Kumiko Yokouchi

Differential neurogenesis and gliogenesis by local and migrating neural stem cells in the olfactory bulb

The rostral migratory stream (RMS) is a unique forebrain structure that provides a long-distance migratory route for the neural stem cells of the periventricular region towards the olfactory bulb (OB). The purpose of the study presented here is to examine the extent of neurogenesis and gliogenesis by the neural stem cells of different origins (periventricular vs. intrabulbar) in the OB. After the RMS had been subjected to injury, the rats received intraperitoneal injections of 5-bromodeoxyuridine (BrdU) and were further reared for 2 weeks. Neuronal and glial differentiations of the BrdU(+) cells in the olfactory bulbar granule cell (OB-GCL) and the olfactory glomerular (OB-GL) layers were examined immunohistochemically using antibodies against neuronal (NeuN, neuronal nuclei) and glial (GFAP, glial fibrillary acidic protein) markers in the OBs with injured and uninjured (control) RMS. In the completely RMS-lesioned OB, where migration of the periventricular neural stem cells was inhibited, a small number of BrdU(+) NeuN(+) cells were found in both the OB-GCL and OB-GL. The BrdU(+) NeuN(+) cells accounted for a much higher percentage of the BrdU(+) cells on the control side (OB-GCL, 36.7%; OB-GL, 8.8%) than on the completely RMS-lesioned side (OB-GCL, 3.7%; OB-GL, 0.6%). The percentage of the BrdU(+) GFAP(+) cells relative to the BrdU(+) cells did not show any major difference between the control and completely RMS-lesioned sides. This study revealed differences in neurogenesis and gliogenesis between the local and migrating neural stem cells in the OB of the adult rodent.

Continual replacement of newly-generated olfactory neurons in adult rats

It has been known that stem cells do exist in the central nervous system, and adult neurogenesis is continually taking place in the olfactory bulb during life. We report here, with the combined method of autoradiography using (3)H-thymidine and immunohistochemistry for a neuronal marker, that 65.3-76.9% of calretinin-immunoreactive bulbar neurons are replaced during the short period of 6 weeks in the adult rodent. The results indicate that neuronal replacement is a common phenomenon in the olfactory bulb during life.

Functionally essential neuronal population of the facial motor nucleus

Cranial nerve impairment is one of the more serious complications in neurosurgery. Nevertheless, the important question of how many neurons are required for cranial nerve functions remains unanswered. The VIIth cranial nerve (facial nerve) in mice was subjected to graded crush injuries. After assessment of the facial function, the number of uninjured, healthy facial motor neurons was quantified with a retrograde neuronal tracer. We report that normal facial function is preserved if intact neurons account for more than 56% of the control value, while complete facial paralysis occurs if intact neurons are reduced to less than 32% of the control value.

Regenerative process of the facial nerve: rate of regeneration of fibers and their bifurcations.

After the main trunk of the mouse facial nerve was injured by crushing, a fiber tracing method was used to quantify the facial motor neurons that extended regenerating nerve fibers to the specific site of the facial nerve branch. The total number of motor neurons retrogradely labeled with a fluorescent tracer, Fluoro-Gold (FG), were 0 on postsurgical days (PSDs) 1 and 2, 75+/-25 on PSD3, 264+/-21 on PSD4, 378+/-19 on PSD6, 428+/-19 on PSD8, 491+/-13 on PSD12 and 532+/-15 on PSD16. Assuming that the FG-positive neurons (535+/-11) of the control mice represent 100%, the FG-labeled neurons accounted for 0, 14, 49, 71, 80, 92 and 99% on the corresponding days. Two different fluorescent tracers were applied to the different facial nerve branches 16 days after facial nerve injuries. Double-labeled neurons were consistently found in the nerve-crushed facial nucleus (3.2%), and their number increased in the nerve-transected facial nucleus (12.2%). The present study indicates that the regenerating facial nerve consists of heterogeneous nerve fibers with varying growth rates and that excessive axonal branching occurs more frequently in the nerve-transected than in the nerve-crushed injuries.

Cell dynamics of calretinin-immunoreactive neurons in the rostral migratory stream after ibotenate-induced lesions in the forebrain

It is now apparent that adult neurogenesis is taking place during life in the olfactory bulb (OB) of the rodent brain. In the olfactory nervous system, the precursor cells of the subventricular zone are known to continually proliferate, migrate through the rostral migratory stream (RMS) and differentiate into the bulbar neurons. The RMS, consisting of heterogeneous cell populations of the neural and neuronal precursor cells, is the unique forebrain structure that provides a long-distance migratory route for the precursor cells. The present study was undertaken to examine whether neuronal regeneration, focusing on calretinin-immunoreactive (+) cells, may proceed in the RMS following lesions induced by an excitotoxin. Two days after ibotenate injections, massive degeneration of calretinin (+) cells occurred in the RMS and its adjacent forebrains. Thereafter, calretinin (+) cells gradually increased in the RMS and reached above their control value 2 weeks after ibotenate injections. Removal of the OB also produced a marked increase in calretinin (+) cells in the RMS. Autoradiographic experiments using (3)H-thymidine showed that calretinin (+) cells were continually generated in the RMS and underwent neuronal turnover within 8 weeks in a normal condition. The results indicate that, in terms of calretinin (+) cells, neuronal differentiation and replacement is continually taking place within the RMS, and that the RMS is capable of repopulating those cells which were injured by ibotenate.

Calretinin-immunoreactive neurons in rostral migratory stream: neuronal differentiation.

The purpose of the present study was to examine immunohistochemically the expression of nestin and polysialylated neural cell adhesion molecule in calretinin-immunoreactive neurons of the rostral migratory stream, the restricted pathway through which neuronal precursor cells migrate towards the olfactory bulb. Using mirror sectioning, calretinin-immunoreactive neurons of the rostral migratory stream were shown to co-express nestin (20.8%) and polysialylated neural cell adhesion molecule (61.8%). The results show that calretinin-immunoreactive neurons in the rostral migratory stream still express immature neural and neuronal phenotypes.

Neurotrophic effect of hepatocyte growth factor on neonatal facial motor neurons.

Abstract The neurotrophic effect of hepatocyte growth factor (HGF) on axotomized facial motor neurons was examined after local application of HGF to the proximal facial nerve stump of the neonatal rat on post-natal day one (P1). Motor neuron survival was expressed as the neuronal cell count on the injured side as a percentage of that on the noninjured side. Motor neuron survival of the control group was 76% on P3, 54% on P5 and 23% on P8, that of the HGF-treated group 78% on P3, 69% on P5 and 31% on P8, and that of the brain-derived neurotrophic factor (BDNF)-treated group 91% on P5 and 45% on P8. The motor neuron survival rates were then adjusted by deducting the facial motor neurons corresponding to the uninjured retroauricular branch (20%) of the facial nerve. The adjusted values were 70% (P3), 42% (P5) and 4% (P8) for the control group, 72% (P3), 61% (P5) and 14% (P8) for the HGF-treated group, and 88% (P5) and 32% (P8) for the BDNF-treated group. These findings demonstrate that HGF has a neuroprotective effect on injured facial motor neurons and suggest that HGF has neurotrophic properties distinct from those of BDNF.

Effects of hypoglossal and facial nerve injuries on milk-suckling.

Functional roles of the perioral anatomical structures involved in breastfeeding were examined in newborn rat pups in which the hypoglossal (XII) and facial (VII) nerves had been resected at the neonatal stage. The XII nerve controls tongue movement and is comprised of two functionally distinct branches: the medial branch related to protrusion of the tongue and the lateral branch related to its retraction. Newborn rat pups with bilateral resection of either of the XII nerve components (main trunk: XII‐trunk; medial branch: XII‐med; lateral branch: XII‐lat) failed to suckle milk and did not survive. Unilateral XII nerve‐resected neonates showed different milk‐suckling capabilities, which thus resulted in differences in survival rate (XII‐trunk: 38%; XII‐med: 24%; XII‐lat: 92%) and postnatal growth during the postnatal 3 weeks until P21. Unilateral and bilateral resections of the VII nerve innervating the buccolabial musculature produced lowered suckling capabilities and retarded postnatal growth, although all pups showed 100% survival. The results indicate a crucial role of the tongue, especially of protruding muscular elements innervated by the XII‐med nerve, in breastfeeding. The results also indicate differential effects of the VII and XII nerve components on suckling capability, survival, and postnatal growth of newborn rat pups.

Determination of functionally essential neuronal population of the olfactory epithelium for nipple search and subsequent suckling behavior in newborn rats.

Neuronal population of the olfactory epithelium required for nipple search and subsequent suckling behavior was examined in newborn rats. After unilateral ablation of the olfactory bulb, different concentrations of ZnSO(4) were contralaterally injected into the nasal cavity to produce varying degrees of neuronal degeneration in the olfactory epithelium. The ZnSO(4)-treatment resulted in two populations of pups. One exhibited suckling while the other did not, and intact olfactory receptor neurons were quantified immunohistochemically using an antibody for olfactory marker protein (OMP), a marker protein for olfactory receptor neurons. The total numbers of the OMP (+) cells in the ZnSO(4)-treated pups with suckling capability ranged between 2457 and 4615, whereas those in the ZnSO(4)-treated pups without suckling capability ranged between 112 and 2398. With the mean value (4969) of the total numbers of the OMP (+) cells of the normal/control pups assumed to represent 100%, the total numbers of the OMP (+) cells accounted for 49-93% in the suckling (+) group and 2-48% in the suckling (-) group. From these findings, we conclude that approximately 50% of neuronal population of the olfactory epithelium is a critical value to distinguish between the two groups with and without suckling capability in the unilateral olfactory system of newborn rats.

Tyrosine hydroxylase-immunoreactive fibers in the human vagus nerve

Sympathetic catecholaminergic fibers in the vagus nerve were immunohistochemically examined in formalin-fixed human cadavers using an antibody against the noradrenalin-synthetic enzyme tyrosine hydroxylase (TH). TH-positive fibers were extensively distributed in the vagal nerve components, including the superior and inferior ganglia, the main trunk and the branches (superior and recurrent laryngeal, superior and inferior cardiac, and pulmonary branches). The inferior ganglion and its continuous cervical main trunk contained numerous TH-positive fibers with focal or diffuse distribution patterns in each nerve bundle. From these findings, we conclude that sympathetic fibers are consistently included in the human vagus nerve, a main source of parasympathetic preganglionic fibers to the cervical, thoracic and abdominal visceral organs.