (Yang et al., 1997a), acting on AMPA and NMDA receptors (Min and Appenteng, 1996; Min et al., 1999, 2002), while the transmitters for the inhibitory inputs are GABA and/or glycine (Yang et al., 1997b), with GABA acting on GABAA and GABAB receptors (Min et al., 1996, 1999, 2002) and glycine on the strychnine-sensitive glycine receptor (Min et al., 1996, 1999; Yang et al., 1996).

In addition to receiving the conventional fast synaptic inputs described above, trigeminal motoneurons also receive noradrenergic (NA) innervation arising from the perikarya of the A7 and A5 nuclei, both of which are located in the midbrain and belong to the lateral tegmental NA groups (Vornov and Sutin, 1983; Grzanna et al., 1987; Lyons and Grzanna, 1988). Early in vivo electrophysiological evidence indicated that noradrenaline release from NA terminals facilitates both the excitatory postsynaptic potentials recorded in motoneurons and the monosynaptic reflex evoked by stimulation of Ia primary afferent axons from the masseter muscle (Morilak and Jacobs, 1985; Vornov and Sutin, 1986; Stafford and Jacobs, 1990). In addition, Katakura and Chandler (1990) reported that application of small quantities of noradrenaline potently facilitates the glutamate- and synaptically mediated excitation of trigeminal motoneurons during rhythmical masticatory-like activity. These physiological studies suggest an important role for noradrenaline in modulating masticatory movement. However, whether NA terminals perform this role by simple nonsynaptic release of neurotransmitter into the extracellular space, thus affecting not only adjacent, but also more distant, motoneurons (the so-called volume transmission) (Beaudet and Descarries, 1978), or by making specific synaptic contacts with motoneurons, is still a matter of debate. A potentially sensitive method for addressing this issue is to examine the ultrastructure of NA terminals using a large number of serial sections covering the entire NA terminal in the Vmot in both adult and developing animals. Although a previous study examined the ultrastructural characteristics of the NA innervation in the Vmot (Card et al., 1986), no information is available on the anatomical organization of the NA system in the developing Vmot or on the ultrastructure of NA terminals obtained using a large number of serial sections. In the present study, we sought to examine the precise pattern of innervation and synaptic organization of this system in the developing and mature rat Vmot using light microscopic (LM) and electron microscopic (EM) immunocytochemistry and antibodies raised against dopamine-β-hydroxylase (DBH), the enzyme catalyzing the conversion of dopamine to noradrenaline, or against tyrosine hydroxylase (TH), the enzyme catalyzing the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Our aims were to provide a detailed description of the NA innervation pattern in the Vmot, how this pattern is established during development, and how the ultrastructural features of noradrenaline-containing synapses change with age by examining serial ultrathin sections cut through entire TH-immunoreactive (IR) boutons.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Perfusion and Fixation

The use of animals in this study was in accordance with the rules for animal research of the Ethical Committee of the National Science Council in Taiwan. Wistar rats of both sexes of various ages were deeply anesthetized with sodium pentobarbitone and perfused via the cardiac-vascular system with normal saline followed by fixative consisting of 4% paraformaldehyde (Merck, Frankfurt, Germany) in 0.1 M phosphate buffer (PB), pH 7.4 (preparations for fluorescence microscopy and LM), or 2.5% glutaraldehyde (Merck) and 1% paraformaldehyde in 0.1 M PB (preparations for EM). The brains were then rapidly removed and placed in the same fixative at 4°C for 3–4 hr and stored overnight in cold (4°C) 0.1 M PB. For preparations for fluorescence microscopy and LM, the brains were then transferred to 30% sucrose in 0.01 M PB for cryoprotection.

Fluorescence Microscopic Immunocytochemistry

Five Wistar rats of both sexes [two P15 and three P60–90 (adult); P0 is day of birth] were used in this study. Serial sagittal brainstem sections (50 μm thick) containing the Vmot and surrounding regions were cut using a frozen sectioning technique. The sections were collected in 0.1 M PB, rinsed in 0.03% Triton X-100 in phosphate-buffered saline (TPBS), and incubated for 1 hr at room temperature in TPBS containing 2% bovine serum albumin (BSA), 10% normal goat serum, and 10% normal horse serum (NHS). The sections were then incubated overnight at 4°C in TPBS containing a 1/1,500 dilution of mouse antibody against DBH and a 1/3,000 dilution of rabbit antibodies against TH (both from Chemicon, Temecula, CA). After rinses in TPBS, the sections were incubated with fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG antibodies and tetramethyl rhodamine (TRITC)-conjugated goat antirabbit IgG antibodies (both from Jackson, PA), both diluted 1/50 in TPBS.

Light Microscopic Immunocytochemistry

Twenty-seven male and female Wistar rats of the following ages were used: P0 (n = 3), P5 (n = 4), P10 (n = 3), P15 (n = 5), P20 (n = 5), P30 (n = 4), and P60–90 (n = 3). Sagittal brainstem sections (50 μm thick), cut using a frozen sectioning technique, were treated for 20 min with 3% H2O2 in 0.1 M PB to quench endogenous peroxidase activity. After rinses in 0.1 M PB and TPBS, the sections were preincubated for 1 hr in 10% NHS in TPBS, then incubated overnight at 4°C with mouse anti-DBH antibody diluted 1/1,500 in TPBS. After rinsing in TPBS, the sections were incubated for 1 hr with biotinylated antimouse IgG antibody (Vector, Burlingame, CA) diluted 1/200 in TPBS and then, after rinses in TPBS, were incubated for 1 hr with avidin-biotin horseradish peroxidase complex (ABC; Vector). Following rinses in TPBS and 0.1 M PB, the sections were immersed in 0.05% 3,3′-diaminobenzidine solution for 10 min, then 0.004% H2O2 was added and the sections incubated for a further 10 min.

Electron Microscopic Immunocytochemistry

Ten male and female Wistar rats of the following ages were used: postnatal day P5 (n = 4), P15 (n = 3), and P60–90 (adult; n = 3). For EM immunocytochemistry, sagittal sections (70 μm thick) were cut with a Vibratome (Ted Pella, CA) and subjected to one cycle of freeze-thawing to increase reagent penetration. The sections were preincubated for 15 min in 0.5% sodium borohydride in 0.1 M PB, rinsed in 0.1 M PB, and incubated for 1 hr in phosphate-buffered saline (PBS) containing 2% BSA and 10% NHS, then for 48 hr at 4°C with mouse anti-TH antibody diluted 1/3,000 in PBS. After rinses in PBS, the sections were incubated for 16–18 hr with biotinylated antimouse IgG antibody diluted 1/200 in PBS and then, after further rinses in PBS, were incubated for 1 hr with ABC reagent. The sections were rinsed in PBS and 0.1 M PB, immersed for 10 min in 0.05% DAB solution, then 0.04% H2O2 was added and the sections incubated for a further 10 min. The sections were postfixed for 1 hr at 4°C with 1% osmium tetroxide in 0.1 M PB, rinsed twice with H2O, dehydrated in graded ethanol, and infiltrated for 20 min at room temperature with a 1:1 mixture of propylene oxide in Durcupan resin (Sigma). The sections were then infiltrated with Durcupan alone for 2 × 30 min at 60°C, flat-embedded under coverslips, and placed in a 60°C oven overnight to cure the resin. The coverslips were peeled off, the sections examined under a dissecting microscope, and the areas containing the Vmot cut out and reembedded in resin moulds. Serial ultrathin sections (60 nm) were mounted on single-slot copper grids coated with 1.5% Formvar in chloroform, stained with uranyl acetate followed by lead citrate, and examined with a JOEL JEM-2000 EXII electron microscope.

Light Microscopic Data Analysis

The density of DBH-IR fibers was examined in four nonconsecutive sections through the Vmot in each brainstem specimen and 3–4 brainstems were examined for each developmental age. The Vmot in each section was divided into nine subareas, and three of these were randomly selected and analyzed. Each of the selected subareas was further divided by 10 horizontal lines (length 235 μm) and 13 vertical lines (length 180 μm) and the number of times DBH-IR fibers crossed a vertical or horizontal line was counted. The average of the number of crossings per 100 μm of line was then calculated to evaluate the density of DBH-IR fibers and their projection direction. The area of the DBH-IR varicosities was measured in 3–4 nonconsecutive sections in each brainstem specimen, 3–4 brainstems being examined for each developmental age. To measure the area of a DBH-IR varicosity, photographs were taken using a 40× objective lens. This magnification allowed the area of an individual DBH-IR varicosity to be precisely outlined and measured using a graphics tablet (model XD-0608-U; Wacom, Japan) and NIH image software (downloaded from the National Institute of Health Web site: www.nih.gov). Measurements were made on about 160 varicosities for each developmental age.

Electron Microscopic Data Analysis

For each 70 μm thick Vibrotome section, up to 30–50 serial ultrathin sections were cut and collected in consecutive 10 grids, 3–5 ultrathin sections being arranged on one grid.

Ultrathin sections were systemically scanned for TH-IR axon varicosities. When a clearly labeled varicosity was encountered, it was followed for its full extent in serial sections to look for evidence of a synapse. TH-IR axon varicosities were collected from 3–4 brainstem specimens for each developmental age. Synapses formed by labeled varicosities were identified by the presence of a restricted zone of parallel pre- and postsynaptic membrane specializations with slight enlargement of the intercellular space, i.e., a visible synaptic cleft, and/or associated postsynaptic thickening, and the accumulation, of synaptic vesicles in the presynaptic profile (Peters and Palay, 1996; Bajic et al., 2001; Latsari et al., 2002). Furthermore, the synaptic junctions were classified as asymmetrical when a prominent plaque of dense material was seen on the cytoplasmic face of the postsynaptic membrane and as symmetrical when a less prominent density was seen on the postsynaptic membrane (Peters and Palay, 1996; Bajic et al., 2001). These structural features used as criteria for judging a synaptic contact are clearly labeled in all EM photographs. Somata were identified by the presence of dense stacks of rough endoplasmic reticulum, ribosomes, Golgi apparatus, mitochondria, and a nucleus. The major dendritic processes were distinguished from axons by containing Nissl bodies (rough endoplasmic reticulum and ribosomes) and abundant microtubules, neurofilaments, and mitochondria. Boutons were identified by the presence of densely packed vesicles and the absence of endoplasmic reticulum. The maximal cross-sectional area, apposition length, and active zone length of synaptic boutons were measured using a graphics tablet and NIH image software as described in the LM section. The apposition length was measured as the distance over which the pre- and postsynaptic plasma membranes were apposed and the active zone was measured as the length of the region showing postsynaptic thickness and aggregation of presynaptic vesicles (Yang et al., 1997b). Examples of these measurements are shown in Figure 4B.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Confocal Microscopic Observations of DBH- and TH-Immunoreactive Terminals in Vmot

We first confirmed the specificity of the anti-DBH and anti-TH antibodies used by examining the profile of their immunoreactivity in the substantia nigra, a region known to contain dopaminergic, but no NA, neurons. All neurons in the substantia nigra were found to be TH-IR (Fig. 1B; labeled with FITC-conjugated second antibody), but not DBH-IR (Fig. 1A; labeled with TRITC-conjugated second antibody). These results are consistent with previous reports (Plenz and Kitai, 1998; Emmett and Greenfield, 2005) and suggested that the two antibodies were specific enough to distinguish dopaminergic and NA neurons in the central nervous system.

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Figure 1. Confocal fluorescence microscopic images showing DBH and TH immunoreactivity in the substantia nigra (A and B), A7 and A5 (D and E), and the Vmot (G and H). C shows a control section incubated without primary antibody. F and I show superposition of TH and DBH labeling in the A7 and A5 cell groups and the Vmot, respectively. Green, TH labeling; red, DBH labeling; yellow, merging of TH and DBH labeling. Scale bars = 200 μ m (A–I); 20 μm (insets).

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We then used the same staining procedures to examine sections containing the Vmot and the surrounding area. Under confocal microscopic examination at low magnification, two populations of positively stained neurons located, respectively, rostrally and ventrally to the Vmot could be identified (Fig. 1D and E). These two groups were, respectively, the A7 and A5 cell groups according to Paxinos and Watson (1998). All of the neurons in these groups were found to be both TH-IR and DBH-IR (Fig. 1F), suggesting that, in rats, these two nuclei consist exclusively of NA neurons, as reported previously (Levitt and Moore, 1979; Grzanna et al., 1987; Lyons and Grzanna, 1988). At high magnification, numerous neuronal fibers could be seen in the Vmot (Fig. 1G and H). The vast majority of these varicosities were TH-IR and DBH-IR (Fig. 1I), although some rare variscosities were only TH-IR. These results suggested that most of the catecholaminergic fibers in the Vmot are NA, rather than dopaminergic. The TH-IR/DBH-IR boutons in the Vmot were visible as en passant swellings along the axons, as bulbous axon terminals, or as a single swelling at the end of fine axon branches (Fig. 1I). Control samples processed without primary antibody showed no staining (Fig. 1C).

Light Microscopic Observations of DBH-IR Axons in Developing and Adult Rats

We next examined the developing patterns of NA fibers in the Vmot. Since we had confirmed that the vast majority of positively stained fibers in the Vmot were both TH-IR and DBH-IR, and since the reaction products of chromogens can provide better resolution of fine structures than fluorescent markers, in this series of experiments, anti-DBH antibody was used to label NA fibers, and DAB was used as chromogen. On the day of birth (P0), DBH-IR fibers in the Vmot were extremely sparse (Fig. 2A). Compared to those in the adult, these fibers were relatively thin and less arborized, with few varicosities. At P5 (Fig. 2B), the distribution of DBH-IR fibers was similar to that at P0, but their density was increased. At this developmental stage, DBH-IR fibers were thicker and there were more varicosities than at P0. A continual increase in the density of labeled fibers in the Vmot was observed at days P10 (Fig. 2C), P15 (Fig. 2D), and P20 (Fig. 2E), followed by a slight decrease at P30 (Fig. 2F), then fiber density leveled off until the adult stage (Fig. 2G). The distribution pattern and fiber density at P15 were similar to those in the adult, suggesting that NA innervation in the Vmot was close to the mature level at P15. In controls, the primary antibody was omitted, and, as expected, this resulted in no staining (Fig. 2H).

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Figure 2. Low-power LM photomicrographs showing the density and distribution of NA innervation of the rat Vmot at various ages. A: Postnatal day (P) 0. B: P5. C: P10. D: P15. E: P20. F: P30. G: Adult. H: Control. The inset in each panel shows the NA varicosities at high magnification. Scale bars = 50 μm (A–H); 3 μm (insets).

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The quantitative data for the density of DBH-IR fibers in the Vmot (Fig. 3) showed that the averaged number of DBH-IR fibers crossing per 100 μm of horizontal lines and vertical lines combined increased up to P20, then fell slightly on P30 and in the adult. In addition, at all developmental ages, the averaged number of DBH-IR fibers crossing per 100 μm of vertical lines was significantly higher than that for the horizontal lines (Fig. 3, cf. white or dark gray columns, respectively), suggesting that, in the Vmot, there was more DBH-IR fiber projection in the horizontal direction than in the vertical direction. A significant increase in horizontal, vertical, and total fiber density was seen between each two consecutive developmental stages up to P15, but not beyond this stage, although fiber density at P20 appeared higher. These results suggest that fiber density might reach the mature level by P15.

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Figure 3. Quantitative analysis of the density of DBH-IR fibers in the Vmot. The white, black, and gray columns show, respectively, the averaged number of DBH-IR fibers crossing per 100 μm of vertical lines, horizontal lines, and both combined (mean ± SD). Note the averaged count for DBH-IR fibers crossing vertical lines is significantly higher than that for fibers crossing horizontal lines at every postnatal age examined. Asterisk, P < 0.05 (Mann-Whiney U-test).

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The area of NA varicosities measured within the Vmot was also age-dependent and increased gradually from P0 to P30, as shown in the insets in Figure 2. The quantitative data for the area of DBH-IR varicosities at the different developmental ages were 0.44 ± 0.01 μm2 at P0, 0.59 ± 0.02 μm2 at P5, 0.70 ± 0.02 μm2 at P10, 0.80 ± 0.02 μm2 at P15, 0.92 ± 0.02 μm2 at P20, 1.03 ± 0.03 μm2 at P30, and 1.02 ± 0.02 μm2 in the adult. A significant increase in varicosity size was found between each two consecutive developmental stages up to P30, but not beyond. These results suggest that the DBH-IR varicosities might be morphologically mature by P30.

Electron Microscopic Observations of TH-IR Boutons

In order to obtain better ultrastructure for electron microscopic observations, a glutaraldehyde-based fixative was used for specimen preparation. In a pilot study, we found that good immunostaining results were obtained with glutaraldehyde-fixed specimens using the anti-TH antiserum, but not the anti-DBH antiserum. Since we had demonstrated that the vast majority of the DBH-IR fibers in the Vmot were also TH-IR, the anti-TH antibody was therefore chosen. We also confirmed that the morphology of the TH-IR terminals was very similar to that of the DBH-IR terminals. However, significantly fewer fibers were labeled; this might be ascribed to the use of glutaraldehyde as fixative, which may partially destroy the antigenicity of TH.

Postnatal day 5

On EM, axonal boutons that contained electron-dense, crystal-like DAB products were assumed to be TH-IR boutons (Fig. 4). Thirty-seven TH-IR boutons, collected from 3–4 brainstems, were analyzed at the EM level at P5 (Table 1). Four of these were found to make axo-somatic synapses (Fig. 4A), 10 made axo-dendritic synapses (Fig. 4C), and 3 were associated with unlabeled axon terminals and appeared to make axo-axon contacts (Fig. 4D). The other 20 boutons did not make synapses with any neuronal structure visible in serial ultrathin sections. Of the 17 TH-IR boutons that formed synaptic contacts, 16 formed typical symmetrical synapses (Fig. 4A, B, and D) and only 1 formed an asymmetrical synapse (Fig. 4C). All 37 of the boutons contained loosely packed synaptic vesicles, which consisted of a mixture of spherical, ovoid, and flatted forms and were classified as pleomorphic, since they contained a roughly equal mixture of all vesicle types (Fig. 4). In addition, 19 of the 37 TH-IR boutons also contained dense-core vesicles. Morphometric measurement of boutons based on serial sections gave a maximal area (mean ± SD) of 0.56 ± 0.29 (range, 0.17–1.65) μm2, a maximal apposition length of 0.86 ± 0.32 (range, 0.60–1.47) μm, and a maximal active zone length of 0.20 ± 0.07 (range, 0.24–0.34) μm.

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Figure 4. EM photomicrographs illustrating TH-IR varicosities in the Vmot at P5. Labeled varicosities were identified by the presence of the dark, crystal-like products of the DAB reaction throughout the labeled varicosity. Synaptic contacts were identified by the presence of a restricted zone of parallel pre- and postsynaptic membrane specializations, as indicated by the two parallel arrows in A–D, with a visible synaptic cleft (see “SC” in A) and/or associated synaptic density (see “SD” in C), and by the accumulation of synaptic vesicles in the presynaptic profile (indicated by “SV” in A–D). Note that dark precipitates of DAB reaction products are sometimes seen on synaptic vesicles. The distance indicated by the two parallel arrows showing postsynaptic thickness and aggregation of presynaptic vesicles was taken as a measure of the active zone. The distance covered by the apposition of the pre- and postsynaptic plasma membranes, as indicated by the dotted lines and arrows in B, was taken as a measure of the apposition length. A and B show two TH-IR boutons forming symmetrical synapses with, respectively, a cell body (A) and a dendrite (B) in the Vmot, because, in both cases, there is a less prominent density (indicated by the two parallel arrows) on the postsynaptic membrane. C shows a TH-IR terminal forming an asymmetrical synapse on a dendrite in the Vmot, because a prominent density can be identified on the face of the postsynaptic membrane (indicated by the two parallel arrows). Note the presence, next to the TH-IR varicosity, of an unlabeled terminal (UT) also forming an asymmetrical synapse with the same dendrite (white arrow). D shows a TH-IR bouton making an axo-axon contact with another unlabeled terminal (UT) in the Vmot. The inset shows a larger magnification. Scale bars = 0.2 μm (A–D); 0.05 μm (inset in D).

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Table 1. Quantitative data for TH-immunoreactive boutons in the trigeminal motor nucleus at different postnatal ages
AgeNo sampledForming SynapseActive zoneaPostsynaptic targetaVesicle typea
NoYesSymmAsymSomaSomatic spineDendriteDendritic spineAxon terminalPdcv
  • a

    Abbreviations : Symm, symmetrical; Asym, asymmetrical; P, pleomorphic; dcv, dense core vesicles.

  • b, c

    Total number of postsynaptic targets (25) exceeds total number of boutons forming synapses (23), since 2 boutons formed separate active zones on two different targets.

  • d, e

    Total number of postynaptic targets (47) exceeds total number of boutons forming synapses (43), since 3 boutons formed separate acitve zones on two different targets.

P53720171614010033719
P15318231943b116c143128
Adult6925443954139d03e6959
Postnatal day 15

Thirty-one TH-IR boutons from 3–4 brainstems were examined at P15 (Table 1). Three were found to make synaptic contact with cell bodies in the Vmot (Fig. 5A) and one of these also formed a second separate synaptic active zone onto a somatic spine. Sixteen of the 31 formed synaptic contacts with dendrites in the Vmot (Fig. 5B and C), 1 of these making contact with a dendritic spine (Fig. 5D). Four of the 31 boutons were associated with unlabeled axon terminals and appeared to make axo-axon contacts, and 1 of these also formed a separate active zone onto a dendrite. The remaining eight boutons did not make synaptic contact with any neuronal structure. Of the 23 TH-IR boutons that made synaptic contacts, 19 formed symmetrical synapses (Fig. 5A–C) and 4 asymmetrical synapses (Fig. 5D). All 31 boutons contained pleomorphic vesicles and the majority (28/31) contained dense-core vesicles (Fig. 5C). Morphometric measurement gave a maximal area of 0.49 ± 0.23 (range, 0.13–1.14) μm2, a maximal apposition length of 0.61 ± 0.31 (range, 0.21–1.33) μm, and a maximal active zone length of 0.25 ± 0.17 (range, 0.10–0.98) μm.

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Figure 5. EM photomicrographs illustrating TH-IR varicosities in the Vmot at P15. AC show three TH-IR terminals making symmetrical synaptic contacts with, respectively, a cell body (A), a dendrite (B), and a distal dendrite (C). Note the less prominent density on the face of the postsynaptic membrane indicated by the two parallel arrows in all three cases. In addition, the bouton in C contains dense-cored vesicles indicated by white arrowheads. D shows a TH-IR bouton and an unlabeled bouton (UT) forming asymmetrical synapses (white arrows) with a dendritic spine. Note the prominent postsynaptic density of the active zone indicated by four arrows. Another unlabeled bouton (UT) also forms a asymmetrical synapses (white arrows) with another dendrite. Scale bars = 0.2 μm.

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Adult

A total number of 69 TH-IR boutons from 3–4 brainstems were examined in adult rats (Table 1). Five made synaptic contacts on cell bodies in the Vmot (Fig. 6A), one being on a somatic spine. Thirty-nine of the 69 made axo-dendritic contacts (Fig. 6B and C) and 3 of these made multiple synaptic contacts on an unlabeled terminal in addition to dendrites (Fig. 6D). Twenty-five of the 69 did not make synaptic contacts on any neuronal structure (Fig. 7). Of the 44 TH-IR boutons that formed synaptic contacts, 39 were identified as symmetrical synapses (Fig. 6A, B, and D) and 5 as asymmetrical synapses (Fig. 6C). All of the 69 TH-IR boutons contained pleomorphic vesicles, with dense-core vesicles being found in 59 (Fig. 6B and C). Morphometry gave a maximal area of 0.57 ± 0.28 (range, 0.12–1.79) μm2, a maximal apposition length of 0.79 ± 0.36 (range, 0.39–1.83) μm, and a maximal active zone length of 0.27 ± 0.12 (range, 0.10–0.62) μm.

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Figure 6. EM photomicrographs illustrating TH-IR varicosities in the Vmot in the adult. A and B show two TH-IR terminals making symmetrical synaptic contacts (black arrows) with a cell body (A) and a dendrite (B). C shows a TH-IR axon profile comprising two varicosities (asterisks) and one intervaricose segment (white arrowheads); the inset is a higher magnification of the small rectangle in the main panel to show the TH-IR varicosity forming asymmetrical synapses (white arrows) with a dendrite. Note that the bouton in A, B, and C contains a dense-cored vesicle (black arrowhead). D: A TH-IR terminal simultaneously forming a symmetrical synapse with a dendrite (large black arrow) and an unlabeled bouton (UT; small black arrow). Scale bars = 0.2 μm (A, B, and D); 0.5 μm (C); 0.13 μm (inset in C).

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Figure 7. AE: Serial ultrathin sections through a TH-IR axon terminal in the adult Vmot, which contains a varicosity (asterisks) and an intervaricose segment (black arrows) and does not make synaptic contacts with any neuronal structure. Note the synaptic vesicles in this TH-IR axon terminal (F and G; white arrows). Scale bar = 0.2 μm (A–E); 0.1 μm (F and G).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Using double fluorescence with primary antibodies against TH and DBH, we demonstrated that the vast majority of catecholaminergic fibers in the rat Vmot were immunoreactive for both enzymes. We therefore presume that the identified fibers and related structures are mainly NAergic and not dopaminergic. The presumed NA fibers and their varicosities appeared as early as the day of birth. These structures showed an age-dependent increase in both fiber density and the size of their varicosities, which, according to morphological criteria, reached mature levels at P15 and P30, respectively. Although a lower density of fibers and fewer varicosities were observed at early postnatal ages (before P15), ultrastructural examination revealed that functional synaptic connections of the presumed NA fibers on somata, dendrites, and other axonal terminals in the Vmot were seen at all postnatal ages examined. These results suggest that the NA system functions immediately after birth and exerts its effect on synaptic transmission, both pre- and postsynaptically, and on global neuronal excitability during the entire postnatal development of neuronal circuits in the Vmot.

Noradrenergic Innervation in Vmot

Although we did not provide any direct evidence for the possible sources of the NA innervation in the Vmot, it is reasonable to presume that it arises from the lateral tegmental catecholaminergic cell groups, referred to as the A5 and A7 catecholamine cell groups by many investigators (Vornov and Sutin, 1983; Grzanna et al., 1987; Lyons and Grzanna, 1988). Consistent with this suggestion, we found that all neurons of the A5 and A7 groups were both TH-IR and DBH-IR. Direct morphological evidence for this idea comes from the report of Vornov and Sutin (1983), who injected horseradish peroxide into the Vmot of normal and noradrenergically hyperinnervated rats and found that the A7 cell group was the sole source of NA afferents to the Vmot. Grzanna et al. (1987) and Lyons and Grzanna (1988) also reported that multiple cell groups in the pons provide noradrenergic innervation to the Vmot. Moreover, these studies suggest that the Vmot receives NA afferents mainly from the A7 cell group and, to a lesser extent, from the A5 cell group. It should be borne in mind that the A7 group is located rostral, and the A5 group ventral, to the Vmot (see also Fig. 1D–F). If the A7 neurons send NA fibers to the Vmot caudally with their fibers running in a horizontal direction, and the A5 neurons send NA fibers to the Vmot dorsally with their fibers running in a vertical direction, this could explain our observation that projection in the horizontal direction was more abundant than that in the vertical direction at all developmental ages in the present study.

Development of Noradrenergic Axon Profiles in Vmot

In this study, we found that both the density and size of varicosities of the NA fibers increased during postnatal development and reached adult levels at approximately P15 and P30, respectively. In rats, maturation of both the distribution pattern and density of the NA innervation by P15 has been reported in many other areas of the central nervous system, including the visual and motor cortex (Latsari et al., 2002) and the septal area (Antonopoulos et al., 2004). Nevertheless, our EM morphometric measurements suggested that the bouton areas showed no age-dependent change. This result seems to conflict with the results of the LM study, which showed an age-dependent increase in varicosity size. One possible explanation could be that the shape of the bouton might resemble a cylinder, with a small diameter and a long axis. It is possible that, during development, the diameter remains relatively constant, while the axis increases in length. Since an ultrathin section only covers a short distance of the axis and reveals mainly the diameter, whereas the LM section covers the entire bouton structure, this would explain the age independence of the EM observations on bouton area and the age dependence of the LM observations. This argument is supported by the fact that fewer serial sections were needed to reveal synaptic contacts at an early postnatal age (P5) than in the adult.

Although there was a lower density of adrenergic fibers and fewer varicosities at early postnatal ages, functional synaptic contacts could be seen as early as P5. This conclusion was based on the observations that several important ultrastructural parameters related to the function of synapses (Peters et al., 1991), including the maximal apposition length, maximal active zone length, and morphology of synaptic vesicles, in early postnatal stages were the same as in the adult.

Ultrastructure of NA Terminals

The ultrastructural characteristics of NA terminals in various regions of the brain have been studied by several investigators (Beaudet and Descarries, 1978; Itakura et al., 1981; Olschowka et al., 1981; Papadopuolos et al., 1989; Hagihira et al., 1990). One important issue that is still controversial is the proportion of NA terminals forming specific synaptic contacts. Studies using autoradiography after topical application of [3H] noradrenaline (Beaudet and Descarries, 1978) or potassium permanganate fixation (Itakura et al., 1981) have suggested that monoaminergic axon terminals do not form synaptic contacts with distinct membrane specializations and that monoamines seem to be released nonsynaptically in the central nervous system, influencing not only adjacent, but also more distant, neurons, the so-called volume transmission (Vizi et al., 2004). However, apart from criticisms of the effectiveness of these two experimental approaches for the study of the fine structure of monoaminergic terminals, the number of consecutive sections examined (only three in each study) was probably inadequate. Since a single section contains only a small portion of a typical varicosity (the diameter of a varicosity is roughly 15 times greater than the thickness of an ultrathin section) and since the site of membrane specialization is a relatively small portion of the total surface area of a varicosity, it is likely that many randomly oriented sections through synaptic varicosities do not contain the specialized zone (Olschowka et al., 1981).

In the present study, the ultrastructure of a TH-IR axon terminal was investigated throughout an entire varicosity in order to judge whether the labeled terminals formed synaptic contacts with any neuronal structures. Our study revealed that more than half (74% at P15 and 62% in the adult) of TH-IR terminals made synaptic contacts with other neuronal elements with clear membrane specialization in the Vmot. Thus, it is reasonable to assume that the difference between the results presented here and those of other workers could be at least in part due to the different number of consecutive sections examined. Consistent with the present results, Arce et al. (1994), Hagihira et al. (1990), and Papadopoulos et al. (1989), who also examined the morphological characteristics of NA-containing axon terminals in serial ultrathin sections, demonstrated that the majority of NA terminals in the superior colliculus, spinal dorsal horn, and visual and frontoparietal cortex form conventional synapses, suggesting that the action of noradrenaline is mainly mediated via a synaptic mechanism. Taken together, the results of the present and other studies based on multiple serial ultrathin sections demonstrate that the action of central noradrenaline is mediated by conventional synaptic transmission and is therefore characterized by a high degree of functional specificity determined by the spatial distribution of specialized junctions between presynaptic varicosities and adrenergic receptors.

However, since we also found that a substantial percentage of NA varicosities did not make contacts with postsynaptic targets, but contained clusters of synaptic vesicle aggregates near the plasma membrane, the possibility that certain central NA varicosities might exert their influence through a nonsynaptic mechanism has to be taken into account. In particular, recent studies have suggested the existence of extrasynaptic receptors and that, in some experimental or pathological conditions, e.g., blockage of transmitter uptake, transmitters could spill over and quickly fill up the extracellular space, allowing the activation of extrasynaptic receptors in the central nervous system (Sykova, 2004; Vizi et al., 2004). As for the peripheral nervous system, it has also been reported that noradrenaline released from nonsynaptic varicosities might diffuse beyond adjacent postsynaptic elements and influence large numbers of neurons (Descarries et al., 1977; Vizi et al., 2004). The effect of noradrenaline released in this diffuse fashion would be relatively unselective and determined primarily by the distribution of adrenergic receptors on nonadjacent neuronal elements.

Regardless of whether or not a TH-IR bouton targeted a postsynaptic element, we found that two types of synaptic vesicles could be identified in most TH-IR terminals, namely, small, pleomorphic, agranular vesicles and prominent dense-core vesicles. It has been suggested that the small pleomorphic vesicles probably contain catecholamines (Doyle and Maxwell, 1991), while the dense-core vesicles contain transmitters other than catecholamines (Hökfelt and Ljungdahl, 1972). Recent evidence indicates that some of these dense-core vesicles might contain neuropeptides (Meright et al., 1989). In fact, the coexistence and corelease of catecholamines and neuropeptides, such as neuropeptide Y (Charnay et al., 1982; Everitt et al., 1984; Yamazoe et al., 1985; Melander et al., 1986) and galanin (Xu et al., 1998; Simpson et al., 2006), have been suggested in the central nervous system.

Functional Implications

The majority of active zones formed by NA terminals in the Vmot were classified as being symmetrical, with a slight accumulation of electron-dense material on the presynaptic and postsynaptic sides of the junction. In agreement with the present results, similar observations have been reported for synapses on the cat lumbosacral spinal dorsal horn using TH as marker (Doyle and Maxwell, 1991) and in the rat visual and motor cortex (Latsari et al., 2002) and septal area (Antonopoulos et al., 2004) using DBH as marker. In our study, we found that the majority of postsynaptic targets were dendrites (59% at P5, 74% at P15, and 89% in the adult) and the remainder cell bodies or unlabeled axon terminals. These results suggest that NA terminals might be directly involved in governing neuronal excitability and in modulating synaptic input onto trigeminal motoneurons, both pre- and postsynaptically. Consistent with this idea are results from previous electrophysiological studies, which showed that noradrenaline released from NA terminals facilitates both the excitatory postsynaptic potentials recorded in trigeminal motoneurons and the monosynaptic reflex evoked by stimulation of group Ia primary afferent axons from the masseter muscle (the masseteric jaw closing reflex) (Morilak and Jacobs, 1985; Vornov and Sutin, 1986; Stafford and Jacobs, 1990). Since the masticatory rhythm is due to alternation of excitatory and inhibitory postsynaptic potentials in jaw-closer motoneurons (Goldberg, 1972; Chandler et al., 1985; Enamoto et al., 1987), the NA innervation could play an important role in the control of rhythmical jaw movements. This view is supported by the demonstration that low-current iontophoretic application of noradrenaline potently facilitates both glutamate-induced motoneuronal discharges and ongoing rhythmical motoneuronal activity during rhythmical jaw movements evoked by repetitive cortical stimulation (Katakura and Chandler, 1990).

In conclusion, our data provide direct morphological evidence that adrenergic innervation modulates rhythmical jaw movement control, as suggested by previous physiological studies. Our results also suggest that the adrenergic innervation might exert its effect via both synaptic and nonsynaptic mechanisms, with the former being more important, and that these mechanisms might operate as early as the day of birth.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Drs. T.F.C. Batten and K. Appenteng for invaluable comments on the article and Dr. Y.-S. Fu for assistance with the EM.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  • Antonopoulos J, Latsari M, Dori I, Chiotelli M, Parnavelas JG, Dinopoulos A. 2004. Noradrenergic innervation of the developing and mature septal area of the rat. J Comp Neurol 476: 8090.
  • Appenteng K, Conyers L, Moore JA. 1989. The monosynaptic excitatory connections of single trigeminal interneurons to the V motor nucleus of the rat. J Physiol (Lond) 417: 91104.
  • Appenteng K. 1990. Jaw muscle spindles and their central connections. In: TaylorA, editor. Neurophysiology of the jaws and teeth. London: Macmillan. p 96141.
  • Appenteng K, Conyers L, Curtis JC, Moore JA. 1990. Monosynaptic connections of single trigeminal interneurons to the contralateral V motor nucleus in anaethetised rats. Brain Res 514: 128130.
  • Arce EA, Bennett-Clarke CA, Rhoades RW. 1994. Ultrastructural organization of the noradrenergic innervation of the superficial gray layer of the hamster's superior colliculus. Synapse 18: 4654.
  • Bajic D, Van Bockstaele EJ, Proudfit HK. 2001. Ultrastructural analysis of ventrolateral periaqueductal gray projections to the A7 catecholamine cell group. Neuroscience 104: 181197.
  • Beaudet A, Descarries L. 1978. The monoamine innervation of rat cerebral cortex: synaptic and nonsynaptic axon terminals. Neuroscience 3: 851860.
  • Card JP, Riley JN, Moore RY. 1986. The motor trigeminal nucleus of the rat: analysis of neuronal structure and the synaptic organization of noradrenergic afferents. J Comp Neurol 250: 469484.
  • Chandler SH, Nielsen SA, Goldberg LJ. 1985. The effects of a glycine antagonist (strychnine) on cortically-induced rhythmical jaw movements in the anaesthetized guinea pig. Brain Res 325: 181186.
  • Charnay Y, Leger L, Dray F, Berod A, Jouvet M, Pujol JF, Dubois PM. 1982. Evidence for the presence of enkephalin in catecholaminergic neurons of cat locus coeruleus. Neurosci Lett 30: 147151.
  • Descarries L, Watkin KC, Lapierre Y. 1977. Noradrenergic axon terminals in the cerebral cortex of rat: III, topometric ultrastructural analysis. Brain Res 133: 197222.
  • Donga R, Lund JP. 1991. Discharge patterns of trigeminal commissural last-order interneurons during fictive mastication in the rabbit. J Neurophysiol 66: 15641578.
  • Doyle CA, Maxwell DJ. 1991. Catecholaminergic innervation of the spinal dorsal horn: a correlated light and electron microscopic analysis of tyrosine hydroxylase-immunoreactive fibers in the cat. Neuroscience 45: 161176.
  • Emmett SR, Greenfield SA. 2005. Correlation between dopaminergic neurons, acetylcholinesterase and nicotinic acetylcholine receptors containing the alpha3- or alpha5-subunit in the rat substantia nigra. J Chemi Neuroanat 30: 3444.
  • Enamoto S, Katakura N, Sunada T, Katayama T, Hirose Y, Ishiwata Y, Nakamura Y. 1987. Cortically induced masticatory rhythm in masseter motoneurons after blocking inhibition by strychnine and tetanus toxin. Neurosci Res 4: 396412.
  • Everitt BJ, Hokfelt T, Terenius L, Tatemoto K, Mutt V, Goldstein M. 1984. Differential co-existence of neuropeptide Y (NPY)-like immunoreactivity with catecholamines in the central nervous system of the rat. Neuroscience 11: 443462.
  • Goldberg LJ. 1972. Excitatory and inhibitory effects of lingual nerve stimulation on reflexes controlling the activity of masseteric motoneurons. Brain Res 39: 95108.
  • Goldberg LJ, Chandler SH. 1990. Central mechanisms of rhythmical trigeminal activity. In: TaylorA, editor. Neurophysiology of the jaws and teeth. London: Macmillan. p 268321.
  • Grzanna R, Chee WK, Akeyson EW. 1987. Noradrenergic projections to brainstem nuclei: evidence for differential projections from noradrenergic subgroups. J Comp Neurol 263: 7691.
  • Hagihira S, Senba E, Yoshida S, Tohyama M, Yoshiya I. 1990. Fine structure of noradrenergic terminals and their synapses in the rat spinal dorsal horn: an immunohistochemical study. Brain Res 526: 7380.
  • Hamos JE, King JS. 1980. The synaptic organization of the motor nucleus of the trigeminal nerve in the opossum. J Comp Neurol 194: 441463.
  • Hökfelt T, Ljungdahl A. 1972. Application of cytochemical techniques to the study of suspected transmitter substances in the nervous system. Adv Biochem Psychopharmac 6: 136.
  • Itakura T, Kasamatsu T, Pettigrew JD. 1981. Norepinephrine-containing terminals in kitten visual cortex: laminar distribution and ultrastructure. Neuroscience 6: 159175.
  • Katakura N, Chandler SH. 1990. An iontophoretic analysis of the pharmacologic mechanisms responsible for trigeminal motoneuronal discharge during masticatory-like activity in the guinea pig. J Neurophysiol 63: 356369.
  • Latsari M, Dori I, Antonopoulos J, Chiotelli M, Dinopoulos A. 2002. Noradrenergic innervation of the developing and mature visual and motor cortex of the rat brain: a light and electron microscopic immunocytochemical analysis. J Comp Nerurol 445: 145158.
  • Levitt P, Moore RY. 1979. Origin and organization of brainstem catecholamine innervation in the rat. J Comp Neurol 186: 505528.
  • Limwongase V, DeSantis M. 1977. Cell body locations and axonal pathways of neurons innervating muscles of mastication in the rat. Am J Anat 149: 477488.
  • Luo P, Li J. 1991. Monosynaptic connections between neurons of trigeminal mesencephalic nucleus and jaw-closing motoneurons in the rat: an ultrastructural horseradish peroxidase labeling study. Brain Res 559: 346350.
  • Lyons WE, Grzanna R. 1988. Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: a double retrograde neuronal labeling study. Neuroscience 26: 681693.
  • Melander T, Hokfelt T, Rokaeus A. 1986. Distribution of galanin-like immunoreactivity in the rat central nervous system. J Comp Neurol 248: 475517.
  • Meright A, Polak JM, Fumagalli G, Theodosis DT. 1989. Ultrastructural localization of neuropeptides and GABA in rat dorsal horn: a comparison of different immunogold labeling techniques. J Histochem Cytochem 37: 529540.
  • Min MY, Appenteng K. 1996. Multimodal distribution of amplitude of miniature and spontaneous EPSPs recorded in rat trigeminal motoneurones. J Physiol (Lond) 494: 171182.
  • Min MY, Appenteng K, Luther A, Curtis JC. 1996. Changes in frequency and amplitude of miniature EPSPs (mEPSPs) in trigeminal motoneurones following application of a GABAB antagonist. J Physiol 483: 28p.
  • Min MY, Appenteng K, Batten TFC, Yang HW. 1999. Inhibitory control of excitatory transmission. In: NakamuraY, editor. Neurobiology of mastication: from molecular to system approach. New York: Elsevier Press. p 6177.
  • Min MY, Appenteng K, Yang HW. 2002. Role of GABAB receptor in the regulation of excitatory synaptic transmission in trigeminal motoneurons. J Biomed Sci 9: 348358.
  • Min MY, Hsu PC, Yang HW. 2003. The physiological and morphological characteristics of interneurons caudal to the trigeminal motor nucleus in rats. Eur J Neurosci 18: 29812998.
  • Morilak DA, Jacobs BL. 1985. Noradrenergic modulation of sensorimotor processes in intact rats: the masseteric reflex as a model system. J Neurosci 5: 13001306.
  • Olschowka JA, Molliver ME, Grzanna R, Rice FL, Coyle JT. 1981. Ultrastructural demonstration of noradrenergic synapses in the rat central nervous system by dopamine-β-hydroxylase immunocytochemistry. J Histochem Cytochem 29: 271280.
  • Papadopoulos GC, Parnavelas JG, Buijs RM. 1989. Light and electron microscopic immunocytochemical analysis of the noradrenaline innervation of the rat visual cortex. J Neurocytol 18: 110.
  • Paxinos G, Watson G. 1998. The rat brain in stereotaxic coordinates. San Diego, CA: Academic Press.
  • Peters A, Palay SL, Webster H. 1991. The fine structure of the nervous system. New York: Oxford University Press.
  • Peters A, Palay SL. 1996. The morphology of synapses. J Neurocytol 25: 687700.
  • Plenz D, Kitai ST. 1998. Regulation of the nigrostriatal pathway by metabotropic glutamate receptors during development. J Neurosci 18: 41334144.
  • Shigenaga Y, Hirose Y, Yoshida A, Fukamim H, Honma S, Bae YC. 2000. Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat. J Comp Neurol 426: 1330.
  • Simpson KL, Waterhouse BD, Lin RCS. 2006. Characterization of neurochemically specific projections from the locus coeruleus with respect to somatosensory-related barrels. Anat Rec 288: 166173.
  • Stafford IL, Jacobs BL. 1990. Noradrenergic modulation of the masseteric reflex in behaving cats: II, physiological studies. J Neurosci 10: 99107.
  • Sykova E. 2004. Extrasynaptic volume transmission and diffusion parameters of the extracellular space. Neuroscience 129: 861876.
  • Taylor A, Durbaba R, Rodgers JF. 1995. Central connectivity patterns of jaw proprioceptors. In: MorimotoT, TatsuyaT, TakadaK, editors. Brain and oral functions: oral motor function and dysfunction. Amsterdam: Elsevier. p 5964.
  • Vizi ES, Kiss JP, Lendvai B. 2004. Nonsynaptic communication in the central nervous system. Neurochem Int 45: 443451.
  • Vornov JJ, Sutin J. 1983. Brainstem projections to the normal and noradrenergically hyperinnervated trigeminal motor nucleus. J Comp Neurol 214: 198208.
  • Vornov JJ, Sutin J. 1986. Noradrenergic hyperinnervation of the motor trigeminal nucleus: alternations in membrane properties and responses to synaptic input. J Nuerosci 6: 3037.
  • Xu ZQ, Shi TJ, Hökfelt T. 1998. Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampus formation and cortex with notes on the galanin-R1 and -R2 receptors. J Comp Neurol 392: 227251.
  • Yamazoe M, Shiosaka S, Emson PC, Tohyama M. 1985. Distribution of neuropeptide Y in the lower brainstem: an immunohistochemical analysis. Brain Res 335: 109120.
  • Yang HW, Min MY, Appenteng K, Batten TFC. 1996. Glycine and GABA as inhibitory transmitters in rat trigeminal motor nucleus. Soc Neurosci Abstr 22: 40.2.
  • Yang HW, Appenteng K, Batten TFC. 1997a. Ultrastructural subtypes of glutamate-immunoreactive terminals on rat trigeminal motoneurones and their relationships with GABA-immunoreactive terminals. Exp Brain Res 114: 99116.
  • Yang HW, Min MY, Appenteng K, Batten TFC. 1997b. Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and GABA-immunoreactive terminals. Brain Res 749: 301319.
  • Yoshida A, Fukami H, Nagase Y, Appenteng K, Honma S, Zhang LF, Bae YC, Shigenaga Y. 2001. Quantitative analysis of synaptic contacts made between functionally identified oralis neurons and trigeminal motoneurons in cats. J Neurosci 21: 62986307.