Hiroshi Izumi

The Identification of Attractive Volatiles in Aged Male Mouse Urine

In many species, older males are often preferred mates because they carry good genes that account for their viability. In some animals, including mice, which rely heavily on chemical communication, there is some indication that an animals age can be determined by its scent. In order to identify the attractants in aged male mouse urine, chemical and behavioral studies were performed. We herein show that aged mice have higher levels of 3,4-dehydro-exo- brevicomin (DB), 2-sec-butyl-4,5-dihydrothiazole (BT), and 2-isopropyl-4,5-dihydrothiazole (IT) and a lower level of 6-hydroxy-6-methyl-3-heptanone relative to adult male mice. We also demonstrate that the attraction of females to the odor of male mouse urine is greater when the urine is from aged males. However, the attraction of aged urine odor was offset by the ultrafiltration of adult and aged mouse urine. When DB, BT, and IT were added to adult urine, the attraction of the urine was enhanced. Our results suggest that inbred aged male mice develop an aging odor that is attractive to female mice in an experimental setting and that this attraction is due to increased mouse pheromone signaling.

Sympathetic attenuation of parasympathetic vasodilatation in oro-facial areas in the cat

1 The present study was designed to examine the interaction between sympathetic and parasympathetic influences on blood flow in oro‐facial areas such as lower lip, palate and submandibular gland (SMG) and in the common carotid artery (CCA) in anaesthetized cats. 2 Section of the ipsilateral superior cervical sympathetic trunk (CST) increased the basal CCA blood flow significantly. The control level with the nerve intact was comparable with that seen at 0.5‐1 Hz CST stimulation, suggesting a spontaneous discharge of around 0.5‐1 Hz in the CST fibres innervating the beds supplied by the CCA. The basal blood flow at all sites examined was reduced by CST stimulation in a frequency‐dependent manner. 3 Electrical stimulation of the central end of the lingual nerve (LN) evoked blood flow increases in the lower lip and palate. These blood flow increases were markedly reduced by concurrent CST stimulation in a manner that was frequency dependent, but not simply related to the vasoconstrictor effect of CST stimulation. This effect of CST stimulation was not observed in tongue or SMG, even though CST stimulation evoked vasoconstriction in these tissues. A significant reduction in the level of CCA blood flow attained during LN stimulation was observed on repetitive CST stimulation only at 10 Hz, indicating that this response behaved in a fashion different from that seen in the lower lip, palate, tongue and SMG. 4 The present study suggests that concurrent repetitive CST stimulation reduces parasympathetically mediated blood flow increases in certain oro‐facial areas (such as the lower lip and palate), but not in the tongue and SMG. This inhibitory action was not a simple additive effect (between vasoconstriction and vasodilatation) and it disappeared rapidly after the cessation of CST stimulation.

Evidence for parasympathetic vasodilator fibres in the rat masseter muscle

The present study was designed to examine (1) whether there are vasodilator fibres in the masseter muscle, and (2) if there are, to establish the neural pathways mediating these responses in urethane‐anaesthetized rats. Electrical stimulation of the central cut end of the lingual nerve (LN) elicited intensity‐ and frequency‐dependent increases of the blood flow in the masseter muscle (MBF) and lower lip (LBF). Increases in both the MBF and LBF evoked by the LN stimulation were reduced by hexamethonium in a dose‐dependent manner (1–10 mg kg−1). Pretreatment with phentolamine or propranolol at a dose of 100 μg kg−1 had no effect on the increases in either MBF or LBF evoked by LN stimulation. Pretreatment with atropine (100 μg kg−1) significantly reduced the MBF increase induced by LN stimulation, but not that in the LBF. The sectioning of the superior cervical sympathetic trunk did not affect the responses. MBF increases occurred with electrical stimulation of the trigeminal ganglion, and these increases were significantly reduced by the administration of hexamethonium and atropine. Lidocaine microinjection into the trigeminal spinal nucleus or salivatory nuclei caused a significant attenuation of the LN‐induced MBF increases. When wheat germ agglutinin–horseradish peroxidase (WGA–HRP) was injected into the masseter muscle, labelled neurones were abundantly observed in the otic ganglion. The present study indicates that there are parasympathetic cholinergic and noncholinergic vasodilator fibres originating from cell bodies in the otic ganglion in the rat masseter muscle. The MBF increase evoked by activation of the parasympathetic fibres occurred via the trigeminal mediated reflex, suggesting that the novel parasympathetic vasodilator response may play an important role in the regulation of the haemodynamics of jaw muscles.

A novel assay system for myeloperoxidase activity in whole saliva

OBJECTIVESnThe application of a novel assay system for the direct measurement of MPO (myeloperoxidase) activity in whole saliva.nnnDESIGN AND METHODSnThe assay system employs a novel sensitive substrate from 3,3-diaminobenzidine (DAB) and guaiacol in the presence of dapsone (4,4-diaminodiphenylsulfone) to determine MPO activity in whole saliva using an original sandwich test-disk (DEAE-cellulose paper and cellulose chromatography paper). The saliva (0.1 mL) was directly applied to the sandwich test-disk, and then 0.1 mL of the substrate solution containing 1 mM dapsone in 0.3 M Tris-HCl buffer (pH 7.5) was added. After incubation for 30 min at room temperature, absorbance on the test-disk was measured at 460 nm with an optical analyzer.nnnRESULTSnThe assay system was shown to distinguish MPO from salivary peroxidase in whole mixed saliva and was sensitive, easy and cheap. The assays revealed that MPO activity in whole saliva from subjects with periodontal disease was significantly higher than in saliva from healthy subjects. There was also a significant positive correlation between MPO activity and the probing depth of subgingival pockets (r=0.736, p<0.001).nnnCONCLUSIONSnThese results indicate that this novel assay system for measurement of MPO is a useful technique for predicting the progression of periodontal disease.

Pyrazine analogues are active components of wolf urine that induce avoidance and freezing behaviours in mice.

Background The common grey wolf (Canis lupus) is found throughout the entire Northern hemisphere and preys on many kinds of mammals. The urine of the wolf contains a number of volatile constituents that can potentially be used for predator–prey chemosignalling. Although wolf urine is put to practical use to keep rabbits, rodents, deer and so on at bay, we are unaware of any prior behavioural studies or chemical analyses regarding the fear-inducing impact of wolf urine on laboratory mice. Methodology/Principal Findings Three wolf urine samples harvested at different times were used in this study. All of them induced stereotypical fear-associated behaviors (i.e., avoidance and freezing) in female mice. The levels of certain urinary volatiles varied widely among the samples. To identify the volatiles that provoked avoidance and freezing, behavioural, chemical, and immunohistochemical analyses were performed. One of the urine samples (sample C) had higher levels of 2,6-dimethylpyrazine (DMP), trimethylpyrazine (TMP), and 3-ethyl-2,5-dimethyl pyrazine (EDMP) compared with the other two urine samples (samples A and B). In addition, sample C induced avoidance and freezing behaviours more effectively than samples A and B. Moreover, only sample C led to pronounced expression of Fos-immunoreactive cells in the accessory olfactory bulb (AOB) of female mice. Freezing behaviour and Fos immunoreactivity were markedly enhanced when the mice were confronted with a mixture of purified DMP, TMP, and EDMP vs. any one pyrazine alone. Conclusions/Significance The current results suggest that wolf urinary volatiles can engender aversive and fear-related responses in mice. Pyrazine analogues were identified as the predominant active components among these volatiles to induce avoidance and freezing behaviours via stimulation of the murine AOB.

Profiles of Volatiles in Male Rat Urine: The Effect of Puberty on the Female Attraction

Rat urine contains many volatile constituents that may be used for chemical communication. The levels of certain urinary volatiles are strongly dependent on the sex and endocrine status (e.g., puberty). We performed chemical and behavioral studies to identify the volatiles in adult male rat urine that attract mature females. Our results demonstrated that adult male rats have higher levels of 2-heptanone (2-HP), 4-methylphenol (4-MP), and 4-ethylphenol (4-EP) than prepubescent male rats; furthermore, female rats are more attracted to the odor of adult male rat urine than that of prepubescent males. When prepubescent rat urine was supplemented with 2-HP, 4-MP, and 4-EP to the levels found in adult male urine, the attractiveness of the urine to females was markedly enhanced. Our results suggested that this attraction is due to an increased level of chemosignaling.

Bulbar pathway for contralateral lingual nerve-evoked reflex vasodilatation in cat palate.

We investigated the brain-stem pathway(s) by which electrical stimulation of the central cut end of the lingual nerve (LN) evokes parasympathetic reflex vasodilatation in the palate contralateral to the stimulated side. This occurs in artificially ventilated, cervically vagosympathectomized cats deeply anesthetized with alpha-chloralose and urethane. For this purpose, we made microinjections within the brain stem to produce nonselective, reversible local anesthesia (lidocaine) or soma-selective, irreversible neurotoxic damage (kainic acid). Local anesthesia of the trigeminal spinal nucleus (Vsp) ipsilateral to the stimulated side produced by microinjection of lidocaine (2%; 1 microl/site) reversibly and significantly reduced the LN stimulus-evoked palatal blood flow (PBF) increases. PBF increases ipsilateral and contralateral to the stimulated nerve were equally affected. In contrast, microinjection of lidocaine into the Vsp contralateral to the stimulated side did not affect these responses. Microinjection of kainic acid (10 mM/site; 1 microl) into the Vsp ipsilateral to the stimulated side led to a bilateral irreversible reduction in reflex vasodilatation in the palate. Microinjection of lidocaine into either superior salivatory nucleus (SSN) attenuated the PBF increase only on the side ipsilateral to the microinjection site. Hexamethonium (1.0 mg/kg iv) significantly reduced the vasodilator responses to electrical stimulation of Vsp by blocking ganglionic transmission on both sides. The simplest interpretation of these results is that the LN-evoked parasympathetic reflex vasodilatation in the contralateral palate depends on activation of a pathway originating from the Vsp ipsilateral to the stimulated nerve and crossing to the contralateral SSN.

Vagal visceral inputs to the nucleus of the solitary tract: Involvement in a parasympathetic reflex vasodilator pathway in the rat masseter muscle

The present study examined whether vagal visceral inputs are involved in parasympathetic reflex vasodilatation in the masseter muscle in urethane-anesthetized and cervically sympathectomized rats. Electrical stimulation of the central cut end of the cervical vagus nerve (VN) including visceral afferent fibers, which consisted of cervical/thoracic branches (heart and lungs) and abdominal branches (entire gastrointestinal tract), elicited intensity- and frequency-dependent increases of blood flow in the masseter muscle (MBF). Activation of the abdominal VN inferior to the diaphragm failed to affect the MBF. MBF increases evoked by cervical VN stimulation were reduced significantly by hexamethonium. Pretreatment with atropine reduced the MBF increase evoked by VN stimulation significantly, whereas pretreatment with either propranolol or phentolamine had no effect on the response. MBF increases occurred with electrical stimulation of nucleus of the solitary tract (NTS), and these increases were significantly reduced by the administration of hexamethonium and atropine. MBF increases also occurred after microinjection of glutamate into the NTS in a dose-dependent manner. Microinjection of muscimol into the NTS caused a significant attenuation of the VN stimulation-induced MBF increases. Our results suggest that vagal visceral inputs passing to the NTS are involved in the parasympathetic reflex vasodilatation in the rat masseter muscle. The MBF increase evoked by the vagal-parasympathetic reflex mechanism occurred via visceral afferents running in the cervical VN, but not in the abdominal VN, suggesting that the vagal visceral afferents derived from cardiovascular and/or respiratory systems may play an important role in the regulation of the MBF.

Difference between male and female rats in cholinergic activity of parasympathetic vasodilatation in the masseter muscle

We compared the changes in blood flow of the masseter muscle (MBF), lower lip (LBF) and common carotid artery (CCABF) evoked by electrical stimulation of the lingual nerve (LN) in order to examine whether high cholinergic activity of parasympathetic vasodilatation in females is specific for the masseter muscle, and whether sex-associated differences in cholinergic parasympathetic vasodilatation affect the regulation of blood flow to the orofacial area from the CCABF in urethane-anaesthetized, vago-sympathectomized male and female rats. Increases in the MBF, LBF and CCABF evoked by LN stimulation appear to be mediated via an activation of parasympathetic reflex vasodilatation since these increases were profoundly reduced by pretreatment with the autonomic cholinergic ganglion blocker hexamethonium (10 mg/kg). Although alpha- and beta-adrenoceptor antagonists (phentolamine and propranolol, 100 microg/kg) had no effect on the LN stimulation-induced blood flow increases in either sex, a marked difference was found between males and females in the effects of the antimuscarinic agent atropine (1-100 microg/kg) on these blood flow increases. Pretreatment with atropine slightly attenuated the increase in the MBF in males, but in females it markedly reduced the increases in all three sites measured, especially in the MBF. Our results suggest that (1) cholinergic activity of the parasympathetic vasodilatation in females is higher than that in males in most orofacial tissues, but particularly in the masseter muscle and (2) cholinergic parasympathetic vasodilatations are more involved in the regulation of blood flow to the orofacial area from the CCABF in females than in males.

Circulating adrenaline released by sympathoadrenal activation elicits acute vasodilatation in the rat masseter muscle

The present study was designed to examine the effects of circulating catecholamines released by sympathoadrenal system on the haemodynamics of the masseter muscle in deeply urethane-anaesthetized, artificially ventilated, cervically vagotomized and sympathectomized rats. Intravenous administration of adrenaline induced a biphasic change of blood flow in the masseter muscle (MBF). The change of blood flow showed an initial marked increase and successive slight decrease in a dose-dependent manner (0.01-1 microg/kg). The administration of noradrenaline had no significant effect on the MBF. The increase in the MBF evoked by exogenously applied adrenaline was markedly reduced by the intravenous administration of propranolol (100 microg/kg), whereas pretreatment with either hexamethonium (10 mg/kg), atropine (100 microg/kg), or phentolamine (1 mg/kg) failed to affect the MBF increase. Electrical stimulation of splanchnic nerve (SPLN) preganglionic neurones projecting to the adrenal medulla elicited frequency-dependent (1-20 Hz) increases in the MBF. The intravenous administration of the beta(2)-adrenergic receptor selective antagonist, ICI 118551 (0.5 mg/kg), almost abolished the MBF increase induced by SPLN stimulation, but pretreatment with the beta(1)-adrenergic receptor selective antagonist, atenolol (1 mg/kg), had no effect on this response. The results of the present study indicate that circulating adrenaline elicits acute vasodilatation through a beta-adrenergic mechanism in the rat masseter muscle. Vascular beta(2)-adrenergic receptors in the masseter muscle may be activated preferentially by adrenaline released from the adrenal medulla, suggesting that the sympathoadrenal system is involved in the marked MBF increase during sympathoexcitation.