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The locus coeruleus projects to the mesencephalic trigeminal nucleus in rats
Neuroscience Research 68 (2010) 103–106
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
The locus coeruleus projects to the mesencephalic trigeminal nucleus in rats Takeshi Takahashi a,b , Masayoshi Shirasu a,b , Mari Shirasu a , Kin-ya Kubo c , Minoru Onozuka d , Sadao Sato b , Kazuo Itoh a , Hiroyuki Nakamura a,∗ a
Department of Morphological Neuroscience, Division of Neuroscience, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu 501-1194, Japan Department Craniofacial Growth and Development Dentistry, Division of Orthodontics, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan Faculty of Care and Rehabilitation, Seijoh University, 2-172 Fukinodai, Tokai, Aichi 476-8588, Japan d Department of Physiology and Neuroscience, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan b c
a r t i c l e
i n f o
Article history: Received 3 February 2010 Received in revised form 21 June 2010 Accepted 24 June 2010 Available online 3 July 2010 Keywords: Noradrenaline Periodontal ligament Single axon tracing Biotinylated dextran amine Aggressive behavior
a b s t r a c t The ganglion-cells in the mesencephalic trigeminal nucleus (Me5) process proprioceptive signals from the masticatory muscles and the periodontal ligaments, and are considered to regulate the rhythm of biting and bite strength. The locus coeruleus (LC) is the major source of noradrenergic projections in the brain and plays an important role in stressful situations and aggressive behavior. The two nuclei are adjacently located to each other in the lateral part of the periaqueductal gray matter of the fourth ventricle. In the present study, a small number of neurons were labeled in the LC with a neuronal tracer biotinylated dextran amine. The labeled single axons were traced from the labeled LC neuronal somata to the ipsilateral Me5 region where they produced terminal-like swellings. Some of the swellings appeared to make contact with the ganglion-cells of the Me5. These results suggest that the LC regulates the bite strength by modifying the ganglion-cell activity in the Me5. Additionally, these findings shed light on the enigma of why the main part of the Me5 at the level of pons is located at the lateral end of the gray matter ventral to the fourth ventricle, instead of at the trigeminal ganglion. © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction The mesencephalic trigeminal nucleus (Me5) is composed of pseudo-unipolar cells like other sensory ganglia and provides primary afferent fibers from the muscle spindles of jaw closers and the periodontal receptors (Alvarado-Mallart et al., 1975; Jacquin et al., 1983; Liem et al., 1991). It projects to the trigeminal motor nucleus and the premotor neurons and is thought to regulate biting rhythm (Shigenaga et al., 1988; Verdier et al., 2004). It is located at the ventrolateral end of the periaqueductal gray matter of the midbrain and at the lateral end of the gray matter of the floor of the fourth ventricle. As a result, at the level of pons, Me5 is located just lateral to the locus coeruleus (LC). The LC is the major source of noradrenaline in the brain and it sends axons to most of the brain regions, including the cerebral cortex, basal forebrain, amygdala, and the hypothalamus (Foote et al., 1983). It is thus associated with emotion-related regions, such as the basal forebrain and the hypothalamus, and plays an important role in aggressive behavior, defense, and defeat (Moore
∗ Corresponding author. Tel.: +81 58 230 6244; fax: +81 58 230 6247. E-mail address: [email protected] (H. Nakamura).
and Bloom, 1979; Miczek et al., 2004). In addition to emotionrelated projections, the LC also projects to a variety of regions throughout the trigeminal somatosensory pathway (Simpson et al., 1997). The synapses on the neuronal somata in the Me5 are immunoreactive to noradrenaline, GABA, dopamine, serotonin, and glutamate (Copray et al., 1990). Therefore, the Me5 receives afferent projections from variety of brain regions that may regulate brain function. Among these, the noradrenergic fibers were suggested to originate from the LC (Copray et al., 1990); however, there has been no direct evidence for projection from the LC to the Me5. In the present study, a small amount of LC cells are labeled with an axonal tracer in order to identify whether the LC neurons directly project to the Me5 or not. 2. Materials and methods 2.1. Animals Three adult male Sprague–Dawley rats weighing 250–300 g were housed individually and allowed free access for food and water. The animals were maintained at an ambient temperature (23 ◦ C) and on a 12 h light–dark cycle (lights on at 08:00 h). All animals were cared in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Regulations for Animal Experiments in Gifu University. This study was approved by the Committee for Animal Research and Welfare of Gifu University.
0168-0102/$ – see front matter © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2010.06.012
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2.2. Tracer injections The animals were deeply anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and were then mounted on a stereotaxic frame (Narishige, Tokyo, Japan). A skin incision and a bone opening was made over the LC and a glass micropipette filled with biotinylated dextran amine (BDA; BDA-10,000, Molecular Probes, Eugene, OR) was inserted according to the stereotaxic atlas (Paxinos and Watson, 1998). The parameters of the tip of the glass micropipette were 0.7 mm posterior from the interaural line, 1 mm lateral from the midline, and 6.7 mm deep from the surface of the cortex. Under intermittent gas pressure using a Picospritzer II (General Valve, Fair Field, NJ), a small amount of BDA was injected into the LC. Four days after the injection, the animals were anesthetized with a lethal dose of sodium pentobarbital (80 mg/kg, i.p.) and were then perfused through the ascending aorta with 300 ml of saline followed by 1 liter of a 4% paraformaldehyde solution in 0.1 M sodium phosphate buffer (PB), pH 7.4. The brain was removed from the skull and was immersed in a chilled solution of 30% sucrose in PB. 2.3. Histology After 2 days, 50-m thick coronal sections were cut frozen and collected in PB. All the sections were processed for BDA visualization. The sections were incubated with 1% hydrogen peroxide in PB for 2 h and then with 0.3% polyoxyethylene octylphenyl ether (Wako, Osaka, Japan) in PB for 3 h. The sections were rinsed and then processed with Elite ABC kit (Vector, Burlingame, CA) at 4 ◦ C overnight. The sections were rinsed and further processed with a 0.1% diaminobenzidine tetrahydrochloride (DAB, Dojin, Kumamoto, Japan) solution with 2.5% ammonium nickel sulfate (Wako), 0.2% -d-glucose (Sigma–Aldrich, St Louis, MO), 0.04% ammonium chloride (Wako), and 0.001% glucose oxidase (Sigma–Aldrich). The sections were mounted consecutively in gelatinized slide glasses, cleared with xylene, and coverslipped using DPX (Fluka, Buchs, Switzerland). 2.4. Observation and analysis We used a bright-field microscope with a phase-contrast condenser (BX50, Olympus, Tokyo) and a differential interference contrast microscope (Axioskop2 MOT, Zeiss, Jena, Germany) to identify the positions and borders of the LC and the Me5. The injection site was photographed using the differential interference contrast microscope equipped with a CCD camera (AxioCam, Zeiss) and software (AxioVision Release 4.6.3, Zeiss). Labeled neurons were outlined under the phase-contrast field. The BDA-labeled axons were traced from the labeled LC cells using a microscope (DPTIPHOTO, Nikon, Tokyo, Japan) equipped with a camera lucida. The axons were reconstructed from the tracings over several sections. Photographs of these sections were taken under the phase-contrast microscope equipped with a CCD camera (CMOS300-USB2, Fortissimo, Tokyo, Japan). The BDA-labeled LC neurons were reconstituted from several photographs with different foci and photographs from different sections. These photographs were aligned using blood vessels and tissue artifacts.
3. Results A total of 33 labeled neurons were identified within the LC. Fig. 1 shows a differential interference contrast photomicrograph of the injection site. The neurons of the LC were seen in the right side of Fig. 1B and the darkly stained labeled cells were localized in the LC. The ganglion-cells of the Me5 were apparent at the left of the
Fig. 1. Injection site showing darkly stained locus coeruleus (LC) neurons that are labeled with biotinylated dextran amine (BDA). (A) A photomicrograph using a low power objective lens. The box indicates the field shown in (B). (B) A photomicrograph using differential interference contrast microscope. Labeled neuronal somata are seen in the lower part of the LC near the border to the locus subcoeruleus. IV, fourth ventricle; bc, brachium conjunctivum; Me5, mesencephalic trigeminal nucleus; PAG, periaqueductal gray; scale bars, 100 m.
figure. The labeled LC cells were multipolar (18%), triangular (48%), fusiform (24%), or oval (9%) in shape. The sizes of the perikarya of the labeled cells varied from 83 m2 to 466 m2 (mean: 252.4 m2 ; SD: 75.9 m2 ) (Fig. 2).
Fig. 2. Distribution of the size of labeled LC cells (gray columns) and that of Me5-projecting LC cells (black columns).
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Fig. 3. Composite bright-field photomicrograph showing BDA-labeled soma of a LC cell, the axonal trajectory, and the synaptic terminal-like swellings on the Me5 cell. Scale bar, 50 m.
Axons of 6 neurons out of the 33 BDA-labeled LC neurons were traced laterally to the Me5 and were found to make synapselike contacts on the Me5 ganglion-cell perikarya (Figs. 3 and 4). The labeled axons were traced to Me5 and produced synaptic bouton-like swellings on the perikarya of the Me5 ganglion-cells. The labeled LC cells that projected onto the ganglion-cells of the Me5 were large (270–338 m2 ; mean: 308 m2 ; SD: 26.3 m2 ) (Fig. 2) and were fusiform (Fig. 4b), triangular (Fig. 4c), or multipolar (Figs. 3 and 4a, d and e) in shape. The sizes of the Me5-projecting LC cells were not significantly larger than the sizes of the labeled LC cells (t = 0.98 < t (37, 0.2)). A total of approximately 100 labeled axons were observed in the Me5, but most of them only produced terminal-like swellings in the Me5 region without synapse-like contact with the Me5 ganglioncell somata. Out of 745 terminal-like swellings in the Me5 regions, only 180 swellings were found in the vicinity of the ganglion-cells (24%). In the brainstem areas other than Me5, BDA-positive axons and bouton-like swellings were also observed in the dorsal raphe, median raphe, ventral tegmental area, substantia nigra, and the trigeminal motor nucleus. 4. Discussion Labeling of the LC neurons revealed the existence of direct projections from the LC to the Me5. Some of the synaptic boutonlike swellings seemed to make contact with the Me5 ganglion-cell somata, whereas others were distributed in the Me5 region without apparent synaptic contacts with the ganglion-cells. Injecting a small amount of BDA resulted in the uptake of the tracer by several LC neurons without any diffusion in and around the nucleus. This selective uptake of BDA by a handful of LC neurons enabled us to trace single axons from the labeled perikarya of the LC to the terminations of the Me5. Six LC neurons were identified to project to the Me5 ganglion-cells. Despite the small sample size, we were able to show that the LC directly projected to the Me5. The synapse-like swellings were observed in the vicinity of the Me5 ganglion-cells; however, further study using an electron microscope is necessary to identify whether the LC axons directly make synapse with the Me5 ganglion-cells. Axons of large LC neurons were traced to the Me5 and made synapse-like swellings on the Me5 ganglion-cell somata. The cells
Fig. 4. Drawing of the BDA-labeled LC neurons that project to Me5. Dots indicate terminal-like swellings.
are multipolar, triangular, or fusiform in shape. As a result, the Me5-projecting LC cells are medium-sized cells that have been previously reported in a study using golgi preparation (Shimizu et al., 1978). These neurons were reported to be the source of noradrenaline containing pathways (Chiba et al., 1976). Moreover, medium-sized LC cells project to the somatosensory cortex and thalamus, and are dopamine--hydroxylase immunoreactive (Simpson et al., 1997). Therefore, it is plausible that the Me5projecting LC neurons were noradrenergic cells. The present results thus suggest that noradrenaline is released from the LC axons in the Me5, and may modify the activity of the Me5 neurons and regulate the rhythm of mastication and bite force.
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In the rat, noradrenergic LC neurons have also been shown to contain serotonin and GABA (Iijima et al., 1992). It is thus possible that Me5-projections from the LC are serotonergic or GABAergic, although it is not apparent if serotonin or GABA is released from LC neurons. It was also reported that vasoactive intestinal polypeptide and neuropeptide Y were also positive in some LC cells and were observed in fibers that contact with Me5 ganglion-cells (Copray et al., 1990). These neuropeptides were considered to co-exist with noradrenaline (Hokfelt et al., 1987). Critical situations, such as encounter of an enemy, are known to activate the LC and elevate noradrenaline release (Pavcovich et al., 1990). It is also known that the elevated release of noradrenaline dampens the response to noxious stimuli (Pertovaara, 2006). In addition to the elevation of the threshold for pain, it suppresses or enhances the proprioceptive signals from the masticatory muscles and periodontal ligaments, and may hence be helpful in increasing the bite force for the impending fight. The conditions that activate noradrenergic neurons were reported to increase the amplitude of the masseteric reflex response (Stafford and Jacobs, 1990b). Microinfusion of noradrenaline in the trigeminal motor nucleus also increased the amplitude of the evoked masseteric reflex response in unanesthetized cats (Stafford and Jacobs, 1990a). This effect is considered to be the result of activation of the lateral tegmental (A5 and A7) noradrenergic neurons because there is a dense innervation of the trigeminal motor nucleus from these neurons, but not from LC (Vornov and Sutin, 1983; Grzanna et al., 1987; Stafford and Jacobs, 1990a). The direct projection from the LC to the Me5 shown in the present study thus suggests the existence of another noradrenergic pathway that may modify masseteric reflex. Acknowledgments This work was supported by Grants-in-Aid for Scientific Research (20592171, to KI and 22592063, to HN) and an Open Research Center subsidy (20592171, to SS) from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan). References Alvarado-Mallart, M.R., Batini, C., Buisseret-Delmas, C., Corvisier, J., 1975. Trigeminal representations of the masticatory and extraocular proprioceptors as revealed by horseradish peroxidase retrograde transport. Exp. Brain Res. 23, 167–179.
Chiba, T., Hwang, B.H., Williams, T.H., 1976. A method for studying glyoxylic acid fluorescence and ultrastructure of monoamine neurons. Histochem. J. 49, 95–106. Copray, J.C., Ter Horst, G.J., Liem, R.S., van Willigen, J.D., 1990. Neurotransmitters and neuropeptides within the mesencephalic trigeminal nucleus of the rat: an immunohistochemical analysis. Neuroscience 37, 399–411. Foote, S.L., Bloom, F.E., Aston-Jones, G., 1983. Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol. Rev. 63, 844–914. Grzanna, R., Chee, W.K., Akeyson, E.W., 1987. Noradrenergic projections to brainstem nuclei: evidence for differential projections from noradrenergic subgroups. J. Comp. Neurol. 263, 76–91. Hokfelt, T., Johansson, O., Holets, V., Meister, B., 1987. Distribution of neuropeptides with special reference to their coexistence with classical transmitters. In: Meltzer, H.Y. (Ed.), Psychopharmacology: The Third Generation of Progress. Raven Press, New York, pp. 401–416 (Chapter 42). Iijima, K., Sato, M., Kojima, N., Ohtomo, K., 1992. Immunocytochemical and in situ hybridization evidence for the coexistence of GABA and tyrosine hydroxylase in the rat locus coeruleus. Anat. Rec. 234, 593–604. Jacquin, M.F., Rhoades, R.W., Enfiejian, H.L., Egger, M., 1983. Organization and morphology of masticatory neurons in the rat. A retrograde HRP study. J. Comp. Neurol. 218, 239–256. Liem, R.S., Copray, J.C., van Willigen, J.D., 1991. Ultrastructure of the rat mesencephalic trigeminal nucleus. Acta Anat. 140, 112–119. Miczek, K.A., Covington III, H.E., Nikulina Jr., E.M., Hammer, R.P., 2004. Aggression and defeat: persistent effects on cocaine self-administration and gene expression in peptidergic and aminergic mesocorticolimbic circuits. Neurosci. Biobehav. Rev. 27, 787–802. Moore, R.Y., Bloom, F.E., 1979. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine systems. Ann. Rev. Neurosci. 2, 113–168. Pavcovich, L., Cancela, L., Volosin, M., Ramirez, O., 1990. Chronic stressinduced changes in locus coeruleus neural activity. Brain Res. Bull. 24, 293–296. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinate, fourth ed. Academic Press, Sydney. Pertovaara, A., 2006. Noradrenergic pain modulation. Prog. Neurobiol. 80, 53–83. Shigenaga, Y., Yoshida, A., Mitsuhiro, Y., Doe, K., Syemune, S., 1988. Morphology of single mesencephalic trigeminal neurons innervating periodontal ligament of the cat. Brain Res. 448, 331–338. Shimizu, N., Ohnishi, S., Satoh, K., Tohyama, M., 1978. Cellular organization of locus coeruleus in the rat as studied by golgi method. Arch. Histol. Jpn. 41, 103–112. Simpson, K.L., Altman, D.W., Wang, L.I., Kirifides, M.L., Kin, R.C.S., Waterhouse, B.D., 1997. Lateralization and functional organization of the locus coeruleus projection to the trigeminal somatosensory pathway in rat. J. Comp. Neurol. 385, 135–147. Stafford, I.L., Jacobs, B.L., 1990a. Noradrenergic modulation of the masseteric reflex in behaving cats. I. Pharmacological studies. J. Neurosci. 10, 91–98. Stafford, I.L., Jacobs, B.L., 1990b. Noradrenergic modulation of the masseteric reflex in behaving cats. II. Physiological studies. J. Neurosci. 10, 99–107. Verdier, D., Lund, J.P., Kolta, A., 2004. Synaptic inputs to trigeminal primary afferent neurons cause firing and modulate intrinsic oscillatory activity. J. Neurophysiol. 92, 2444–2455. Vornov, J.J., Sutin, J., 1983. Brainstem projections to the normal and noradrenergically hyper-innervated trigeminal motor nucleus. J. Comp. Neurol. 412, 198–208.
Contents lists available at ScienceDirect
Neuroscience Research journal homepage: www.elsevier.com/locate/neures
The locus coeruleus projects to the mesencephalic trigeminal nucleus in rats Takeshi Takahashi a,b , Masayoshi Shirasu a,b , Mari Shirasu a , Kin-ya Kubo c , Minoru Onozuka d , Sadao Sato b , Kazuo Itoh a , Hiroyuki Nakamura a,∗ a
Department of Morphological Neuroscience, Division of Neuroscience, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu 501-1194, Japan Department Craniofacial Growth and Development Dentistry, Division of Orthodontics, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan Faculty of Care and Rehabilitation, Seijoh University, 2-172 Fukinodai, Tokai, Aichi 476-8588, Japan d Department of Physiology and Neuroscience, Kanagawa Dental College, Yokosuka, Kanagawa 238-8580, Japan b c
a r t i c l e
i n f o
Article history: Received 3 February 2010 Received in revised form 21 June 2010 Accepted 24 June 2010 Available online 3 July 2010 Keywords: Noradrenaline Periodontal ligament Single axon tracing Biotinylated dextran amine Aggressive behavior
a b s t r a c t The ganglion-cells in the mesencephalic trigeminal nucleus (Me5) process proprioceptive signals from the masticatory muscles and the periodontal ligaments, and are considered to regulate the rhythm of biting and bite strength. The locus coeruleus (LC) is the major source of noradrenergic projections in the brain and plays an important role in stressful situations and aggressive behavior. The two nuclei are adjacently located to each other in the lateral part of the periaqueductal gray matter of the fourth ventricle. In the present study, a small number of neurons were labeled in the LC with a neuronal tracer biotinylated dextran amine. The labeled single axons were traced from the labeled LC neuronal somata to the ipsilateral Me5 region where they produced terminal-like swellings. Some of the swellings appeared to make contact with the ganglion-cells of the Me5. These results suggest that the LC regulates the bite strength by modifying the ganglion-cell activity in the Me5. Additionally, these findings shed light on the enigma of why the main part of the Me5 at the level of pons is located at the lateral end of the gray matter ventral to the fourth ventricle, instead of at the trigeminal ganglion. © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
1. Introduction The mesencephalic trigeminal nucleus (Me5) is composed of pseudo-unipolar cells like other sensory ganglia and provides primary afferent fibers from the muscle spindles of jaw closers and the periodontal receptors (Alvarado-Mallart et al., 1975; Jacquin et al., 1983; Liem et al., 1991). It projects to the trigeminal motor nucleus and the premotor neurons and is thought to regulate biting rhythm (Shigenaga et al., 1988; Verdier et al., 2004). It is located at the ventrolateral end of the periaqueductal gray matter of the midbrain and at the lateral end of the gray matter of the floor of the fourth ventricle. As a result, at the level of pons, Me5 is located just lateral to the locus coeruleus (LC). The LC is the major source of noradrenaline in the brain and it sends axons to most of the brain regions, including the cerebral cortex, basal forebrain, amygdala, and the hypothalamus (Foote et al., 1983). It is thus associated with emotion-related regions, such as the basal forebrain and the hypothalamus, and plays an important role in aggressive behavior, defense, and defeat (Moore
∗ Corresponding author. Tel.: +81 58 230 6244; fax: +81 58 230 6247. E-mail address: [email protected] (H. Nakamura).
and Bloom, 1979; Miczek et al., 2004). In addition to emotionrelated projections, the LC also projects to a variety of regions throughout the trigeminal somatosensory pathway (Simpson et al., 1997). The synapses on the neuronal somata in the Me5 are immunoreactive to noradrenaline, GABA, dopamine, serotonin, and glutamate (Copray et al., 1990). Therefore, the Me5 receives afferent projections from variety of brain regions that may regulate brain function. Among these, the noradrenergic fibers were suggested to originate from the LC (Copray et al., 1990); however, there has been no direct evidence for projection from the LC to the Me5. In the present study, a small amount of LC cells are labeled with an axonal tracer in order to identify whether the LC neurons directly project to the Me5 or not. 2. Materials and methods 2.1. Animals Three adult male Sprague–Dawley rats weighing 250–300 g were housed individually and allowed free access for food and water. The animals were maintained at an ambient temperature (23 ◦ C) and on a 12 h light–dark cycle (lights on at 08:00 h). All animals were cared in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Regulations for Animal Experiments in Gifu University. This study was approved by the Committee for Animal Research and Welfare of Gifu University.
0168-0102/$ – see front matter © 2010 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2010.06.012
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2.2. Tracer injections The animals were deeply anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and were then mounted on a stereotaxic frame (Narishige, Tokyo, Japan). A skin incision and a bone opening was made over the LC and a glass micropipette filled with biotinylated dextran amine (BDA; BDA-10,000, Molecular Probes, Eugene, OR) was inserted according to the stereotaxic atlas (Paxinos and Watson, 1998). The parameters of the tip of the glass micropipette were 0.7 mm posterior from the interaural line, 1 mm lateral from the midline, and 6.7 mm deep from the surface of the cortex. Under intermittent gas pressure using a Picospritzer II (General Valve, Fair Field, NJ), a small amount of BDA was injected into the LC. Four days after the injection, the animals were anesthetized with a lethal dose of sodium pentobarbital (80 mg/kg, i.p.) and were then perfused through the ascending aorta with 300 ml of saline followed by 1 liter of a 4% paraformaldehyde solution in 0.1 M sodium phosphate buffer (PB), pH 7.4. The brain was removed from the skull and was immersed in a chilled solution of 30% sucrose in PB. 2.3. Histology After 2 days, 50-m thick coronal sections were cut frozen and collected in PB. All the sections were processed for BDA visualization. The sections were incubated with 1% hydrogen peroxide in PB for 2 h and then with 0.3% polyoxyethylene octylphenyl ether (Wako, Osaka, Japan) in PB for 3 h. The sections were rinsed and then processed with Elite ABC kit (Vector, Burlingame, CA) at 4 ◦ C overnight. The sections were rinsed and further processed with a 0.1% diaminobenzidine tetrahydrochloride (DAB, Dojin, Kumamoto, Japan) solution with 2.5% ammonium nickel sulfate (Wako), 0.2% -d-glucose (Sigma–Aldrich, St Louis, MO), 0.04% ammonium chloride (Wako), and 0.001% glucose oxidase (Sigma–Aldrich). The sections were mounted consecutively in gelatinized slide glasses, cleared with xylene, and coverslipped using DPX (Fluka, Buchs, Switzerland). 2.4. Observation and analysis We used a bright-field microscope with a phase-contrast condenser (BX50, Olympus, Tokyo) and a differential interference contrast microscope (Axioskop2 MOT, Zeiss, Jena, Germany) to identify the positions and borders of the LC and the Me5. The injection site was photographed using the differential interference contrast microscope equipped with a CCD camera (AxioCam, Zeiss) and software (AxioVision Release 4.6.3, Zeiss). Labeled neurons were outlined under the phase-contrast field. The BDA-labeled axons were traced from the labeled LC cells using a microscope (DPTIPHOTO, Nikon, Tokyo, Japan) equipped with a camera lucida. The axons were reconstructed from the tracings over several sections. Photographs of these sections were taken under the phase-contrast microscope equipped with a CCD camera (CMOS300-USB2, Fortissimo, Tokyo, Japan). The BDA-labeled LC neurons were reconstituted from several photographs with different foci and photographs from different sections. These photographs were aligned using blood vessels and tissue artifacts.
3. Results A total of 33 labeled neurons were identified within the LC. Fig. 1 shows a differential interference contrast photomicrograph of the injection site. The neurons of the LC were seen in the right side of Fig. 1B and the darkly stained labeled cells were localized in the LC. The ganglion-cells of the Me5 were apparent at the left of the
Fig. 1. Injection site showing darkly stained locus coeruleus (LC) neurons that are labeled with biotinylated dextran amine (BDA). (A) A photomicrograph using a low power objective lens. The box indicates the field shown in (B). (B) A photomicrograph using differential interference contrast microscope. Labeled neuronal somata are seen in the lower part of the LC near the border to the locus subcoeruleus. IV, fourth ventricle; bc, brachium conjunctivum; Me5, mesencephalic trigeminal nucleus; PAG, periaqueductal gray; scale bars, 100 m.
figure. The labeled LC cells were multipolar (18%), triangular (48%), fusiform (24%), or oval (9%) in shape. The sizes of the perikarya of the labeled cells varied from 83 m2 to 466 m2 (mean: 252.4 m2 ; SD: 75.9 m2 ) (Fig. 2).
Fig. 2. Distribution of the size of labeled LC cells (gray columns) and that of Me5-projecting LC cells (black columns).
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Fig. 3. Composite bright-field photomicrograph showing BDA-labeled soma of a LC cell, the axonal trajectory, and the synaptic terminal-like swellings on the Me5 cell. Scale bar, 50 m.
Axons of 6 neurons out of the 33 BDA-labeled LC neurons were traced laterally to the Me5 and were found to make synapselike contacts on the Me5 ganglion-cell perikarya (Figs. 3 and 4). The labeled axons were traced to Me5 and produced synaptic bouton-like swellings on the perikarya of the Me5 ganglion-cells. The labeled LC cells that projected onto the ganglion-cells of the Me5 were large (270–338 m2 ; mean: 308 m2 ; SD: 26.3 m2 ) (Fig. 2) and were fusiform (Fig. 4b), triangular (Fig. 4c), or multipolar (Figs. 3 and 4a, d and e) in shape. The sizes of the Me5-projecting LC cells were not significantly larger than the sizes of the labeled LC cells (t = 0.98 < t (37, 0.2)). A total of approximately 100 labeled axons were observed in the Me5, but most of them only produced terminal-like swellings in the Me5 region without synapse-like contact with the Me5 ganglioncell somata. Out of 745 terminal-like swellings in the Me5 regions, only 180 swellings were found in the vicinity of the ganglion-cells (24%). In the brainstem areas other than Me5, BDA-positive axons and bouton-like swellings were also observed in the dorsal raphe, median raphe, ventral tegmental area, substantia nigra, and the trigeminal motor nucleus. 4. Discussion Labeling of the LC neurons revealed the existence of direct projections from the LC to the Me5. Some of the synaptic boutonlike swellings seemed to make contact with the Me5 ganglion-cell somata, whereas others were distributed in the Me5 region without apparent synaptic contacts with the ganglion-cells. Injecting a small amount of BDA resulted in the uptake of the tracer by several LC neurons without any diffusion in and around the nucleus. This selective uptake of BDA by a handful of LC neurons enabled us to trace single axons from the labeled perikarya of the LC to the terminations of the Me5. Six LC neurons were identified to project to the Me5 ganglion-cells. Despite the small sample size, we were able to show that the LC directly projected to the Me5. The synapse-like swellings were observed in the vicinity of the Me5 ganglion-cells; however, further study using an electron microscope is necessary to identify whether the LC axons directly make synapse with the Me5 ganglion-cells. Axons of large LC neurons were traced to the Me5 and made synapse-like swellings on the Me5 ganglion-cell somata. The cells
Fig. 4. Drawing of the BDA-labeled LC neurons that project to Me5. Dots indicate terminal-like swellings.
are multipolar, triangular, or fusiform in shape. As a result, the Me5-projecting LC cells are medium-sized cells that have been previously reported in a study using golgi preparation (Shimizu et al., 1978). These neurons were reported to be the source of noradrenaline containing pathways (Chiba et al., 1976). Moreover, medium-sized LC cells project to the somatosensory cortex and thalamus, and are dopamine--hydroxylase immunoreactive (Simpson et al., 1997). Therefore, it is plausible that the Me5projecting LC neurons were noradrenergic cells. The present results thus suggest that noradrenaline is released from the LC axons in the Me5, and may modify the activity of the Me5 neurons and regulate the rhythm of mastication and bite force.
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In the rat, noradrenergic LC neurons have also been shown to contain serotonin and GABA (Iijima et al., 1992). It is thus possible that Me5-projections from the LC are serotonergic or GABAergic, although it is not apparent if serotonin or GABA is released from LC neurons. It was also reported that vasoactive intestinal polypeptide and neuropeptide Y were also positive in some LC cells and were observed in fibers that contact with Me5 ganglion-cells (Copray et al., 1990). These neuropeptides were considered to co-exist with noradrenaline (Hokfelt et al., 1987). Critical situations, such as encounter of an enemy, are known to activate the LC and elevate noradrenaline release (Pavcovich et al., 1990). It is also known that the elevated release of noradrenaline dampens the response to noxious stimuli (Pertovaara, 2006). In addition to the elevation of the threshold for pain, it suppresses or enhances the proprioceptive signals from the masticatory muscles and periodontal ligaments, and may hence be helpful in increasing the bite force for the impending fight. The conditions that activate noradrenergic neurons were reported to increase the amplitude of the masseteric reflex response (Stafford and Jacobs, 1990b). Microinfusion of noradrenaline in the trigeminal motor nucleus also increased the amplitude of the evoked masseteric reflex response in unanesthetized cats (Stafford and Jacobs, 1990a). This effect is considered to be the result of activation of the lateral tegmental (A5 and A7) noradrenergic neurons because there is a dense innervation of the trigeminal motor nucleus from these neurons, but not from LC (Vornov and Sutin, 1983; Grzanna et al., 1987; Stafford and Jacobs, 1990a). The direct projection from the LC to the Me5 shown in the present study thus suggests the existence of another noradrenergic pathway that may modify masseteric reflex. Acknowledgments This work was supported by Grants-in-Aid for Scientific Research (20592171, to KI and 22592063, to HN) and an Open Research Center subsidy (20592171, to SS) from MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan). References Alvarado-Mallart, M.R., Batini, C., Buisseret-Delmas, C., Corvisier, J., 1975. Trigeminal representations of the masticatory and extraocular proprioceptors as revealed by horseradish peroxidase retrograde transport. Exp. Brain Res. 23, 167–179.
Chiba, T., Hwang, B.H., Williams, T.H., 1976. A method for studying glyoxylic acid fluorescence and ultrastructure of monoamine neurons. Histochem. J. 49, 95–106. Copray, J.C., Ter Horst, G.J., Liem, R.S., van Willigen, J.D., 1990. Neurotransmitters and neuropeptides within the mesencephalic trigeminal nucleus of the rat: an immunohistochemical analysis. Neuroscience 37, 399–411. Foote, S.L., Bloom, F.E., Aston-Jones, G., 1983. Nucleus locus coeruleus: new evidence of anatomical and physiological specificity. Physiol. Rev. 63, 844–914. Grzanna, R., Chee, W.K., Akeyson, E.W., 1987. Noradrenergic projections to brainstem nuclei: evidence for differential projections from noradrenergic subgroups. J. Comp. Neurol. 263, 76–91. Hokfelt, T., Johansson, O., Holets, V., Meister, B., 1987. Distribution of neuropeptides with special reference to their coexistence with classical transmitters. In: Meltzer, H.Y. (Ed.), Psychopharmacology: The Third Generation of Progress. Raven Press, New York, pp. 401–416 (Chapter 42). Iijima, K., Sato, M., Kojima, N., Ohtomo, K., 1992. Immunocytochemical and in situ hybridization evidence for the coexistence of GABA and tyrosine hydroxylase in the rat locus coeruleus. Anat. Rec. 234, 593–604. Jacquin, M.F., Rhoades, R.W., Enfiejian, H.L., Egger, M., 1983. Organization and morphology of masticatory neurons in the rat. A retrograde HRP study. J. Comp. Neurol. 218, 239–256. Liem, R.S., Copray, J.C., van Willigen, J.D., 1991. Ultrastructure of the rat mesencephalic trigeminal nucleus. Acta Anat. 140, 112–119. Miczek, K.A., Covington III, H.E., Nikulina Jr., E.M., Hammer, R.P., 2004. Aggression and defeat: persistent effects on cocaine self-administration and gene expression in peptidergic and aminergic mesocorticolimbic circuits. Neurosci. Biobehav. Rev. 27, 787–802. Moore, R.Y., Bloom, F.E., 1979. Central catecholamine neuron systems: anatomy and physiology of the norepinephrine systems. Ann. Rev. Neurosci. 2, 113–168. Pavcovich, L., Cancela, L., Volosin, M., Ramirez, O., 1990. Chronic stressinduced changes in locus coeruleus neural activity. Brain Res. Bull. 24, 293–296. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinate, fourth ed. Academic Press, Sydney. Pertovaara, A., 2006. Noradrenergic pain modulation. Prog. Neurobiol. 80, 53–83. Shigenaga, Y., Yoshida, A., Mitsuhiro, Y., Doe, K., Syemune, S., 1988. Morphology of single mesencephalic trigeminal neurons innervating periodontal ligament of the cat. Brain Res. 448, 331–338. Shimizu, N., Ohnishi, S., Satoh, K., Tohyama, M., 1978. Cellular organization of locus coeruleus in the rat as studied by golgi method. Arch. Histol. Jpn. 41, 103–112. Simpson, K.L., Altman, D.W., Wang, L.I., Kirifides, M.L., Kin, R.C.S., Waterhouse, B.D., 1997. Lateralization and functional organization of the locus coeruleus projection to the trigeminal somatosensory pathway in rat. J. Comp. Neurol. 385, 135–147. Stafford, I.L., Jacobs, B.L., 1990a. Noradrenergic modulation of the masseteric reflex in behaving cats. I. Pharmacological studies. J. Neurosci. 10, 91–98. Stafford, I.L., Jacobs, B.L., 1990b. Noradrenergic modulation of the masseteric reflex in behaving cats. II. Physiological studies. J. Neurosci. 10, 99–107. Verdier, D., Lund, J.P., Kolta, A., 2004. Synaptic inputs to trigeminal primary afferent neurons cause firing and modulate intrinsic oscillatory activity. J. Neurophysiol. 92, 2444–2455. Vornov, J.J., Sutin, J., 1983. Brainstem projections to the normal and noradrenergically hyper-innervated trigeminal motor nucleus. J. Comp. Neurol. 412, 198–208.