Yasushige Ohmori

Kisspeptin neurons mediate reflex ovulation in the musk shrew (Suncus murinus)

The present study investigated whether kisspeptin–G protein-coupled receptor 54 (GPR54) signaling plays a role in mediating mating-induced ovulation in the musk shrew (Suncus murinus), a reflex ovulator. For this purpose, we cloned suncus Kiss1 and Gpr54 cDNA from the hypothalamus and found that suncus kisspeptin (sKp) consists of 29 amino acid residues (sKp-29). Injection of exogenous sKp-29 mimicked the mating stimulus to induce follicular maturation and ovulation. Administration of several kisspeptins and GPR54 agonists also induced presumed ovulation in a dose-dependent manner, and Gpr54 mRNA was distributed in the hypothalamus, showing that kisspeptins induce ovulation through binding to GPR54. The sKp-29–induced ovulation was blocked completely by pretreatment with a gonadotropin-releasing hormone (GnRH) antagonist, suggesting that kisspeptin activates GnRH neurons to induce ovulation in the musk shrew. In addition, in situ hybridization revealed that Kiss1-expressing cells are located in the medial preoptic area (POA) and arcuate nucleus in the musk shrew hypothalamus. The number of Kiss1-expressing cells in the POA or arcuate nucleus was up-regulated or down-regulated by estradiol, suggesting that kisspeptin neurons in these regions were the targets of the estrogen feedback action. Finally, mating stimulus largely induced c-Fos expression in Kiss1-positive cells in the POA, indicating that the mating stimulus activates POA kisspeptin neurons to induce ovulation. Taken together, these results indicate that kisspeptin–GPR54 signaling plays a role in the induction of ovulation in the musk shrew, a reflex ovulator, as it does in spontaneous ovulators.

Projections of visceral and somatic primary afferents to the sacral spinal cord of the domestic fowl revealed by transganglionic transport of horseradish peroxidase.

The termination of visceral and somatic primary afferent fibers in the sacral spinal cord of the domestic fowl was studied using transganglionic transport of horseradish peroxidase. Nerve terminals of visceral afferent fibers were found in the lateral edge and base of the dorsal horn and in the dorsal gray commissure in close proximity to parasympathetic preganglionic neurons. Somatic afferents terminated in laminae 2 and 3. The results demonstrate that visceral and somatic afferent fibers in this avian species terminate in different areas of the dorsal horn.

Immunohistochemical localization of serotonin, galanin, cholecystokinin, and methionine-enkephalin in adrenal medullary cells of the chicken

The identification of adrenaline- (A) and noradrenaline- (NA) containing cells in the adrenal medulla of the chicken and colocalization of serotonin and neuropeptides with A or NA in medullary cells were investigated with the use of immunohistochemical methods. Antisera against tyrosine hydroxylase and phenylethanolamine-N-methyltransferase were used as markers for catecholamine- and A-synthesizing cells, respectively. About 70% of catecholamine-synthesizing cells also exhibited immunoreactivity for phenylethanolamine-N-methyltransferase antiserum. Therefore, these cells are A-containing ones and the rest of cells seem to be NA-containing cells. Immunoreactivity with serotonin antiserum was observed in almost all medullary cells. Galanin-immunoreactivity was also found throughout the adrenal medulla, but was stronger in A-containing cells than in NA-containing ones. Cholecystokinin-immunoreactivity was restricted to A-containing cells. Methionine-enkephalin-immunoreactivity was seen in both A- and NA-containing cells, but in about half of medullary cells. From these results, it is suggested that serotonin, galanin, cholecystokinin, and methionine-enkephalin may be co-released with A and/or NA from adrenal medullary cells of the chicken.

Localization of the Motoneurons Innervating the Hindlimb Muscles in the Spinal Cord of the Domestic Fowl

The origins of the motor nerve fibers supplying the individual forelimb muscles were elucidated in the fowl by the retrograde degeneration method. Chromatolytic cells were seen in the ipsilateral lamina 9 of the cervical enlargement. Motoneurons innervating the individual forelimb muscles occupy the restricted area in the lamina 9. The muscles acting on the shoulder joint are supplied by motoneurons of the more cranial segments and the others acting on the elbow and the more distal joints by those of the more caudal ones. The origin of the dorsal cord is situated laterally in the lamina 9 and that of the ventral cord is located medially in it.

Immunohistochemical studies on the intestinal nerve of remak in the male chicken

A peroxidase anti-peroxidase method was used to investigate and compare the distribution of neuropeptide and catecholamine synthesizing enzyme immunoreactive (IR) ganglion cells and nerve fibres in the intestinal nerve of Remak (INR) of male chickens. In the INR there were three kinds of ganglion cells: tyrosine hydroxylase (TH)-, aromatic L-amino acid decarboxylase (AADC)- and phenylethanolamine-N-methyltransferase (PNMT)-IR cells; AADC- and PNMT-IR but TH-immunonegative cells; and ganglion cells being immunoreactive for methionine enkephalin (mENK)- and somatostatin (SOM). The first one was distributed throughout the INR. The second was restricted in the ileojejunal region, and the last was localized in the rectal region. Substance P- and vasoactive intestinal polypeptide-IR nerve fibres were distributed in common but variable in number around three kinds of ganglion cells. Then TH-IR cells were characterized by the distribution of many calcitonin gene related peptide- and a few cholecystokinin-IR fibres. mENK and SOM-IR cells, and TH-immunonegative cells were distinguished by the distribution of SOM- and galanin-IR fibres. In addition, TH-immunonegative cells were characterized by the distribution of mENK- and neuropeptide Y-IR nerve fibres which were very few in number. Fig. 21 summarizes the connections described in the present study.

Immunohistochemical studies on the intrinsic pancreatic nerves in the chicken.

A peroxidase anti-peroxidase method or an avidin-biotinylated complex method was used to visualize neural elements immunostained for several neuropeptides in the chicken pancreas. Pancreatic ganglion cells were only immunoreactive with vasoactive intestinal polypeptide (VIP), galanin and substance P (SP) antisera. VIP-immunoreactive (IR) ganglion cells were the most numerous, and most of them also showed the distinct immunoreaction with galanin. VIP- and galanin-IR nerve fibers were observed in the exocrine portion, the adventitia of the artery and the connective tissue of the ductal wall. The number and distribution of the VIP- and galanin-IR nerve fibers around the artery and duct were similar. SP-IR nerve fibers were found mainly close to the blood vessel. SP- and CGRP-IR nerve fibers were detected in the VIP-IR ganglion and extrapancreatic nerve bundle. Tyrosine hydroxylase (TH)- and aromatic L-amino acid decarboxylase (AADC)-IR nerve fibers were observed as nerve bundles in the interlobular space or extrapancreatic nerves. Consequently, VIP and galanin coexist in the intrinsic neural elements. SP is partially located in the intrinsic neural elements, but most of it seems likely to originate from the extrinsic ganglion. It is probable that calcitonin gene related peptide (CGRP)-, TH- and AADC-IR nerve fibers have an extrinsic origin.

Afferent and Efferent Connections of the Nucleus Rotundus Demonstrated by WGA‐HRP in the Chick

Organization of the fibre connections in the chick nucleus rotundus (Rt) was investigated by an axonal tracing method using wheat germ agglutinin conjugated to horseradish peroxidase (WGA‐HRP). After an injection of WGA‐HRP into the Rt, labelled neurones were observed in the striatum griseum centrale (SGC) in both sides of the tectum (TO) and in the ipsilateral nucleus subpretectalis/nucleus interstito‐pretecto‐subpretectalis (SP/IPS). Labelled fibres and terminals were also found in the ipsilateral ectostriatum (Ect). These fibre connections were topographically organized rostrocaudally. In the TO‐Rt projection, the rostral and the dorsocaudal parts of the Rt received afferents from the superficial part of the SGC, the middle part of the Rt received afferents from the intermediate part of the SGC, and the ventrocaudal part of the Rt received mainly fibres from the deep part of the SGC. These topographic projections were accompanied by a considerable number of diffuse projections to the thalamic regions surrounding the Rt. In addition, the rostral and middle caudal parts of the Rt received afferents from the lateral and medial parts of the SP/IPS, respectively, and respective parts of the Rt sent efferents to the lateral and medial parts of the Ect.

Afferent and Efferent Connections of the Nucleus Geniculatus Lateralis Ventralis Demonstrated by WGA-HRP in the Chick

Fibre connections of the chick nucleus geniculatus lateralis ventralis (GLv) were investigated using the axonal tracing method with wheat germ agglutinin conjugated to horseradish peroxidase (WGA‐HRP). After an injection of WGA‐HRP into the GLv, many labelled neurons were observed in layer i of the stratum griseum et fibrosum superficiale (SGFS) in the ipsilateral tectum opticum (TO) and in the nucleus lentiformis mesencephali (LM). In the TO‐GLv projection, cells of origin were located in the deeper part of layer i of the TO and were topographically distributed along the direction from the rostrodorsal part to the caudoventral part of the TO relating to a rostrocaudal axis of the GLv. In the LM‐GLv connection, the dorsal and ventral parts of the LM connected reciprocally with the rostral and caudal halves of the GLv, respectively. In contrast, in the GLv efferent connection, labelled axon terminals spread widely in the ipsilateral area pretectalis without any clear topographical arrangement.

Projections of neurons in the intestinal nerve of Remak to the chicken intestine

To date, the exact site of the extrinsic postganglionic neurons innervating the intramural plexuses in the chicken intestine is unknown. In this study, neurons in the intestinal nerve of Remak (INR) were immunohistochemically labelled by injecting cholera toxin subunit B (CTb) into each one of the jejunal, ileal, cecal and rectal walls. The CTb-immunoreactive (IR) neurons were counted, and some sections from the rectal portion of INR (rectal INR) were also immunostained for either tyrosine hydroxylase (TH) or methionine enkephalin (mENK). Following injection of CTb into the jejunum or ileum, only a few CTb-IR neurons were found in the jejunal or ileal part of INR caudal to the injection site. Following injection into the more caudal intestine, CTb-IR neurons were seen in the rectal INR. Of the 3490 CTb-IR neurons that were counted in the rectal INR, 40% projected into the rostral rectum, 24% into the caudal rectum, 17% into the ileum, 10% into the cecum and only 9% into the middle rectum. Rostrocaudally dividing the rectal INR into three parts, one third of the CTb-IR neurons in the rostral part projected into the rostral rectum, the majority of CTb-IR ones in the middle part innervated the rostral rectum, and half of CTb-IR neurons in the caudal part ran into the caudal rectum. Consequently, the rostral rectum received the densest innervation, and almost all the neurons in the rectal INR exhibited an ascending projection. By double labelling, CTb-IR neurons containing TH or mENK were observed in the rectal INR after the rectal injection. The mENK-IR neurons localized in middle and caudal parts of rectal INR amounted to one third of the total CTb-IR neurons, and mainly projected into the rostral and caudal rectum. TH-and mENK-immunonegative neurons were restricted to the rostral part of rectal INR and the more rostral level.

Spinal Nerve Innervation to the Sonic Muscle and Sonic Motor Nucleus in Red Piranha, Pygocentrus nattereri (Characiformes, Ostariophysi)

The red piranha, Pygocentrus nattereri, produces sounds by rapid contractions of a pair of extrinsic sonic muscles. The detailed innervation pattern of the sonic muscle of the red piranha was investigated. The sonic muscle is innervated by branches (sonic branches) of the third (S3so), fourth (S4so), and fifth (S5so) spinal nerves. The average total number of nerve fibers contained in the right sonic branches (n = 5; standard length, SL, 71–85 mm) was 151.8 (standard deviation, SD, 28.3). The occipital nerve did not innervate the sonic muscle. The sonic motor nucleus (SMN) in the piranha was identified by tracer methods using wheat germ agglutinin-conjugated horseradish peroxidase; labeled sonic motor neurons were only observed on the side ipsilateral to the sonic muscle injected with the tracer. In the transverse sections, the labeled sonic motor neurons were located in the dorsal zone (mainly large and medium neurons) and in the ventral zone (mainly small neurons) of the ventral horn. In the horizontal sections, the labeled neurons formed a rostrocaudally elongated SMN from the level of the caudal part of the second spinal nerve root to the intermediate region between the fifth and sixth spinal nerve roots. The average number of the labeled neurons (n = 5; SL, 64–87 mm) was 152.6 (SD, 7.3). We conclude that the sonic muscles of the piranha are innervated by approximately 300 sonic motor neurons located only in the spinal cord.