Role of medullary GABA signal transduction on parasympathetic reflex vasodilatation in the lower lip

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Research Report

Role of medullary GABA signal transduction on parasympathetic reflex vasodilatation in the lower lip So Kawakamia , Hiroshi Izumib , Eiji Masakia , Satoshi Kuchiiwac , Kentaro Mizutaa,⁎ a

Division of Dento-oral Anesthesiology, Department of Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba, Sendai, 980-8575, Japan b Division of Physiology, Department of Oral Biology, School of Dentistry, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido, 061-0293, Japan c Department of Neuroanatomy, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, 890-8520, Japan

A R T I C LE I N FO

AB S T R A C T

Article history:

In the orofacial area, noxious stimulation of the orofacial structure in the trigeminal region

Accepted 13 December 2011

evokes parasympathetic reflex vasodilatation, which occurs via the trigeminal spinal

Available online 20 December 2011

nucleus (Vsp) and the inferior/superior salivatory nucleus (ISN/SSN). However, the neurotransmitter involved in the inhibitory synaptic inputs within these nuclei has never

Keywords:

been described. This parasympathetic reflex vasodilatation is suppressed by GABAergic

Parasympathetic reflex

action of volatile anesthetics, such as isoflurane, sevoflurane, and halothane, suggesting

Vasodilatation

that medullary GABAergic mechanism exerts its inhibitory effect on the parasympathetic

GABA

reflex via an activation of GABA receptors. The aim of the present study was to determine

Trigeminal spinal nucleus

the role of GABAA and GABAB receptors in the Vsp and the ISN in regulating the lingual

Inferior salivatory nucleus

nerve (LN)-evoked parasympathetic reflex vasodilatation in the lower lip. Under urethane

Lower lip

anesthesia (1 g/kg), change in lower lip blood flow elicited by electrical stimulation of the LN was recorded in cervically vago-sympathectomized rats. Microinjection of GABA (10 μM; 0.3 μl/site) into the Vsp or the ISN significantly and reversibly attenuated the LNevoked parasympathetic reflex vasodilatation. Microinjection of the GABAA receptorselective agonist muscimol (100 μM; 0.3 μl/site) or the GABAB receptor-selective agonist baclofen (100 μM; 0.3 μl/site) into the Vsp or the ISN significantly and irreversibly reduced this reflex vasodilatation, and these effects were attenuated by pretreatment with microinjection of each receptor-selective antagonists [GABAA receptor selective antagonist bicuculline methiodide (1 mM; 0.3 μl/site) or GABAB receptor selective antagonist CGP-35348 (1 mM; 0.3 μl/site)] into the Vsp or the ISN. Microinjection of these antagonists alone into the Vsp or the ISN had no significant effect on this reflex vasodilatation. In addition, microinjection (0.3 μl/site) of the mixture of muscimol (100 μM) and baclofen (100 μM) into the Vsp or the ISN also significantly reduced this reflex vasodilatation. These results suggest that medullary GABA signal transduction inhibits the parasympathetic reflex vasodilatation in the rat lower lip via GABAA and GABAB receptors in the Vsp and the ISN. © 2011 Elsevier B.V. All rights reserved.

⁎ Corresponding author. Fax: + 81 22 717 8404. E-mail address: [email protected] (K. Mizuta). 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2011.12.023

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1.

Introduction

Noxious stimulation of orofacial areas, such as the tooth-pulp, tongue and nasal mucosa, as well as electrical stimulation of the lingual or inferior alveolar nerve, evokes salivary secretions from the submandibular gland, and blood flow increases in the tissues of the orofacial region, such as lower lip, palate, tongue, salivary gland, gingiva and masseter muscle in both anesthetized animals and humans (Gonzalez et al., 1975; Ishii et al., 2005; Izumi and Karita, 1992b, 1993; Kemppainen et al., 1994, 2001; Mizuta et al., 2000; Satoh-Kuriwada et al., 2003; Yasui et al., 1997). These are thought to be mediated via activation of an autonomic reflex mechanism serving to maintain hemodynamic and metabolic homeostasis, the socalled somato-parasympathetic reflex mechanisms (Izumi, 1999). Previous findings have suggested that somatoparsympathetic reflex in orofacial area occurs via both the trigeminal spinal nucleus (Vsp) and the salivatory nucleus (Izumi et al., 2003; Koeda et al., 2009; Mizuta and Izumi, 2004; Mizuta et al., 2002). However, a neurotransmitter involved in the synaptic inputs within these nuclei has been unclear. GABA is a major inhibitory neurotransmitter with numerous actions in mammalian central nervous system (CNS), and stimulates two distinct types of receptors, ligand-gated ionotropic GABAA receptors and Gi protein-linked metabotropic GABAB receptors. Several lines of evidence suggested that the parasympathetic reflex vasodilatation in the orofacial area is regulated by GABA signal transduction in both the Vsp and the inferior/superior salivatory nucleus (ISN/SSN). First, GABAA and/or GABAB receptors in CNS have been reported to modulate a variety of reflexes (Suzuki et al., 1999; Zubcevic and Potts, 2010), particularly in the medulla (Ishii et al., 2010). Second, localization of GABA (Avendano et al., 2005) and expression of GABAA receptors (Matsushima et al., 2009) have been reported in the Vsp and the SSN, respectively. Third, we previously demonstrated that the GABAergic action of volatile anesthetics, including isoflurane, sevoflurane, and halothane, suppressed the LN-evoked parasympathetic reflex vasodilatation in the lower lip, submandibular gland, and palate, and these inhibitory effects have been deduced to act on the medulla (Ito et al., 1998; Izumi et al., 1997; Mizuta et al., 2006). These findings led us to hypothesize that the GABA receptors within the Vsp or the salivatory nucleus receive GABAergic inhibitory inputs and exert the inhibitory effects on parasympathetic reflex vasodilatation in the orofacial area. The purpose of the present study was to investigate whether the GABA receptor subtypes located in the Vsp or the salivatory nucleus exert its inhibitory effects on the parasympathetic reflex vasodilatation in the rat lower lip.

2.

Results

2.1. Confirmation of cannula implantation and microinjection in the Vsp or the ISN To confirm whether the cannula was implanted in the Vsp or the ISN, coronal brain sections were made at the end of the study. Photomicrographs of representative coronal sections

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showing sites used for electrical stimulation or microinjection of GABAergic agents into the Vsp or the ISN are shown in Fig. 1. A histological marker (pontamine sky blue, 0.3 μl, 2%) was microinjected into the Vi or the ISN to indicate how far the injections might have spread (Figs. 1C and D). The spread of pontamine sky blue was approximately 0.4–0.5 mm in radius (this was also the spread in the rostral/caudal direction). This spread suggests that the effects produced by microinjecting drugs into the Vsp or the ISN were not a consequence of spread of these drugs to the other site (i.e., Vsp injection did not spread to ISN). Previous studies have suggested that the preganglionic parasympathetic neurons participating in the innervation of the lower lip via the glossopharyngeal nerve originate from the ISN, whereas neurons innervating the submandibular gland via the facial nerve originate from the SSN (Izumi and Karita, 1992b; Nicholson and Severin, 1981). However, other studies using morphological techniques have reported that the distribution of these nuclei overlaps within the reticular formation and that the ISN and the SSN are not clearly distinguishable (Nicholson and Severin, 1981; Rezek et al., 2008). In the present study, hexamethonium, a cholinergic autonomic ganglion blocker, was used to verify the autonomic nature of the lower lip vasodilatation elicited by electrical stimulation of the reticular formation, and this was used as an indicator of correct ISN cannula implantation. Pretreatment with hexamethonium at 1 mg/kg and 10 mg/kg markedly decreased ISN-evoked vasodilator responses in the lower lip (1 mg/kg: n = 7, p < 0.01; 10 mg/kg: n = 5, p < 0.001), and the responses recovered in a time-dependent manner (Fig. 2). Therefore, the cannula was implanted in the ISN rather than SSN.

2.2. Effects of microinjection of GABA into the Vsp or the ISN on parasympathetic reflex vasodilatation in the lower lip GABA was microinjected (10 μM; 0.3 μl/site) into the Vsp or the ISN to examine whether the GABA in the Vsp or the ISN exerts an inhibitory effect on parasympathetic reflex vasodilatation in the lower lip. Microinjection of GABA into the Vsp or the ISN significantly attenuated the LN-evoked parasympathetic reflex vasodilatation in the lower lip (Vsp: p < 0.05, n = 4, 5 min after the microinjection, ISN: p < 0.05, n = 3, 10–15 min after the microinjection), and the responses recovered in a timedependent manner (Fig. 3). Typical microinjection sites are shown in Fig. 1.

2.3. Effects of microinjection of GABAA receptor agonist into the Vsp or the ISN on parasympathetic reflex vasodilatation in the lower lip The GABAA receptor agonist muscimol hydrobromide was microinjected (100 μM; 0.3 μl/site) into the Vsp or the ISN to examine whether the GABAA receptors in the Vsp or the ISN exerts any effect on parasympathetic reflex vasodilatation in the lower lip. The LN-evoked parasympathetic reflex vasodilatation in the lower lip was attenuated by microinjection of muscimol hydrobromide into the Vsp (p < 0.001 vs. control lower lip blood flow (LBF) response, n = 5) (Figs. 4A and B) or the ISN (p < 0.001 vs. control LBF response, n = 5) (Fig. 4C), while systemic arterial blood pressure was not affected by

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Fig. 1 – (A–D) Photographs of representative coronal sections through the medulla oblongata of the rat showing typical sites (arrowheads) at which electrical stimulation and microinjection of GABAergic agents were delivered to the Vsp (A, C) and inferior salivatory nucleus (ISN) (B, D). (A, B) Coronal sections after the thionin staining. (C, D) Coronal sections before the thionin staining to show the microinjection site stained by pontamine sky blue (2%, 0.3 μl). The area inside the dashed lines indicates the area stained by pontamine sky blue (2%, 0.3 μl) delivered from the microinjection cannula tip. Scale bars represent 1 mm. (E, F) Schematic representation of brain stem transverse sections at 11.0, 11.5 and 14.0 mm caudal to Bregma. The location of the sites at which microinjection of GABAergic agents was delivered into the Vsp (E: filled circle) or the ISN (F: filled triangle). Inferior salivatory nucleus scattered localized within the reticular formation. 7 N, facial nucleus; 8, cochlear nucleus; 12, hypoglossal nucleus; Amb, ambiguous nucleus; Cu, cuneate nucleus; Ecu, external cuneate nucleus; icp, inferior cerebellar peduncle; IO, inferior olive; mlf, medial longitudinal fasciculus; MV, medial vestibular nucleus; py, pyramidal tract; Rt, reticular formation; Sol, nucleus of the solitary tract; Vdm, dorsomedial trigeminal spinal nucleus; Vi, trigeminal spinal nucleus interpolaris; Vo, trigeminal spinal nucleus oralis; Vt, trigeminal spinal tract.

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the ISN exert its inhibitory effect on parasympathetic reflex vasodilatation in the lower lip. The LN-evoked parasympathetic reflex vasodilatation was irreversibly inhibited by microinjection of baclofen into the Vsp (p < 0.001 vs. control LBF response, n = 5) (Fig. 5B) or the ISN (p < 0.01 vs. control LBF response, n = 6) (Fig. 5C), while systemic arterial blood pressure was not affected by microinjection of baclofen into Vsp (Fig. 5A). These effects reached maximum reduction 60 min after microinjection of baclofen, and were significantly blocked by microinjection of the GABAB receptor antagonist CGP35348 (1 mM; 0.3 μl/site) into the Vsp or the ISN 30 min before microinjection of baclofen (Figs. 5B and C). Microinjection of CGP35348 alone into the Vsp or the ISN had no effect on LNevoked parasympathetic reflex vasodilatation in the lower lip (Vsp: ns, n = 5; ISN: ns, n = 5; paired t-test) (Fig. 6B). Typical microinjection sites are shown in Fig. 1.

2.5. Effect of microinjection of mixture of GABAA and GABAB receptor agonists into the Vsp or the ISN on parasympathetic reflex vasodilatation in the lower lip

Fig. 2 – (A) Representative examples of the effect produced by intravenous administration of hexamethonium (1.0 mg/kg and 10 mg/kg) on lip blood flow (LBF) increase [in arbitrary units (a.u.)] elicited by electrical stimulation of the ISN. Electrical stimulation was at 100 μA, 20 Hz, 2-ms pulse duration for 20 s. (B) Time course of the effect of intravenous administration of hexamethonium [1.0 mg/kg (open circles) and 10 mg/kg (filled circles)] on the LBF increase elicited by electrical stimulation of the ISN. Each value is expressed as a percentage of the pretreatment response (at time 0) and is given as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control (at time 0). The number of experiments is shown in parentheses.

microinjection of muscimol into Vsp (Fig. 4A). These effects were irreversible, reached a maximum reduction 60 min after microinjection of muscimol hydrobromide, and were significantly blocked by microinjection of the GABAA receptor antagonist bicuculline methiodide (1 mM; 0.3 μl/site) into the Vsp or the ISN 30 min before microinjection of muscimol hydrobromide (Figs. 4B and C). Microinjection of bicuculline methiodide alone into the Vsp or the ISN had no effect on LN-evoked parasympathetic reflex vasodilatation in the lower lip (Vsp: ns, n = 5; ISN: ns, n = 6, paired t-test) (Fig. 6A). Typical microinjection sites are shown in Fig. 1.

2.4. Effect of microinjection of GABAB receptor agonist into the Vsp or the ISN on parasympathetic reflex vasodilatation in the lower lip The GABAB receptor agonist baclofen was microinjected (100 μM; 0.3 μl/site) into the Vsp (Figs. 5A and B) or the ISN (Fig. 5C) to determine whether GABAB receptors in the Vsp or

The mixture of muscimol (100 μM) and baclofen (100 μM) was microinjected (0.3 μl/site) into the Vsp (Fig. 7A) or the ISN (Fig. 7B) to determine whether the activation of both GABAA and GABAB receptors in the Vsp or the ISN exerts any effect on parasympathetic reflex vasodilatation in the lower lip. The LN-evoked parasympathetic reflex vasodilatation was irreversibly inhibited by microinjection of the mixture of muscimol and baclofen into the Vsp (p < 0.001 vs. control LBF response, n = 5) (Fig. 7A) or the ISN (p < 0.001 vs. control LBF response, n = 6) (Fig. 7B). These effects reached maximum reduction 60 min after microinjection. Typical microinjection sites are shown in Fig. 1.

3.

Discussion

Results of the present study demonstrate that both GABAA and GABAB receptors in the Vsp and the ISN inhibited parasympathetic reflex vasodilatation in the lower lip. Vsp is cytoarchitectonically and functionally subdivided, from rostral to caudal, into the subnuclei of principalis (Vp), oralis (Vo), interpolaris (Vi) and caudalis (Vc) (Sessle, 2000; Takemura et al., 2006). The rostral nuclei, including Vp, Vo, and Vi, are involved in intra-oral nociception and/or sensorimotor reflexive function (Azerad et al., 1982; Shigenaga et al., 1986). We have previously reported that parasympathetic reflex vasodilatation in the lower lip is mediated predominantly via Vi (Koeda et al., 2009; Mizuta et al., 2002). Our present results showed that microinjection of GABAA or GABAB receptors agonist into Vi attenuated the parasympathetic reflex vasodilatation in the rat lower lip, which was reversed by pretreatment with the microinjection of each receptor selective antagonists into Vi (Figs. 4 and 5), being in harmony with other histological studies that systemic administration of the GABAA agonist muscimol or the GABAB receptor agonist baclofen inhibits trigeminal ganglion-evoked c-Fos expression in Vi (Takemura et al., 2000, 2001). Although the present study did not provide direct information regarding the sources of the GABAergic inhibitory input to Vi neurons, it has been

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Fig. 3 – Representative example and time course of the effect produced by microinjection of GABA (10 μM; 0.3 μl/site) into the Vsp (A, C) or the ISN (B, D) on the LBF increase (in a.u.) elicited by electrical stimulation of the central cut end of the LN (ipsilateral to the microinjection site). Each value is expressed as a percentage of the pretreatment blood flow increase (at time 0) and is given as means ± SEM. *p < 0.05 compared with control (at time 0). The number of experiments is shown in parentheses.

demonstrated that GABA is located predominantly in interneurons in the trigeminal spinal nuclei in all four subnuclei (Avendano et al., 2005). In addition, the majority of vibrissa afferents in Vi are modulated presynaptically by interneurons immunopositive for GABA (Moon et al., 2008). These findings indicate that GABAergic interneurons located in Vi inhibit parasympathetic reflex vasodilatation in the lower lip. We have reported that the salivatory nucleus is also involved in parasympathetic reflex vasodilatation in the orofacial area (Ishii et al., 2005; Mizuta and Izumi, 2004; Mizuta et al., 2002). Several studies have indicated that the salivatory nuclei are regulated by GABA signal transduction. Neurons in the ISN have been reported to receive inhibitory afferent input mediated by GABA (Suwabe et al., 2008), and GABAA receptors and glutamic acid decarboxylase (GAD)-positive nerve terminals are abundantly expressed in SSN neurons, suggesting that SSN neurons undergo strong GABAergic inhibition (Matsushima et al., 2009). In addition, neurons in the ISN and the SSN overlap and are difficult to distinguish (Contreras et al., 1980). These pieces of evidence support results of the present study showing that stimulation of GABA

receptors in the ISN inhibited parasympathetic reflex vasodilatation in the lower lip. Previous studies have indicated that preganglionic parasympathetic neurons participating in innervation of the lower lip via the glossopharyngeal nerve originate from the ISN, whereas neurons innervating the submandibular gland and palate via the facial nerve originate from the SSN (Izumi and Karita, 1992b, 1995; Mizuta and Izumi, 2004; Mizuta et al., 2002; Nicholson and Severin, 1981). However, as mentioned above, the ISN and the SSN overlap and are not clearly distinguishable. For example, Contreras et al. (1980) applied the retrograde tracer horseradish peroxidase to the otic ganglion, but could not clearly separate the ISN neurons from the SSN neurons. Rezek et al. (2008) also demonstrated that the submandibular subnucleus of the SSN joins rostrally the ISN, whereas the lacrimal subnucleus of the SSN is located ventrally and laterally to the caudal portion of the ISN. For this reason, it is not possible to selectively stimulate the ISN without stimulating the SSN, although the injection cannula and electrode was implanted in the area corresponding to the ISN (Fig. 1) (Paxinos and Watson, 2005; Rezek et al., 2008). Despite the overlapping of the ISN

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Fig. 4 – (A) Representative example of the effects produced by microinjection of muscimol hydrobromide (100 μM; 0.3 μl/site) into the Vsp on LBF increase (in a.u.) elicited by electrical stimulation of the central cut end of the LN (ipsilateral to the microinjection site). (a1) Effect of microinjection of muscimol into the Vsp. (a2) Effect of microinjection of muscimol into the Vsp after a 30-min pretreatment by microinjection of bicuculline methiodide (1 mM; 0.3 μl/site) into the Vsp. (B, C) Time course of the effect produced by microinjection of muscimol hydrobromide (100 μM; 0.3 μl/site) into the Vsp (B) or the ISN (C) on LBF increase elicited by electrical stimulation of the central cut end of the LN. Open circles: effect of microinjection of muscimol into the Vsp (B) or the ISN (C). Filled circles: effect of microinjection of muscimol into the Vsp (B) or the ISN (C) after a 30-min pretreatment by microinjection of bicuculline methiodide (1 mM; 0.3 μl/site) into the Vsp (B) or the ISN (C). Each value is expressed as a percentage of the pretreatment LBF increase (at time 0) and is given as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control (at time 0). #p < 0.05, ##p < 0.01 and ##p < 0.001 compared between “bicuculline methiodide pretreatment group” and “no pretreatment group” at the same time. The number of experiments is shown in parentheses.

and the SSN, we showed that electrical stimulation of the area corresponding to the ISN evoked vasodilatation in the lower lip (Fig. 1). This vasodilatation appeared to be mediated via a parasympathetic mechanism because this vasodilatation was attenuated by the autonomic ganglion-blocker hexamethonium (all rats were cervically sympathectomized) (Fig. 2). In addition, microinjection (0.3 μl/site) of 4% lidocaine into the ISN inhibited LN-evoked reflex vasodilatation in the lower lip (data not shown). Taken together, these results indicated that the microinjection of GABAergic drugs and electrical stimulation were delivered to the ISN. The distribution and dominance of GABAA and GABAB receptors in the CNS varies among the nuclei (Bowery et al.,

1987). In the nucleus tractus solitarii (NTS), both GABAA and GABAB receptors inhibit the activity of NTS neurons, and modulate the response to stimulation of vagal and baroreceptor afferents (Wang et al., 2010). Similarly, in the present study, microinjection of either muscimol or baclofen into the Vsp or the ISN attenuated the parasympathetic reflex vasodilatation to the same extent, suggesting that neurons in the Vsp and the ISN received the GABAergic inhibition to the same extent via GABAA and GABAB receptors. The GABAB receptor mediated inhibition is slower in onset, given that these receptors are associated with the regulation of K+ and/ or Ca2 + channel conductances. In contrast, GABAA receptormediated inhibition is faster in onset (termed phasic

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Fig. 5 – (A) Representative example of the effects produced by microinjection of baclofen (100 μM; 0.3 μl/site) into the Vsp on LBF increase (in a.u.) elicited by electrical stimulation of the central cut end of the LN (ipsilateral to the microinjection site). (a1) Effect of microinjection of baclofen into the Vsp. (a2) Effect of microinjection of baclofen into the Vsp after a 30-min pretreatment with microinjection of CGP35348 (1 mM; 0.3 μl/site) into the Vsp. (B, C) Time course of the effect produced by microinjection of baclofen (100 μM; 0.3 μl/site) into the Vsp (B) or the ISN (C) on LBF increase elicited by electrical stimulation of the central cut end of the LN. Open circles: effect of microinjection of baclofen into the Vsp (B) or the ISN (C). Filled circles: effect of microinjection of baclofen into the Vsp (B) or the ISN (C) after a 30-min pretreatment by microinjection of CGP35348 (1 mM; 0.3 μl/site) into the Vsp (B) or the ISN (C). Each value is expressed as a percentage of the pretreatment LBF increase (at time 0) and is given as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control (at time 0). #p < 0.05, ##p < 0.01 and ### p < 0.001compared between “CGP35348 pretreatment group” and “no pretreatment group” at the same time course. The number of experiments is shown in parentheses.

inhibition), given that these receptors are associated with an increase in Cl− conductance (Wang et al., 2010). Recently, however, GABAA receptors were also found to induce a much slower inhibition (termed ‘tonic’ inhibition), resulting from persistent activation of GABAA receptors (Semyanov et al., 2004). In the present study, microinjection of GABA into the Vsp or the ISN produced phasic reduction in the reflex vasodilatation (Fig. 3), while its synthetic agonists (muscimol or baclofen) tonically attenuated this reflex vasodilatation (Figs. 4 and 5). These findings suggest that GABA is easily metabolized in the medulla, while GABA synthetic agonists persistently bind to their receptors and were not metabolized. Interestingly, the microinjection of the mixture of both

GABAA and GABAB receptor agonists into the Vsp or the ISN attenuates the reflex vasodilatation at the same degree of the effect produced by the microinjection of single GABA receptor agonist (Fig. 7), suggesting that GABA is not the sole inhibitory neurotransmitter in either Vsp or ISN on this reflex. We previously reported that volatile anesthetics (e.g., isoflurane, sevoflurane, and halothane) tonically inhibit the parasympathetic reflex vasodilatation in the orofacial area, and these inhibitory effects were deduced to act on the reflex center in the medulla (Izumi et al., 1997; Mizuta et al., 2006). Volatile anesthetics potentiate GABAergic synaptic transmission by activation/potentiation of GABAA receptors (Franks and Lieb, 1998; Tanelian et al., 1993), and enhance basal

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Fig. 6 – (A) Effects produced by microinjection (0.3 μl/site) of (A) bicuculline methiodide (1 mM) or (B) CGP35348 (1 mM) into either the Vsp or the ISN on the LBF increase elicited by electrical stimulation of the central cut end of the LN (ipsilateral to the microinjection site). Mean values (± SEM) of the LN-evoked LBF increases (30 min after microinjection) are expressed as a percentage of the control LBF response. The number of experiments is shown in parentheses.

GABA release (Westphalen and Hemmings, 2006). Taken together, the GABAergic action of volatile anesthetics would potentiate GABAergic synaptic transmission and basal GABA release within the Vsp and ISN, which in turn would inhibit parasympathetic reflex vasodilatation in the orofacial area. GABA-mediated blood flow reduction seems to be of considerable importance for reducing the bleeding during the orofacial surgery under the general anesthesia. This is because volatile anesthetics would reduce the parasympathetic reflex vasodilatation in the orofacial structure elicited by noxious surgical stimuli to orofacial area. However, further studies are needed to clarify whether volatile anesthetics exert its inhibitory effect on parasympathetic reflex vasodilatation in the orofacial area via these mechanisms. In conclusion, the present study revealed that parasympathetic reflex vasodilatation in the orofacial area receives GABAergic inhibitory input through both GABAA and GABAB receptors located in the Vsp and the ISN. These findings provide new and relevant information regarding the regulation

of orofacial blood flow by anesthetics that act on GABA receptors and may have implications for the use of volatile anesthetics during orofacial surgery.

4.

Experimental procedures

4.1.

Materials

Isoflurane was obtained from Abbott (Tokyo, Japan). Pancuronium bromide was obtained from Organon (Oss, The Netherlands). Lidocaine hydrochloride was obtained from Nacalai Tesque (Tokyo, Japan). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. 4.2.

Preparation of animals

The experimental protocol was reviewed by the Committee on the Ethics of Animal Experiments of the Tohoku University

Fig. 7 – Time course of the effect produced by microinjection the mixture (0.3 μl/site) of muscimol (100 μM) and baclofen (100 μM) into the Vsp (A) or the ISN (B) on LBF increase elicited by electrical stimulation of the central cut end of the LN. Each value is expressed as a percentage of the pretreatment LBF increase (at time 0) and is given as means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with control (at time 0). The number of experiments is shown in parentheses.

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Graduate School of Dentistry, and was carried out in accordance with the recommendations in the current National Research Council guide. Experiments were performed on 80 adult male Wistar rats weighing 350–480 g. After anesthesia induction with volatile anesthetic (isoflurane), urethane (100 mg/100 g body weight) was injected subcutaneously, and was supplemented as necessary throughout the experiment (see below) to produce basal anesthesia. Urethane was selected because it can induce deep anesthesia with minimal effects on the circulatory dynamics (Farber et al., 1995; Saito et al., 1995). The femoral vein was cannulated to allow for drug injection, and the femoral artery was also cannulated and connected to a Statham pressure transducer to monitor the systemic arterial blood pressure (SABP). Anesthetized animals were intubated, paralyzed by an intravenous injection of pancuronium bromide (0.6 mg/kg initially, supplemented with 0.4 mg/kg/h continuously), and artificially ventilated via a tracheal cannula with a mixture of 50% air and 50% O2. The ventilator (SN-480-7; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 1.5 ml/100 g body weight at a rate of 30–40 breaths/min, and the end-tidal concentration of CO2 was measured with the anesthesia gas monitor (M1026A; Hewlett Packard, Palo Alto, CA). The end-tidal CO2 was kept at 35–40 mm Hg. Rectal temperature was maintained at 37–38 °C with the use of a heating pad. The criterion used to determine whether the depth of anesthesia was adequate, was to determine if a reflex elevation of systemic arterial blood pressure occurred in response to a noxious stimulus (pinching the digit for approximately 2 s). If the depth of anesthesia was considered inadequate, additional urethane (i.e., intermittent doses of 100 mg/kg intravenously) was administered. Once an adequate depth of anesthesia was attained, pancuronium bromide was administered continuously to maintain immobilization during the periods of stimulation. In all experiments, the cervical vagi and superior cervical sympathetic trunks were cut bilaterally in the neck prior to any stimulation to eliminate the reflex actions of the vagus nerve on the cardiovascular system and the effects of sympathetic vasoconstrictor fibers on the orofacial area, respectively. This ensured that only non-vagal parasympathetic effects were being studied. 4.3.

Measurement of blood flow in the lower lip

Blood flow in the lower lip was monitored with a laserDoppler flowmeter (OMEGAFLO FLO-C1; Omegawave, Tokyo, Japan). Lower lip blood flow was selected to monitor LNevoked reflex vasodilatation because its neural pathways are well characterized (Izumi and Karita, 1992a, b; Mizuta et al., 2002). The neural pathways would require at least four synapses: the trigeminal afferent neuron, Vsp neurons which synapse with the ISN neurons directly or with other interneurons in Vsp, the parasympathetic preganglionic neuron with the cell body located in the ISN and the otic postganglionic neuron (Izumi, 1999; Izumi and Karita, 1991, 1992b; Mizuta et al., 2002; Shigenaga et al., 1986; Spencer et al., 1990). The probe was placed against the lower lip without exerting any pressure on the tissue. The laser-Doppler flowmeter values obtained in this way represent the blood flow

in the superficial vessels in the tissue (Edwall et al., 1987; Kim et al., 1990). Electrical calibration for zero blood flow was performed for all recordings. Several gain levels could be selected, and the maximum output of a particular gain level (defined electrically) was set as 100%. The output of the equipment does not give absolute values, but shows relative changes in blood flow [for technical details and an evaluation of the LDF method see Stern et al. (1977)]. The output from the devices was continuously displayed on the four-channel chart recorder (Powerlab 4/20, ADInstruments, Colorado Springs, CO). The blood flow changes were assessed by measuring the height of the response on the chart and are expressed in arbitrary units. We regarded increases in lower lip blood flow as significant when the ratio between the magnitude of the blood flow increase and the amplitude of the baseline fluctuations (signal-to-noise ratio) was more than 3 when the lingual nerve was stimulated at supramaximal intensity. 4.4.

Electrical stimulation of the lingual nerve

To elicit a parasympathetic reflex vasodilatation in lower lip, the central cut end of the lingual nerve (LN) was electrically stimulated with a bipolar electrode. The routine stimulus parameters were as follows: a 20-s train of 2-ms rectangular pulses at a frequency of 20 Hz and at supramaximal intensity (10 V), as described (Mizuta et al., 2000) using an electrical stimulator (SEN-7203; Nihon Kohden, Tokyo, Japan). During the electrical stimulation of the central cut end of the LN, the lower lip blood flow was increased as well as its vascular conductance (lower lip vascular conductance = lower lip blood flow/mean arterial blood pressure), while the mean arterial blood pressure was not significantly changed. These findings indicate that increase in the lower lip blood flow was not produced by increase in the systemic arterial blood pressure but predominantly evoked by vasodilatation in the lower lip. 4.5. Identification of lower lip vasodilatation sites in the Vsp and the ISN The animal was mounted in the flat-scull position (Paxinos and Watson, 2005; Paxinos et al., 1985) in a stereotaxic frame (Narishige, Tokyo, Japan), and a partial craniotomy was performed. A guide cannula (0.4 mm outer diameter) was positioned within the Vsp [posterior (P), 14.0; lateral (L), 2.5–3.0; height (H), 10.0–11.0 mm] or the ISN (P, 11.0–11.5; L, 1.2; H, 9.0–10.0 mm) based on the position of the bregma according to the rat brain atlas (Paxinos and Watson, 2005) via a small burr-hole in the skull. This was achieved with the aid of a micromanipulator and without removing any part of the brain. A concentric monopolar electrode (IMB-9002; 0.2 mm outer diameter; Inter Medical, Tokyo, Japan) insulated with enamel except at the tip was inserted through the guide cannula. A lower lip vasodilatation site in the Vsp or the ISN was identified by lowering the stimulating electrode until a maximal vasodilatation response was elicited by electrical stimulation, as described previously (Mizuta et al., 2002). Quite small movements of the stimulating electrode in the vertical direction (i.e., 0.2 mm) often markedly altered the response to stimulation, indicating that current spread from the electrode tip

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was not excessive. For electrical stimulation of the Vsp or the ISN, we routinely used a 20-s train of rectangular pulses generated by an electrical stimulator (SEN-7203; Nihon Kohden, Tokyo Japan) through an isolation unit (SS-202J; Nihon Kohden, Tokyo, Japan), usually with a current of 100 μA and pulse duration of 2 ms at a frequency of 20 Hz. To confirm that the vasodilatation response elicited by LN stimulation was mediated via the Vsp or the ISN, 4% lidocaine was microinjected into the Vsp or the ISN at a volume of 0.3 μl/site via an injection cannula (0.2 mm outer diameter) inserted through the previously implanted guide cannula. The stimulating electrode was interchangeable with the injection cannula. Both were of equal length and each extended 1.0 mm beyond the tip of the guide cannula. Thus, microinjection and electrical stimulation were carried out at the same sites. Saline (0.3 μl/ site) was used for control injections, and it never produced any significant effect on LN-evoked vasodilatation or on resting cardiovascular parameters. 4.6.

Autonomic ganglion blocking agent

Hexamethonium, a cholinergic autonomic ganglion blocker, was used to verify the autonomic nature of the lower lip vasodilatation elicited by electrical stimulation of the ISN, and this was used as an indicator of correct ISN cannula implantation. Hexamethonium was administered (1.0 mg/kg or 10 mg/kg intravenously), and stimulation was repeated, beginning 5 min later. The magnitude of the responses obtained was expressed as a percentage of the response elicited by electrical stimulation of the ISN before hexamethonium treatment (means ± SEM). 4.7.

Medullary microinjection of GABAergic agents

To evaluate the role of medullary GABA receptor on this reflex vasodilatation, 0.3 μl/site of GABA (10 μM), muscimol (GABAA receptor agonist; 100 μM), or baclofen (GABAB receptor agonist; 100 μM) was microinjected into the Vsp or the ISN at sites at which microinjection of lidocaine evoked a marked decrease in vasodilatation response evoked by stimulation of the ipsilateral LN. Representative microinjection sites were shown in Fig. 1. In the separate experiments, mixture of muscimol (100 μM) and baclofen (100 μM) was microinjected (0.3 μl/site) into the Vsp or the ISN. A volume of 0.3 μl of GABAergic drugs was selected because the effective radial spread of microinjected drug (lidocaine) is approximately 0.4 mm when 0.3 μl of drug is microinjected in the brain (Tehovnik and Sommer, 1997), and the radial spread of 0.4 mm from the implant cannula tip positioned in the Vi or the ISN covered the majority of the regions of the Vi or the ISN (Paxinos and Watson, 2005). Since the implanted cannula positioned in Vi was at least 1.0 mm away from the reticular formation, which contains the ISN, 0.3 μl of GABAergic drugs microinjected into Vi did not affect the ISN, and vice versa (Fig. 1). In the separate experiments, 0.3 μl of either bicuculline methiodide (GABAA receptor antagonist; 1 mM) or CGP35348 (GABAB receptor antagonist; 1 mM) was microinjected 30 min before the microinjection of either muscimol or baclofen. Experiments were started 20–30 min after the LN-evoked response had completely recovered from the effects of lidocaine.

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The magnitude of the LN-evoked response obtained after microinjection of a given agent was expressed as a percentage of the control response recorded before its administration (mean ± SEM). The sites of electrical stimulation and those at which microinjections were made were examined histologically, as described below. 4.8.

Histology

At the end of the experiment, animals were prepared for histological analyses of electrical stimulation and injection sites. Injection sites in the Vsp and ISN were marked with 0.3-μl microinjections of pontamine sky blue dye (2%) after the experiments. Then, all the rats were given an overdose of pentobarbital sodium (approximately 100 mg) by intravenous infusion, and perfused through the ascending aorta with 200 ml of saline, followed immediately by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline at pH 7.4. The brainstem and upper cervical spinal cord were then removed and stored for 4–7 days in cold buffer containing 30% sucrose at 4 °C. After storage, 50-μm-thick sections were cut on a freezing microtome (CM3000 cryostat, Leica, Wetzlar, Germany) and collected in 0.1 M phosphate buffer (pH 7.4). Sections were mounted on MAS-coated microscope slide glass (Matsunami Glass Industry, Osaka, Japan) and stained with thionin. 4.9.

Statistical analysis

Statistical analysis was performed using repeated measures of ANOVA, followed by Bonferroni post test comparison using GraphPad Instat 3.0.6 software (GraphPad Software Inc., San Diego, CA). Comparisons between the bicuculline methiodide + muscimol group and the muscimol group or between the CGP35348 + baclofen group and the baclofen group were carried out using unpaired t-test. Data are presented as means ± SEM, and p < 0.05 was considered statistically significant.

Acknowledgments This work was supported by Grants-in-Aid for Young Scientists (A) [20689036 (KM)] and Research Fellowships for Young Scientists [No. 163019 (KM)] from the Japan Society for the Promotion of Science.

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