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Effect of norepinephrine receptors on trigeminal rhythm generation in newborn rats
Brain Research Bulletin, Vol. 53, No. 2, pp. 171–174, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/00/$–see front matter
PII S0361-9230(00)00322-1
Effect of norepinephrine receptors on trigeminal rhythm generation in newborn rats Mikihiko Kogo,* Atsuyuki Mori, Hidehiko Koizumi, Koji Ishihama, Seji Iida, Susumu Tanaka and Tokuzo Matsuya The First Department of Oral and Maxillofacial Surgery, Osaka University, Faculty of Dentistry, Osaka, Japan [Received 26 July 1999; Accepted 2 June 2000] ABSTRACT: N-methyl-D,L-aspartate acid and bicuculline are required to enhance the trigeminal rhythmic activities in an in vitro isolated brainstem block preparation. In this study, we analyzed the effect of norepinephrine on the trigeminal neural circuit underlying rhythmic jaw movements. Rhythmic trigeminal activity is observed in brainstem preparations (inferior colliculus to obex) only following blockade of ␣2adrenoceptors with idazoxan. This observation, combined with the inhibition of rhythm by ␣2-adrenoceptor agonists suggests endogenous ␣2-adrenoceptor mediated inhibition of trigeminal networks. A complex noradrenergic modulation of trigeminal systems is further supported by the prazosinsensitive potentiation of rhythm by bath application of the ␣1-adrenoceptor agonist phenylephrine. © 2000 Elsevier Science Inc.
rhythmic trigeminal activities in response to N-methyl-D-aspartate (NMDA) receptor agonist, N-methyl-D,L-aspartate (NMA) and GABAA receptor antagonist, bicuculline (BIC), but only after removal of tissue caudal to the rostral border of the facial nucleus [9]. The A5 noradrenergic cell group is located in the vicinity of the facial nucleus [1] and may provide this inhibitory input. Thus, the goals of this study were to examine the role of NE in modulating trigeminal motor systems and test the hypothesis that inhibition of rhythmic trigeminal activity arising from the caudal brainstem is mediated by NE acting at ␣2adrenoceptors. MATERIALS AND METHODS Sprague–Dawley rats (0 –2 d old) were used. Under deep Halothane anesthesia, the brainstem (inferior colliculus to the obex) was taken out and pinned down on sylgard resin in an acrylic chamber filled with modified Kreb’s ringer solution. Three sizes of brainstem preparation were prepared and used, the whole brainstem (inferior colliculus to the obex), a medium brainstem block (inferior colliculus to the caudal end of the facial nucleus), and a minimum brainstem block (inferior colliculus to the level of Y-crossing point at the rhomboidal fossa) (Fig. 1). These dissections were performed under a microscope. The transections were performed by hand using a fine cutting blade (maximum 100-m thick). The transection line was checked histologically after each experiment. The motor output was recorded from the trigeminal nerve with a glass suction electrode. The data were rectified, filtered (300 Hz–3 kHz, 3 dB), and recorded on DAT tape. The data were then acquired and analyzed with appropriate microcomputer and data acquisition/analysis software (MacLabs, AD instruments, Castle Hill, New South Wales, Australia). The amplitude of bursts and cycle frequencies were calculated automatically by the computer. Chemical applications and recordings were performed under static conditions (maximum 30 min) where the chamber was continuously oxygenated with a 95%O2–5%CO2 mixture. To initiate oral motor activity, we used NMA combined with BIC (NMA-BIC) as we described in our previous studies [4,5,9]. To analyze the effect of NE and agonists, phenylephrine (PE) (␣1-adrenoceptor), UK14304 (␣2adrenoceptor) and antagonists, prazocin (␣1-adrenoceptor), and
KEY WORDS: Mastication, Trigeminal motor activity, In vitro brainstem preparation, Norepinephrine, Rhythm generation.
INTRODUCTION The motor command underlying rhythmical jaw movements, such as sucking and mastication, is thought to be generated by the central pattern generator in the brainstem and modulated by sensory information. In our previous study [4], we were able to induce trigeminal rhythmical activity in a very limited area of the brainstem in vitro. The trigeminal motor nucleus and the surrounding tissue was able to show trigeminal rhythmical activities. Therefore, the rhythm generation circuit must exist in this small block. This study also showed that a coronal transection located caudal to the trigeminal motor nucleus is needed to enhance rhythmical activities. It is suggested that there is some modulatory circuit caudal to the trigeminal motor nucleus. Rhythmical jaw movements are controlled by input from the corticobulbar area and sensory input from the oral regions and subject to a large amount of modulation [6,7]. In this study, we examine the role of norepinephrine (NE), acting at ␣1 and ␣2-adrenoceptors, in modulating rhythmic trigeminal activity. Noradrenergic neurons are located throughout the brainstem [8] and have widespread projections to many regions of the brain including the trigeminal nucleus [2]. Second, an in vitro brainstem preparations from neonatal rat, extending from the rostral boundary of the inferior colliculus, can be induced to generate
* Address for correspondence: Mikihiko Kogo, D.D.S., Ph.D, The First Department of Oral and Maxillofacial Surgery, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan. Fax: ⫹81-6-6876-5298; E-mail: [email protected]
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FIG. 1. Transection lines through a sagittal (left) and a horizontal (right) view of the rat brainstem. Abbreviations: VII, facial nucleus; Amb, nucleus ambiguous; IC, inferior colliculus; MoV, motor trigeminal nucleus; SC, superior colliculus.
idazoxan (␣2-adrenoceptor), these drugs were added to the stagnant chamber, after addition of the NMA-BIC. The chamber was filled with standard artificial cerebrospinal fluid solution; NaCl, 128.0, MgSO4, 1.0, KCl, 3.0, NaHCO3, 24.0, NaH2PO4, 0.5, CaCl2, 1.5, and glucose, 30.0 (mM) (buffered to pH 7.4). A gravity-based perfusion was performed during the preparation (1 ml/s) and recovery from chemical stimulation (10 ml/s). The temperature in the chamber was maintained at 25–27°C. The preparation did not alter frequency significantly with each application of the same chemicals. All experiments were reviewed and approved by the Osaka University Faculty of Dentistry Intramural Animal Care and Use Committee. RESULTS Application of NMA (20 M) and BIC (10 M) could not induce trigeminal rhythmical activities in the whole brainstem, while these activities (3– 6Hz) were clearly recorded with the addition of NE (20 M) to this chemical application (n ⫽ 20/30) (Fig. 2B). On the other hand, most of the NMA-BIC applications could induce trigeminal rhythmical activities in the minimum block preparation (n ⫽ 8/10)(Fig. 2A). Effect of the Receptor Agonist and Antagonist on the Enhancement of Trigeminal Rhythmical Activities
␣1-Adrenoceptor. An application of the ␣1-adrenoceptor agonist, PE (20 M), to an NMA-BIC bath induced rhythmical activities in the whole block preparation (n ⫽ 6/7) (Fig. 3A). After the transection at the caudal end of the facial nucleus or Y-crossing point, the application of PE combined with NMABIC, also showed rhythmical activities (n ⫽ 6/7, 5/6, respectively) (Fig. 3B). An application of the ␣1-adrenoceptor antagonist, prazosin (1–100 M) to NMA-BIC bath, inhibited rhythmical activities of all preparations, even the minimum block preparation (n ⫽ 6/7, 7/7, 7/7) (Fig. 3). Only tonic discharges was apparent in the presence of the particular agent. ␣2-Adrenoceptor. Addition of the ␣2-adrenoceptor agonist, UK14304 (1–100 M) to NMA-BIC, inhibited rhythmical activities of all preparations, even the minimum block preparation (n ⫽ 6/7, 7/7, 7/7) (Fig. 2). Only tonic discharges were recognized.
FIG. 2. (A) After coronal transection at the Y-crossing point, bicuculline (BIC) and N-methyl-D,L-aspartate (NMA) application induced trigeminal rhythmical activities. (B) Rhythmical trigeminal motor activity was not induced in the whole brainstem preparation by chemical application of BIC-NMA, while rhythmical trigeminal motor activity was induced by an application of BIC-NMA combined with norepinephrine (NE). Abbreviation: IC, inferior colliculus.
An additional application of the ␣2-adrenoceptor antagonist, idazoxan (20 M), to the NMA-BIC bath induced rhythmical activities in the whole brainstem block preparation (n ⫽ 6/7). Application of idazoxan (20 M), NMA, and BIC also induced rhythmical activities in the medium and minimum brainstem block (n ⫽ 6/7, 5/6, respectively) (Figs. 3, 4). Idazoxan did not inhibit the rhythm generation with NMA-BIC in the minimum brainstem block. Cycle Frequency of Trigeminal Rhythmical Activities The cycle frequency of rhythmical activities(CFRA) were analyzed. In the minimum block preparation, as compared with CFRA induced with NMA-BIC, the rhythm induced with PENMA-BIC or NE-NMA-BIC showed a significantly high cycle frequency (p ⬍ 0.01). While, in the whole brainstem block preparation, NE or idazoxan combined with NMA-BIC induced rhythmical activities with a significantly lower cycle frequency. Only the rhythm with PE-NMA-BIC, showed a significantly high cycle frequency (p ⬍ 0.01) in the whole brainstem block preparation (Fig. 4B). DISCUSSION Many NE neurons were found in the brainstem. Noradrenergic projections from A7 and A5 cell groups to trigeminal motor nucleus have been reported [8]. We tested the effect of noradrenergic receptor activation on the small isolated neural circuit, serving as a fundamental building block from which more complex rhythmical oral-motor behaviors, such as suckling and chewing, arise. The present study suggests the NE can enhance rhythm generation via ␣1-adrenoceptor activation and that ␣2-adrenoceptors are involved in a circuit inhibiting trigeminal rhythmical activities. Effect of ␣1-Adrenoceptor Activation on Rhythm Generators The application of the ␣1-adrenoceptor agonist, PE, increased the frequency of rhythmical activities. PE showed this
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FIG. 3. (A) Application of the ␣1-adrenoceptor agonist and ␣2-adrenoceptor antagonist of norepinephrine (NE) induced rhythmical trigeminal motor activity in the whole brainstem preparation, while application of the ␣1-adrenoreceptor antagonist and ␣2adrenoceptor agonist did not. (B) Application of the ␣1-adrenoceptor agonist and ␣2-adrenoceptor antagonist induced rhythmical trigeminal motor activity in the brainstem preparation containing a facial nucleus, while application of the ␣1-adrenoceptor antagonist and ␣2-adrenoceptor agonist did not. (C) A section drawn up after the end of an experiment that contained a facial nucleus. Abbreviations: IC, inferior colliculus; MoV, motor trigeminal nucleus; VII nu, facial nucleus.
effect in both the minimum and whole block brainstem preparations. The minimum block included only the trigeminal motor nucleus and a small amount of surrounding tissue [9]. The trigeminal motoneurons, premotoneurons, and only some other neurons, including rhythm generator, should relate to these activities. It was previously shown that BIC and NMA induces masticatory-like jaw movement using jaw attached same block preparation [5]. It is likely that ␣1-adrenoceptors can control this cycle frequency. It is also supported by the present result that the ␣1-adrenoceptor antagonist, prazosin inhibits the trigeminal rhythmical activities in the minimum block. The prazosin might block the action of endogenous NE in this preparation. NE receptors are involved in enhancement of the digastric muscles (jaw opener) with stimulation to the oral membrane, dental pulp, and corticomasticatory area [3]. However, no evidence suggesting this effect of ␣1-adrenoceptor activation regarding to the cycle frequency has been found in previous studies. Inhibition of Rhythmical Activities Our previous study demonstrated that it is difficult to induce trigeminal rhythmical activities in the whole brainstem block
preparation [4]. In the present study, the addition of the ␣2adrenoceptor antagonist idazoxan to the BIC-NMA bath induced trigeminal rhythmical activities in the same block. We therefore suggest that an ␣2-adrenoceptor mediated pathway underlies a tonic inhibition of the rhythm. We did not isolate the source of this inhibitory input (which was blocked with idazoxan) in the present study. However, a serial sectioning experiment suggested it is located in the vicinity of the facial nucleus and may therefore correspond to the A5 noradrenergic cell group which projects to the trigeminal motor nucleus. Conclusion This study was performed to analyze the effects of NE receptors on the genesis of trigeminal rhythmical activities in the brainstem, using an isolated brainstem block preparation in vitro. 1. The ␣2-adrenoceptors are involved in the inhibitory circuit that exists caudal to the trigeminal motor nucleus. 2. The ␣1-adrenoceptor agonist can increase the cycle frequency of the trigeminal motor nucleus.
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KOGO ET AL. It is concluded that NE neurons have a close relationship with trigeminal rhythmogenesis. ACKNOWLEDGEMENTS
This work was supported by Grants-in-Aid for Scientific Research A-10307050 and B-09832006 from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Byrum, C. E.; Guyenet, P. G. Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J. Comp. Neurol. 261:529 –542; 1987. 2. Grzanna, R.; Chee, W. K.; Akeyson, E. W. Noradrenergic projections to brainstem nuclei: Evidence for differential projections from noradrenergic subgroups. J. Comp. Neurol. 263:76 –91; 1987. 3. Katakura, N.; Chandler, S. H. Iontophoretic analysis of the pharmacologic mechanisms responsible for initiation and modulation of trigeminal motoneuronal discharge evoked by intra-oral afferent stimulation. Brain Res. 549:66 –77; 1991. 4. Kogo, M.; Funk, G. D.; Chandler, S. H. Rhythmical oral-motor activity recorded in an in vitro brainstem preparation. Somatosens. Mot. Res. 13:39 – 48; 1996. 5. Kogo, M.; Tanaka, S.; Chandler, S. H.; Matsuya, T. Examination of the relationships between jaw opener and closer rhythmical muscle activity in an in vitro brainstem jaw-attached preparation. Somatosens. Mot. Res. 15:200 –210; 1988. 6. Kurasawa, I.; Hirose, Y.; Sunada, T.; Nakamura, Y. Phase-linked modulation of excitability of presynaptic terminals of low threshold afferent fibers in the inferior alveolar nerve during cortically induced fictive mastication in the guinea pig. Brain Res. 446:113– 120; 1988. 7. Lund, J. P. Mastication and its control by the brain stem. Crit. Rev. Oral Biol. Med. 2:33– 64; 1991. 8. Lyons, W. E.; Grzanna, R. Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: A double retrograde neuronal labeling study. Neuroscience 26:681– 693; 1988. 9. Tanaka, S.; Kogo, M.; Chandler, S. H.; Matsuya, T. Localization of oral-motor rhythmogenic circuits in the isolated rat brainstem preparation. Brain Res. 821:190 –199; 1999.
FIG. 4. (A) Rhythmical trigeminal motor activity was induced by the application of norepinephrine (NE), the ␣1-adrenoceptor agonist, the ␣2-adrenoceptor antagonist, and a combination with bicuculline (BIC) and N-methyl-D,L-aspartate (NMA). Furthermore, it was also induced by BIC-NMA stimulation alone and inhibition was not recognized in the minimum brainstem preparation. (B) Bar graphs indicate the cycle frequency of rhythmical trigeminal motor activity of the minimum brainstem and whole brainstem preparations as induced by NE and those agents related with BIC-NMA, respectively. Control: The cycle frequency of trigeminal rhythmical activities induced by BIC-NMA in the minimum brainstem block. In the whole brainstem, the rhythm induced by the ␣1-adrenoceptor agonist, phenylephrine, was faster than the control. A lower cycle frequency as compared to the control was generally recognized in the whole brainstem, whereas when NE or phenylephrine was applied to the minimum brainstem preparation, the rhythm was faster than the control: *significantly different from control (p ⬍ 0.01). Abbreviation: IC, inferior colliculus.
PII S0361-9230(00)00322-1
Effect of norepinephrine receptors on trigeminal rhythm generation in newborn rats Mikihiko Kogo,* Atsuyuki Mori, Hidehiko Koizumi, Koji Ishihama, Seji Iida, Susumu Tanaka and Tokuzo Matsuya The First Department of Oral and Maxillofacial Surgery, Osaka University, Faculty of Dentistry, Osaka, Japan [Received 26 July 1999; Accepted 2 June 2000] ABSTRACT: N-methyl-D,L-aspartate acid and bicuculline are required to enhance the trigeminal rhythmic activities in an in vitro isolated brainstem block preparation. In this study, we analyzed the effect of norepinephrine on the trigeminal neural circuit underlying rhythmic jaw movements. Rhythmic trigeminal activity is observed in brainstem preparations (inferior colliculus to obex) only following blockade of ␣2adrenoceptors with idazoxan. This observation, combined with the inhibition of rhythm by ␣2-adrenoceptor agonists suggests endogenous ␣2-adrenoceptor mediated inhibition of trigeminal networks. A complex noradrenergic modulation of trigeminal systems is further supported by the prazosinsensitive potentiation of rhythm by bath application of the ␣1-adrenoceptor agonist phenylephrine. © 2000 Elsevier Science Inc.
rhythmic trigeminal activities in response to N-methyl-D-aspartate (NMDA) receptor agonist, N-methyl-D,L-aspartate (NMA) and GABAA receptor antagonist, bicuculline (BIC), but only after removal of tissue caudal to the rostral border of the facial nucleus [9]. The A5 noradrenergic cell group is located in the vicinity of the facial nucleus [1] and may provide this inhibitory input. Thus, the goals of this study were to examine the role of NE in modulating trigeminal motor systems and test the hypothesis that inhibition of rhythmic trigeminal activity arising from the caudal brainstem is mediated by NE acting at ␣2adrenoceptors. MATERIALS AND METHODS Sprague–Dawley rats (0 –2 d old) were used. Under deep Halothane anesthesia, the brainstem (inferior colliculus to the obex) was taken out and pinned down on sylgard resin in an acrylic chamber filled with modified Kreb’s ringer solution. Three sizes of brainstem preparation were prepared and used, the whole brainstem (inferior colliculus to the obex), a medium brainstem block (inferior colliculus to the caudal end of the facial nucleus), and a minimum brainstem block (inferior colliculus to the level of Y-crossing point at the rhomboidal fossa) (Fig. 1). These dissections were performed under a microscope. The transections were performed by hand using a fine cutting blade (maximum 100-m thick). The transection line was checked histologically after each experiment. The motor output was recorded from the trigeminal nerve with a glass suction electrode. The data were rectified, filtered (300 Hz–3 kHz, 3 dB), and recorded on DAT tape. The data were then acquired and analyzed with appropriate microcomputer and data acquisition/analysis software (MacLabs, AD instruments, Castle Hill, New South Wales, Australia). The amplitude of bursts and cycle frequencies were calculated automatically by the computer. Chemical applications and recordings were performed under static conditions (maximum 30 min) where the chamber was continuously oxygenated with a 95%O2–5%CO2 mixture. To initiate oral motor activity, we used NMA combined with BIC (NMA-BIC) as we described in our previous studies [4,5,9]. To analyze the effect of NE and agonists, phenylephrine (PE) (␣1-adrenoceptor), UK14304 (␣2adrenoceptor) and antagonists, prazocin (␣1-adrenoceptor), and
KEY WORDS: Mastication, Trigeminal motor activity, In vitro brainstem preparation, Norepinephrine, Rhythm generation.
INTRODUCTION The motor command underlying rhythmical jaw movements, such as sucking and mastication, is thought to be generated by the central pattern generator in the brainstem and modulated by sensory information. In our previous study [4], we were able to induce trigeminal rhythmical activity in a very limited area of the brainstem in vitro. The trigeminal motor nucleus and the surrounding tissue was able to show trigeminal rhythmical activities. Therefore, the rhythm generation circuit must exist in this small block. This study also showed that a coronal transection located caudal to the trigeminal motor nucleus is needed to enhance rhythmical activities. It is suggested that there is some modulatory circuit caudal to the trigeminal motor nucleus. Rhythmical jaw movements are controlled by input from the corticobulbar area and sensory input from the oral regions and subject to a large amount of modulation [6,7]. In this study, we examine the role of norepinephrine (NE), acting at ␣1 and ␣2-adrenoceptors, in modulating rhythmic trigeminal activity. Noradrenergic neurons are located throughout the brainstem [8] and have widespread projections to many regions of the brain including the trigeminal nucleus [2]. Second, an in vitro brainstem preparations from neonatal rat, extending from the rostral boundary of the inferior colliculus, can be induced to generate
* Address for correspondence: Mikihiko Kogo, D.D.S., Ph.D, The First Department of Oral and Maxillofacial Surgery, Osaka University Faculty of Dentistry, 1-8 Yamadaoka, Suita, Osaka, 565-0871, Japan. Fax: ⫹81-6-6876-5298; E-mail: [email protected]
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FIG. 1. Transection lines through a sagittal (left) and a horizontal (right) view of the rat brainstem. Abbreviations: VII, facial nucleus; Amb, nucleus ambiguous; IC, inferior colliculus; MoV, motor trigeminal nucleus; SC, superior colliculus.
idazoxan (␣2-adrenoceptor), these drugs were added to the stagnant chamber, after addition of the NMA-BIC. The chamber was filled with standard artificial cerebrospinal fluid solution; NaCl, 128.0, MgSO4, 1.0, KCl, 3.0, NaHCO3, 24.0, NaH2PO4, 0.5, CaCl2, 1.5, and glucose, 30.0 (mM) (buffered to pH 7.4). A gravity-based perfusion was performed during the preparation (1 ml/s) and recovery from chemical stimulation (10 ml/s). The temperature in the chamber was maintained at 25–27°C. The preparation did not alter frequency significantly with each application of the same chemicals. All experiments were reviewed and approved by the Osaka University Faculty of Dentistry Intramural Animal Care and Use Committee. RESULTS Application of NMA (20 M) and BIC (10 M) could not induce trigeminal rhythmical activities in the whole brainstem, while these activities (3– 6Hz) were clearly recorded with the addition of NE (20 M) to this chemical application (n ⫽ 20/30) (Fig. 2B). On the other hand, most of the NMA-BIC applications could induce trigeminal rhythmical activities in the minimum block preparation (n ⫽ 8/10)(Fig. 2A). Effect of the Receptor Agonist and Antagonist on the Enhancement of Trigeminal Rhythmical Activities
␣1-Adrenoceptor. An application of the ␣1-adrenoceptor agonist, PE (20 M), to an NMA-BIC bath induced rhythmical activities in the whole block preparation (n ⫽ 6/7) (Fig. 3A). After the transection at the caudal end of the facial nucleus or Y-crossing point, the application of PE combined with NMABIC, also showed rhythmical activities (n ⫽ 6/7, 5/6, respectively) (Fig. 3B). An application of the ␣1-adrenoceptor antagonist, prazosin (1–100 M) to NMA-BIC bath, inhibited rhythmical activities of all preparations, even the minimum block preparation (n ⫽ 6/7, 7/7, 7/7) (Fig. 3). Only tonic discharges was apparent in the presence of the particular agent. ␣2-Adrenoceptor. Addition of the ␣2-adrenoceptor agonist, UK14304 (1–100 M) to NMA-BIC, inhibited rhythmical activities of all preparations, even the minimum block preparation (n ⫽ 6/7, 7/7, 7/7) (Fig. 2). Only tonic discharges were recognized.
FIG. 2. (A) After coronal transection at the Y-crossing point, bicuculline (BIC) and N-methyl-D,L-aspartate (NMA) application induced trigeminal rhythmical activities. (B) Rhythmical trigeminal motor activity was not induced in the whole brainstem preparation by chemical application of BIC-NMA, while rhythmical trigeminal motor activity was induced by an application of BIC-NMA combined with norepinephrine (NE). Abbreviation: IC, inferior colliculus.
An additional application of the ␣2-adrenoceptor antagonist, idazoxan (20 M), to the NMA-BIC bath induced rhythmical activities in the whole brainstem block preparation (n ⫽ 6/7). Application of idazoxan (20 M), NMA, and BIC also induced rhythmical activities in the medium and minimum brainstem block (n ⫽ 6/7, 5/6, respectively) (Figs. 3, 4). Idazoxan did not inhibit the rhythm generation with NMA-BIC in the minimum brainstem block. Cycle Frequency of Trigeminal Rhythmical Activities The cycle frequency of rhythmical activities(CFRA) were analyzed. In the minimum block preparation, as compared with CFRA induced with NMA-BIC, the rhythm induced with PENMA-BIC or NE-NMA-BIC showed a significantly high cycle frequency (p ⬍ 0.01). While, in the whole brainstem block preparation, NE or idazoxan combined with NMA-BIC induced rhythmical activities with a significantly lower cycle frequency. Only the rhythm with PE-NMA-BIC, showed a significantly high cycle frequency (p ⬍ 0.01) in the whole brainstem block preparation (Fig. 4B). DISCUSSION Many NE neurons were found in the brainstem. Noradrenergic projections from A7 and A5 cell groups to trigeminal motor nucleus have been reported [8]. We tested the effect of noradrenergic receptor activation on the small isolated neural circuit, serving as a fundamental building block from which more complex rhythmical oral-motor behaviors, such as suckling and chewing, arise. The present study suggests the NE can enhance rhythm generation via ␣1-adrenoceptor activation and that ␣2-adrenoceptors are involved in a circuit inhibiting trigeminal rhythmical activities. Effect of ␣1-Adrenoceptor Activation on Rhythm Generators The application of the ␣1-adrenoceptor agonist, PE, increased the frequency of rhythmical activities. PE showed this
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FIG. 3. (A) Application of the ␣1-adrenoceptor agonist and ␣2-adrenoceptor antagonist of norepinephrine (NE) induced rhythmical trigeminal motor activity in the whole brainstem preparation, while application of the ␣1-adrenoreceptor antagonist and ␣2adrenoceptor agonist did not. (B) Application of the ␣1-adrenoceptor agonist and ␣2-adrenoceptor antagonist induced rhythmical trigeminal motor activity in the brainstem preparation containing a facial nucleus, while application of the ␣1-adrenoceptor antagonist and ␣2-adrenoceptor agonist did not. (C) A section drawn up after the end of an experiment that contained a facial nucleus. Abbreviations: IC, inferior colliculus; MoV, motor trigeminal nucleus; VII nu, facial nucleus.
effect in both the minimum and whole block brainstem preparations. The minimum block included only the trigeminal motor nucleus and a small amount of surrounding tissue [9]. The trigeminal motoneurons, premotoneurons, and only some other neurons, including rhythm generator, should relate to these activities. It was previously shown that BIC and NMA induces masticatory-like jaw movement using jaw attached same block preparation [5]. It is likely that ␣1-adrenoceptors can control this cycle frequency. It is also supported by the present result that the ␣1-adrenoceptor antagonist, prazosin inhibits the trigeminal rhythmical activities in the minimum block. The prazosin might block the action of endogenous NE in this preparation. NE receptors are involved in enhancement of the digastric muscles (jaw opener) with stimulation to the oral membrane, dental pulp, and corticomasticatory area [3]. However, no evidence suggesting this effect of ␣1-adrenoceptor activation regarding to the cycle frequency has been found in previous studies. Inhibition of Rhythmical Activities Our previous study demonstrated that it is difficult to induce trigeminal rhythmical activities in the whole brainstem block
preparation [4]. In the present study, the addition of the ␣2adrenoceptor antagonist idazoxan to the BIC-NMA bath induced trigeminal rhythmical activities in the same block. We therefore suggest that an ␣2-adrenoceptor mediated pathway underlies a tonic inhibition of the rhythm. We did not isolate the source of this inhibitory input (which was blocked with idazoxan) in the present study. However, a serial sectioning experiment suggested it is located in the vicinity of the facial nucleus and may therefore correspond to the A5 noradrenergic cell group which projects to the trigeminal motor nucleus. Conclusion This study was performed to analyze the effects of NE receptors on the genesis of trigeminal rhythmical activities in the brainstem, using an isolated brainstem block preparation in vitro. 1. The ␣2-adrenoceptors are involved in the inhibitory circuit that exists caudal to the trigeminal motor nucleus. 2. The ␣1-adrenoceptor agonist can increase the cycle frequency of the trigeminal motor nucleus.
174
KOGO ET AL. It is concluded that NE neurons have a close relationship with trigeminal rhythmogenesis. ACKNOWLEDGEMENTS
This work was supported by Grants-in-Aid for Scientific Research A-10307050 and B-09832006 from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. Byrum, C. E.; Guyenet, P. G. Afferent and efferent connections of the A5 noradrenergic cell group in the rat. J. Comp. Neurol. 261:529 –542; 1987. 2. Grzanna, R.; Chee, W. K.; Akeyson, E. W. Noradrenergic projections to brainstem nuclei: Evidence for differential projections from noradrenergic subgroups. J. Comp. Neurol. 263:76 –91; 1987. 3. Katakura, N.; Chandler, S. H. Iontophoretic analysis of the pharmacologic mechanisms responsible for initiation and modulation of trigeminal motoneuronal discharge evoked by intra-oral afferent stimulation. Brain Res. 549:66 –77; 1991. 4. Kogo, M.; Funk, G. D.; Chandler, S. H. Rhythmical oral-motor activity recorded in an in vitro brainstem preparation. Somatosens. Mot. Res. 13:39 – 48; 1996. 5. Kogo, M.; Tanaka, S.; Chandler, S. H.; Matsuya, T. Examination of the relationships between jaw opener and closer rhythmical muscle activity in an in vitro brainstem jaw-attached preparation. Somatosens. Mot. Res. 15:200 –210; 1988. 6. Kurasawa, I.; Hirose, Y.; Sunada, T.; Nakamura, Y. Phase-linked modulation of excitability of presynaptic terminals of low threshold afferent fibers in the inferior alveolar nerve during cortically induced fictive mastication in the guinea pig. Brain Res. 446:113– 120; 1988. 7. Lund, J. P. Mastication and its control by the brain stem. Crit. Rev. Oral Biol. Med. 2:33– 64; 1991. 8. Lyons, W. E.; Grzanna, R. Noradrenergic neurons with divergent projections to the motor trigeminal nucleus and the spinal cord: A double retrograde neuronal labeling study. Neuroscience 26:681– 693; 1988. 9. Tanaka, S.; Kogo, M.; Chandler, S. H.; Matsuya, T. Localization of oral-motor rhythmogenic circuits in the isolated rat brainstem preparation. Brain Res. 821:190 –199; 1999.
FIG. 4. (A) Rhythmical trigeminal motor activity was induced by the application of norepinephrine (NE), the ␣1-adrenoceptor agonist, the ␣2-adrenoceptor antagonist, and a combination with bicuculline (BIC) and N-methyl-D,L-aspartate (NMA). Furthermore, it was also induced by BIC-NMA stimulation alone and inhibition was not recognized in the minimum brainstem preparation. (B) Bar graphs indicate the cycle frequency of rhythmical trigeminal motor activity of the minimum brainstem and whole brainstem preparations as induced by NE and those agents related with BIC-NMA, respectively. Control: The cycle frequency of trigeminal rhythmical activities induced by BIC-NMA in the minimum brainstem block. In the whole brainstem, the rhythm induced by the ␣1-adrenoceptor agonist, phenylephrine, was faster than the control. A lower cycle frequency as compared to the control was generally recognized in the whole brainstem, whereas when NE or phenylephrine was applied to the minimum brainstem preparation, the rhythm was faster than the control: *significantly different from control (p ⬍ 0.01). Abbreviation: IC, inferior colliculus.