The development of the emetic reflex in the house musk shrew, Suncus murinus

The emetic (retching and vomiting) reflex is an important component of the body's defence system against accidentally ingested toxins and emesis is also a common symptom of disease and a side-effect of a number of pharmacological therapies. The development of the reflex has been the subject of few systematic studies. The aim of this study was to characterise the development of the emetic reflex in Suncus murinus (the house musk shrew) using emetic stimuli acting via three different afferent pathways: motion via the vestibular system, pyrogallol via abdominal vagal afferents and resiniferatoxin (a capsaicin analog) via the brainstem. The emetic reflex was not present to any stimulus prior to postnatal day 10 but the onset of the response to motion lagged behind that to the other stimuli in not being present until postnatal day 15. Body weight was not a determinant of the presence of the reflex. It is proposed that the delayed presence of the emetic reflex in Suncus makes it an ideal species in which to investigate factors regulating its development.     

Cardiorespiratory reflexes in a working heart-brainstem preparation of the house musk shrew, Suncus murinus

In this study, we adapted the working heart-brainstem preparation (WHBP) from rodents to the Insectivore, Suncus murinus. Suncus WHBPs had a baseline heart rate of 333 +/- 8 beats min(-1), a perfusion pressure of 69 +/- 2 mm Hg and a respiratory cycle length of 6.5 +/- 0.7 s. Administration of atropine produced an increase in heart rate of 26 +/- 9 beats min(-1) indicative of the presence of cardiac vagal tone. Activation of baroreceptors produced pressure-dependent reflex falls in heart rate and reduced respiratory cycle length. The baroreceptor reflex sensitivity in Suncus WHBP was a decrease in heart rate of 8.1 +/- 1.4 beats min(-1) mm Hg(-1). Activation of peripheral chemoreceptors with aortic injections of sodium cyanide (0.1-12.5 microg) produced a dose-dependent reflex fall in heart rate and reduced respiratory cycle length. The reflex falls in heart rate evoked by baroreceptor and peripheral chemoreceptor stimulation were both atropine-sensitive. We conclude that viable WHBP can be prepared from Suncus and that Suncus WHBP is a novel non-rodent model in which to study brainstem-mediated reflexes.     

Contractile effect of tachykinins on Suncus murinus (house musk shrew) isolated ileum

Recent studies used Suncus murinus to investigate the anti-emetic potential of NK(1) tachykinin receptor antagonists. However, the pharmacology of tachykinin receptors in this species has not been fully characterized. In the present studies, therefore, we examined a range of tachykinin receptor agonists for a capacity to induce contractions of the isolated ileum. The tachykinin NK1 receptor preferring agonists substance P, septide and [Sar9Met(O2)11] substance P, and the tachykinin NK2 preferring agonists neurokinin A and GR 64349 (Lys-Asp-Ser-Phe-Val-Gly-R-gamma-lactam-Leu-Met-NH2) caused concentration dependent contractions with EC50 values in the nanomolar range. However, the tachykinin NK3 preferring agonists neurokinin B and senktide (1nM-1microM) induced only weak contractions. The action of senktide, but not [Sar9Met(O2)11] substance P, septide, or GR 64349, was antagonized significantly by atropine (P<0.05); tetrodotoxin and hexamethonium were inactive. The tachykinin NK1 receptor antagonist CP-99,994 ((+)-[(2S,3S)-3-(2-methoxy-benzyl-amino)-2-phenylpiperidine]) (10-100nM) inhibited substance P- and septide-induced contractions non-competitively. The pA2 value estimated for CP-99,994 against septide was 7.3+/-0.1. It also non-competitively antagonized the contractile responses induced by [Sar9Met(O2)11] substance P with a pA2 of 7.4+/-0.1. CP-99,994 also had a slight inhibitory action on neurokinin A-induced contractions, but did not modify the action of GR 64349. Conversely, the tachykinin NK2 receptor antagonist, saredutant, competitively antagonized GR 64349-induced contractions with a pA2 of 7.34+/-0.02. On the other hand, the presence of both CP-99,994 and saredutant competitively antagonized substance P-induced contraction. The present studies indicate that tachykininNK1 and NK2 receptors exist in the ileum of S. murinus and are involved in mediating contractions directly on smooth muscle, whereas tachykinin NK3 receptors may play a minor role involving a release of acetylcholine.     

Neural regulation of esophageal striated muscle in the house musk shrew (Suncus murinus)

In the present study, we characterized the neural regulation of esophageal striated muscle in Suncus murinus (a house musk shrew; "suncus" used as a laboratory name), which was compared with that in the rat. The tunica muscularis consists of striated muscle in the suncus esophagus. An isolated segment of the suncus esophagus was placed in an organ bath and the contractile responses were recorded using a force transducer. Electrical stimulations to vagus nerves induced contractile responses in the esophageal segment. Treatment with α-bungarotoxin, a blocker of nicotinic acetylcholine receptors, blocked the vagally mediated contractions of the suncus esophagus. D-tubocurarine and succinylcholine, typical antagonists of nicotinic acetylcholine receptors, also inhibited the suncus esophageal contractions, while higher concentrations of the agents were required rather than concentrations for producing an equivalent block in the rat. We used capsaicin, a stimulator of small-caliber afferent neurons, for activating the peripheral neural network. The reagent inhibited the vagally mediated twitch contractions of striated muscle in the suncus esophagus, which was reversed by pretreatment with a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester. Application of a nitric oxide donor, diethylamine NONOate diethylammonium salt, mimicked capsaicin-induced inhibition. The results suggest that motility of the suncus esophagus, which consists of striated muscles, is regulated by vagal cholinergic neurons. The local neural network including capsaicin-sensitive neurons and intrinsic nitrergic neurons can modify the vagally mediated motility in the suncus esophagus. In addition, nicotinic acetylcholine receptors of the suncus esophagus might be pharmacologically distinct from those of rodent esophagi.     

Retinal projections in the house musk shrew, Suncus murinus, as determined by anterograde transport of WGA-HRP.

Retinal projections in the house musk shrew (Suncus murinus) were determined by the anterograde transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Unilateral injection of WGA-HRP into the vitreous body resulted in the terminal labeling of the optic projections in the suprachiasmatic nucleus (SCH), the ventral (CGLv) and dorsal (CGLd) lateral geniculate nuclei, the intergeniculate leaflet (IGL), the pretectum, the superficial layers of the superior colliculus (CS), and the dorsal terminal nucleus (DTN) of the accessory optic system (AOS). Labeling of the SCH was bilateral, with ipsilateral predominance, and covered the whole dorsoventral extent of the nucleus. Immunohistochemical studies revealed that VIP-like immunoreactive neurons and fibers were present in almost all parts of the SCH. No hypothalamic regions other than the SCH received the optic fibers. The ipsilateral projections to the CGLv, CGLd, and IGL were sparse, and a considerable number of uncrossed retinal fibers were found in the pretectal olivary nucleus. No retinal projections to the lateral posterior thalamic nucleus (LP) were found. Ipsilateral optic fibers projected sparsely to the medial part of the CS. The AOS consisted of a small DTN with a very few crossed retinal projections but no lateral and medial terminal nuclei. In addition, the AOS had no inferior fascicle.     

Myenteric neural network activated by motilin in the stomach of Suncus murinus (house musk shrew)

Background It has been shown in human and canine studies that motilin, a gastroprokinetic hormone, induces gastric phase III contractions via the enteric nervous; however, the center of motilin action in the stomach has not been clearly revealed. In the present study, we investigated the neural pathway of motilin‐induced gastric contraction by using Suncus murinus, a new animal model for motilin study. Methods An isolated suncus stomach was used in vitro to determine the mechanism of motilin action through the myenteric plexus. Synthetic suncus motilin (10^?11–10^?7 mol L^?1) was added to an organ bath, and the spontaneous contraction response was expressed as a percent of ACh (10^?5 mol L^?1) responses. Motilin‐induced contractions were also studied by a pharmacological method using several receptor antagonists and enzyme inhibitor. Key Results Suncus motilin induced a concentration‐dependent gastric contraction at concentrations from 10^?9 to 10^?7 mol L^?1. The responses to suncus motilin in the stomach were completely abolished by atropine and tetrodotoxin treatment and significantly suppressed by administration of hexamethonium, verapamil, phentolamine, yohimbine, ondansetron, and naloxone, whereas ritanserin, prazosin, timolol, and FK888 did not affect the action of motilin. Additionally, N‐nitro l ‐arginine methylester slightly potentiated the contractions induced by motilin. Conclusions & Inferences The results indicate that motilin directly stimulates and modulates suncus gastric contraction through cholinergic, adrenergic, serotonergic, opioidergic, and NO neurons in the myenteric plexus.     

Calbindin-D28k and calretinin immunoreactive neurons in the olfactory bulb of the musk shrew, Suncus murinus.

The distribution, morphological features, and postnatal development of calbindin-D28k (CB) and calretinin (CR) immunoreactive neurons in the main olfactory bulb (MOB) of the musk shrew, Suncus murinus, were studied by immunostaining to determine the degree of colocalization of CB and CR, and the relationship of CB and CR to neuron development in the MOB of animals of the order Insectivora. In adults, CB-positive neurons were identified as periglomerular and perinidal cells in the periglomerular region, as superficial short-axon cells in the external plexiform layer, and as four types of interneurons (Cajal, horizontal, Golgi, and bitufted cells) in the mitral cell, internal plexiform, and granule cell layers. CR-positive neurons were identified as projection neurons (tufted and mitral cells) and interneurons (periglomerular, perinidal, and granule cells). On postnatal days 1 and 3, CB-positive neurons revealed numerous processes finely arborized near the somata, and were morphologically unidentifiable. At the same time, CR-positive neurons were identified as young periglomerular and granule cells, and as migrating bipolar cells extending leading processes with growth cones in each layer of the MOB and the subependymal layer between the anterior lateral ventricle and the center of the MOB. On postnatal day 28, mature CB-positive and CR-positive interneurons were distributed in their corresponding layers, whereas migrating CR-positive bipolar cells were rarely detected. No cells colocalized CB and CR. The results suggest that perinidal cells in the shrew MOB may develop postnatally, together with glomerular and granule cells. We suggest that CB is associated with mechanisms of the outgrowth of neuronal processes, whereas CR is involved in mechanisms of cell migration and outgrowth of neuronal processes, in some types of neurons in the developing stage of the shrew MOB.     

Nidus and tasseled cell: distinctive neuronal organization of the main olfactory bulb of the laboratory musk shrew (Suncus murinus)

We revealed the structural features of particular synaptic regions, nidi, and newly found neurons, tasseled cells, in the main olfactory bulb (MOB) of the laboratory musk shrew (Suncus murinus). Nidi were intensely immunoreactive for glutamic acid decarboxylase (GAD) and calbindin D28k (CB), were 30-80 microm in diameter, and were located beneath glomeruli, appearing to make glomerulus-nidus unit-like complexes. In contrast to glomeruli, they contained few or no olfactory nerves. Nidi were distributed throughout the whole MOB and made a distinctive layer, nidal layer. Tasseled cells were located in the mitral cell layer and in the middle of the external plexiform layer (EPL) and extended single primary dendrites to the nidus, where their small tuft-like complicated branches intermingled with processes of perinidal cells surrounding nidi. Primary dendrites of mitral/tufted cells also penetrated nidi but passed to glomeruli. In the outer half of the EPL, columnar structures were seen, where CB- and GAD-positive elements appeared to associate with bundles of cylindrical dendrites of presumed mitral/tufted and tasseled cells. By electron microscopic examinations, nidi were confirmed to be particular synaptic areas where GAD-positive processes made symmetrical synapses to GAD-negative presumed tasseled and mitral/tufted cell dendrites and received asymmetrical synapses from the latter. Retrograde tracings revealed that tasseled cells, in addition to mitral/tufted cells, projected their axons to the lateral olfactory tract, indicating that there were two parallel projection systems in the shrew MOB, which might interact with each other via various types of gamma-aminobutyric acid (GABA)ergic interneurons. The present study clearly showed that the neuronal organization of the shrew MOB was distinctly different from that in rodents.     

Induction of Fos protein in neurons in the medulla oblongata after motion- and X-irradiation-induced emesis in musk shrews (Suncus murinus)

To clarify the anatomical location of medullary neurons associated with vomiting, the musk shrew (Suncus murinus), a small animal used as a model for emesis, was exposed to various emetic stimuli and patterns of neuronal excitation were investigated by Fos immunohistochemistry. In motion experiments, musk shrews were shaken for 30 min on a tabletop shaker (displacement=25 mm and frequency=1.2 Hz). Ten of fifteen animals vomited frequently (Mo-FV group); the other five animals did not vomit (Mo-NV group). In radiation experiments, X-ray irradiation (10 Gy) of the whole body caused frequent vomiting in all of seven experimental animals (Ra-FV group). In the Mo-FV group, many Fos-immunoreactive (Fos-ir) neurons were detected in the nucleus of the solitary tract (NTS) and the reticular formation. The distribution pattern of Fos-ir neurons in the Mo-NV group was similar to that in the Mo-FV group, but the Mo-NV group had significantly fewer positive neurons in the NTS and the reticular formation around the nucleus ambiguus. In the Ra-FV group, numerous Fos-ir neurons were observed in the area postrema, an area containing no positive neurons in the motion-stimulated animals. The number of Fos-ir neurons in the NTS of the Ra-FV group was not statistically different from that of the Mo-NV group. In the Mo-FV and Ra-FV groups, Fos-ir neurons were clustered in the reticular formation at the dorsal-dorsomedial edge of the nucleus ambiguus at the level of the rostral medulla, while few such clusters were observed in the Mo-NV group. These neurons may play a role in the regulation of the vomiting response.     

Role of the autonomic nervous system in emetic and cardiovascular responses in Suncus murinus

To clarify the role of the autonomic nervous system in cardiovascular and emetic responses, we studied the influence of drugs that act on autonomic nervous function on emetic and cardiovascular responses induced by chemical or mechanical stimulation to the stomach in two strains of Suncus murinus, Jic:SUN-Her and Jic:SUN-Ler. Latency to the first retching in Jic:SUN-Her was significantly shorter than that in Jic:SUN-Ler to both mechanical and chemical stimulation. This result indicated that there are different sensitivities to mechanical and chemical stimulation to the stomach in these two strains of suncus. However, the numbers of emetic episodes were almost the same in these two strains. Mean blood pressure significantly increased from baseline prior to retching in both strains. Heart rate decreased in Jic:SUN-Her and increased in Jic:SUN-Ler prior to retching, suggesting that a different baroreflex responsiveness might exist in these two strains of suncus. Administration of acetylcholine and phenylephrine affected emetic response induced by mechanical and chemical stimulation. Although the baseline values of mean blood pressure and heart rate after administration of these drugs were different, changes in mean blood pressure and heart rate prior to retching were unaffected. This result suggested that the state of autonomic activity before the emetic response might be important in the development of the emetic response. Pretreatment with hexamethonium suppressed the cardiovascular response prior to retching and prolonged the latency to the first retching. This result indicated that there was an interaction between the mechanisms involved in cardiovascular and emetic responses. The change in autonomic function during the emetic response, especially enhancement of sympathetic activity prior to retching, may be relevant to emetic and cardiovascular responses. Moreover, these results suggest that different autonomic function or different baroreflex responsiveness in Jic:SUN-Her and Jic:SUN-Ler may be involved in emetic responses.     

Neural distribution of estrogen receptor immunoreactive cells in the female musk shrew

In this report we describe the neural distribution of estrogen receptor immunoreactivity (ER-IR) in the female musk shrew (Suncus murinus). The highest concentrations of neurons containing ER-IR were found in the preoptic areas, the lateral septum, the anterior arcuate and ventromedial nuclei of the hypothalamus, the medial nuclei of amygdala, and the midbrain central grey. Additional preoptic, hypothalamic, limbic and midbrain nuclei also contained ER-IR cells. The distribution of ER-IR was similar to that described in other mammals and birds, with several important differences. In the female musk shrew, there was little difference in ER-IR intensity or distribution when brains from gonadally intact and ovariectomized musk shrews were compared. In addition, long-term treatment with a supraphysiological dose of estradiol was required to detect a decrease in ER-IR intensity. Finally ER-IR was noted in both nuclear and cytoplasmic regions of cells in ovariectomized and gonadally intact musk shrews. The dense ER-IR noted in intact females as well as the presence of cytoplasmic stain may be due to the unusual relationship between estradiol, ovulation and sexual receptivity in this species.     

The effects of naloxone on the neural control of the urinary bladder of the cat

Naloxone in doses ranging from 0.5 to 512μg/kg i.v., enhanced reflex contractions of the urinary bladder of the cat. At the lowest doses (threshold, 0.5–5 μg/kg) the drug increased the frequency of spontaneous bladder contractions. In large doses (10–100 μg/kg) the drug produced an initial tonic contraction of bladder lasting 15–40 min followed by a period of high frequency rhythmic activity. Multiunit firing in parasympathetic postganglionic nervs on the surface of the urinary bladder was also enhanced. Bursts of firing which in untreated animals occured during large bladder contractions continously during the entire sustained contraction of the bladder following large doses of naloxone. Various evidence indicates that the site of action of naloxone is in the central nervous system. These findings suggest that the parasympathetic reflex pathway to the urinary bladder may be subject to tonic enkephalinergic inhibitory control.     

Computerized detection and analysis of cancer chemotherapy-induced emesis in a small animal model, musk shrew

Vomiting is a common side effect of cancer chemotherapy and many drug treatments and diseases. In animal studies, the measurement of vomiting usually requires direct observation, which is time consuming and often lacks temporal precision. Musk shrews have been used to study the neurobiology of emesis and have a rapid emetic episode (～1 s for a sequence of retching and expulsion). The aim of the current study was to develop a method to automatically detect and characterize emetic episodes induced by the cancer chemotherapy agent cisplatin. The body contour in each video frame was tracked and normalized to a parameterized shape basis. The tracked shape was projected to a feature space that maximized the shape variations in the consecutive frames during retching. The resulting one dimensional projection was sufficient to detect most emetic episodes in the acute (peak at 2h) and delayed (peak at 54 h) phases after cisplatin treatment. Emetic episodes were relatively invariant in the number of retches (～6.2), duration (～1.2s), inter-retch interval (～198 ms), and amplitude during the 72 h after cisplatin treatment. This approach should open a new vista into emesis research to permit tracking and analysis of emesis in a small animal model and facilitate the development of new antiemetic therapies. These results also yield a better understanding of the brain's central pattern generator for emesis and indicate that the retching response in the musk shrew (at ～5.4 Hz) is the fastest ever recorded in a free-moving animal.     

In vivo motor effects of substance P on the rat urinary bladder

Summary Intravesical pressure recordings of the urinary bladder in anesthetized rats were performed and the role of substance P (SP) in the motor control of this organ was evaluated. Regional injection of SP (0.4 nmoles i.a.) into the superior vesical artery elicited a prompt bladder contraction; this motor response was dosedependent. The detrusor contraction could be completely inhibited by a SP-analogue, (D-Pro², D-Trp^{7, 9})-SP (45–90 nmoles i.a.). Further-more, the detrusor contraction evoked by preganglionic stimulation of the pelvic nerves was partially inhibited by the same antagonist in a higher dose (65% reduction at a total dose of 150–300 nmoles). The contractile response to SP (0.5 nmoles i.a.) was also significantly reduced after blockade of muscarinic receptors with atropine (50% reduction at 1 mg/kg i.a.) or after ganglionic blockade with hexamethonium (75% reduction at 25 mg/kg i.v. +50 mg/kg hr i.a.). Immunocytochemical studies demonstrated the occurrence of SP-immunopositive nerve terminals in the detrusor part of the rat urinary bladder. Based on these findings it is suggested that SP may act as a neurotransmitter/modulator in this organ. The mechanism of action for SP on the detrusor seems to be complex and may involve ganglionic transmission via both types of cholinoceptors as well as direct activation of smooth muscle.     

Role of autonomic nervous system for development and suppression of motion sickness in Suncus murinus.

To clarify the role of autonomic nervous function in motion sickness, the effect of agents that act on the autonomic nervous system on the motion stimuli-induced emesis was studied in two strains of Suncus murinus (Jic:SUN-Her and Jic:SUN-Ler) with congenitally different sensitivity to veratrine sulfate. We demonstrated significant differences between the two strains in sensitivity to motion stimuli. Isoproterenol (2.5 mg kg(-1), s.c.) significantly prolonged the latency to the first emetic episode induced by motion stimuli and significantly decreased the number of emetic episodes in Jic:SUN-Her suncus. Hexamethoium (2.0 mg kg(-1), s.c.) tended to shorten the latency in Jic:SUN-Ler. Acetylcholine (1.2 mg kg(-1), s.c.) enhanced the emetic response in Jic:SUN-Ler, but atropine (4.0 mg kg(-1), s.c.) suppressed motion stimuli-induced emetic response in Jic:SUN-Her. These results suggest that the predominance of parasympathetic nervous activity is relevant to the enhancement of motion stimuli-induced emetic response, whereas the predominance of sympathetic nervous activity suppresses motion stimuli-induced emetic response. Norepinephrine (0.8 mg kg(-1), s.c.) enhanced motion stimuli-induced emesis contrary to isoproterenol in Jic:SUN-Ler although both drugs are adrenergic agents. However, atropine pretreatment (4.0 mg kg(-1), s.c.) inhibits norepinephrine-induced emetic response. It was considered that norepinephrine-induced emetic response might be dependent on a secondary increase of parasympathetic nervous activity due to bororeflex. Moreover, the different emetic response in Jic:SUN-Her and Jic:SUN-Ler suncus to motion stimuli and drug administration mentioned above indicated that different participation of autonomic nervous activity and/or afferent information from the baroreceptor in the emetic response may exist between these animal groups.