Stereological quantification of olfactory receptor neurons in mice.

The total number of olfactory receptor neurons (ORNs) in the mouse main olfactory epithelium (MOE) was estimated using stereological sampling. Noses and skulls of male and female 8-week-old C57BL/6J mice were de-calcified, embedded in paraffin, cut into 10-µm-thick sections serially at 100-µm intervals, and processed for immunohistochemistry for the olfactory marker protein (OMP), a specific marker for ORNs. The number of OMP (+) receptor neurons was measured using an optical fractionator with the Stereo-Investigator software. The mean values of the total number of OMP (+) receptor neurons in the unilateral MOE were 5,140,000±380,000 in males and 5,210,000±380,000 in females, with no significant differences between the sexes. We concluded that the total number of ORNs in the unilateral MOE is approximately 5×10(6) in mice.

A transverse ligament located anterosuperiorly in the lower orbital fat space restricts lower eyelid retraction in the Mongoloid eye.

We previously reported that a lower positioned transverse ligament, which was identified just above the fusional line between the orbital septum and the levator aponeurosis or anteroinferiorly in the upper orbital fat space, determines the lower position of the preaponeurotic fat, and restricts the upper eyelid retraction causing the external features that define the appearance of the Mongoloid eye. Because the narrow palpebral fissure of the Mongoloid eye is composed of the restricted upper eyelid and the restricted lower eyelid, we surmised that the ligamentous tissue, which is analogous to the lower positioned transverse ligament in the upper eyelid, might also exist in the lower eyelid.

Differential effects of hypoglossal and facial nerve injuries on survival and growth of rats at different developmental stages.

The hypoglossal (XII) nerve is made up of functionally different nerve branches: the medial branch related to protrusion of the tongue and the lateral branch related to its retraction. The present study was performed to determine the effects of facial (VII) and XII nerve injuries on the survival and growth of rats in which the unilateral or bilateral VII and XII nerve components (main trunk, XII-trunk; medial branch, XII-med; lateral branch, XII-lat) had been resected at different developmental stages. In the suckling period, unilateral as well as bilateral injuries in the XII-trunk or XII-med nerve produced disturbed milk intake, lower survival rates and growth retardation in the nerve-injured rats. In the transition and mastication periods, only bilateral injury in the XII-trunk or XII-med nerve produced disturbed food intake followed by lower survival rates and growth retardation in those animals. The unilateral XII-lat nerve injury did not have significant effects on milk and food intake, whereas the bilateral injury caused disturbance in milk intake especially at the early neonatal stage. The unilateral VII nerve injury at the early neonatal stage caused deteriorating effects on food intake resulting in lower survival rate and severe growth retardation in the nerve-injured rats. The results indicate that the survival and growth of XII and VII nerve-resected rats differ considerably depending on the nerves injured and the developmental ages of the animals at the time of nerve insult.

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.

Effects of bilateral resection of facial nerves on suckling in developing rats.

The purpose of the present study is to investigate functional role of the facial nerve on suckling in developing rats. The bilateral resection of facial nerves on postnatal day 1 (P1) resulted in cell loss of facial motoneurons and complete facial paralysis without any whisker movement or nictitating reflex at the end of the postnatal 3 weeks. Although the body weight of the nerve-resected rats increased gradually for the postnatal 3 weeks, they weighed less than the control rats without nerve resection. The nerve-resected rats contained less milk (0.25 +/- 0.02 g) than the control rats (0.35 +/- 0.02 g) in the stomach on P17. On P21, the body weight of the nerve-resected rats (25.33 +/- 0.32 g) was decreased by 28% compared to that of the control rats (35.08 +/- 0.57 g). Although their growth was substantially more retarded than that of the control animals, most (92%) of the nerve-resected pups could survive without facial nerve innervation. The orofacial musculature innervated by the facial nerve plays an important role in breastfeeding, but the present study shows that these muscles are not essential for the survival of neonatal rats.

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

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.

Great potentiality of neonatal facial motor neurons for neural plasticity as determined by functionally essential neuronal population.

The present study was undertaken to determine the neuronal population essential for normal and minimal facial function of young adult rats that had received various degrees of crush injuries to the facial nerve in the neonatal period. Using a neuronal tracer, it was found in young adult rats receiving neonatal injuries that the minimum number of tracer-labeled facial motor neurons necessary for normal facial function corresponded to 13-14% of the neurons (2540+/-64) of the age-matched control animals, whereas the minimum number of neurons necessary for minimal facial function corresponded to 5%. On the other hand, the minimum numbers of tracer-labeled facial motor neurons necessary for normal and minimal facial function of young adult rats that received various degrees of crush injuries corresponded to 61 and 27-30%, respectively, of the neurons (2540+/-64) of the uninjured control animals. These results indicate that the facial function of animals with nerves crushed at the neonatal stage can be adequately maintained by a very small population of neurons, implying a great potential of neonatal neurons for neural plasticity.

Proliferating cell populations in experimentally-induced hydrocephalus in developing rats.

To examine the fate of proliferating brain cells in hydrocephalus (Hydro), experimental Hydro was induced in neonatal rats by intracisternal injection of kaolin and, 3 weeks later, the rats were injected with bromodeoxyuridine (BrdU). The BrdU (+) cells were immunohistochemically analyzed by using antibodies against neural (nestin), neuronal (NeuN) and glial (GFAP and MBP) markers in the posterior cerebrum. The percentage of nestin expression for the BrdU (+) cells was 8% in control and increased from 17% in the Hydro to 33% in the Hydro at an earlier stage after the shunt procedure, but was restored to 6% in the Hydro at a later stage after the shunt procedure. The percentages of GFAP expression showed a similar tendency to those of nestin expression. The BrdU (+) cells did not express either NeuN or MBP throughout the experiments.

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.

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.

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.

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.

The minimum number of neurons in the central olfactory pathway in relation to its function: a retrograde fiber tracing study.

The present study was aimed at determining the functionally essential size of the neuronal population in the central olfactory nervous system. Using conditioned rats who had learnt to avoid repellent (cycloheximide) solution by olfaction, varying degrees of injuries were made to the lateral olfactory tract, a major central olfactory pathway connecting the olfactory bulb to the olfactory cortex. After examining their olfactory ability to discriminate cycloheximide solution from water, intact bulbar projection neurons (mitral cells) with fiber connections to the olfactory cortex were quantified using a retrograde fiber tracing technique. The numbers of retrogradely labeled mitral cells from the rats with normal olfaction ranged between 20 and 92% of the control value, while those numbers from the anosmic rats ranged between 0 and 22%. We conclude that the functionally essential neuronal population is approximately one-fifth of the total in the central olfactory pathway, a presumed threshold value in terms of the ability to avoid cycloheximide solution by olfactory discrimination.

Stimulation of the regrowth of MPP+-damaged dopaminergic fibers by the treatment of mesencephalic cultures with basigin.

Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily and widely expressed in the central nervous system. To elucidate functional role of Bsg in the central nervous system, the effects of its glutathione-S-transferase (GST) fusion protein on the number and neurite outgrowth of cultured rat mesencephalic dopaminergic neurons were measured. The fusion protein was not able to promote the survival and neurite outgrowth of tyrosine hydroxylase (TH)-positive neurons under serum-free condition. However, the treatment of 1-methyl-4-phenylpyridinium (MPP+)-exposed cultures with the fusion protein resulted in stimulation of the regrowth of damaged TH-positive fibers. Basic fibroblast growth factor (bFGF) also stimulated the regrowth of neurites in damaged neurons. These results indicate that Bsg may play an important role in the regrowth of damaged dopaminergic fibers.

Onset of calbindin-D 28K and parvalbumin expression in the lateral geniculate complex and olivary pretectal nucleus during postnatal development of the rat.

The onset and distribution of calbindin (CB) and parvalbumin (PV) immunoreactivity were investigated in the lateral geniculate nuclear complex and the olivary pretectal nucleus (OPT) in developing rats. CB expression occurred early (before eye-opening) in the relay neurons of the intergeniculate leaflet, parvocellular portion of the ventral lateral geniculate nucleus and OPT relating to ambient vision mediated by W-like retinal ganglion cells. On the contrary, PV expression occurred late (after eye-opening) in the relay neurons of the magnocellular portion of the ventral lateral geniculate nucleus (VLGMC) and OPT relating to focal vision mediated by Y-like retinal ganglion cells. A unilateral eye enucleating experiment indicated that the VLGMC and OPT received dense input from PV-positive Y-like retinal ganglion cells. The results show the different onsets of CB and PV expressions in the retino-recipient thalamic and pretectal nuclei receiving inputs from different kinds of retinal ganglion cells.

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.

Plume-tracking robots: a new application of chemical sensors.

Many animals have the ability to search for odor sources by tracking their plumes. Some of the key features of this search behavior have been successfully transferred to robot platforms, although the capabilities of animals are still beyond the current level of sensor technologies. The examples described in this paper are (1) incorporating into a wheeled robot the upwind surges and casting used by moths in tracking pheromone plumes, (2) extracting useful information from the response patterns of a chemical sensor array patterned after the spatially distributed chemoreceptors of some animals, and (3) mimicking the fanning behavior of silkworm moths to enhance the reception of chemical signals by drawing molecules from one direction. The achievements so far and current efforts are reviewed to illustrate the steps to be taken toward future development of this technology.

An anatomic examination of the suprascapular flap.

To investigate the potential utility of a suprascapular flap, the authors examined dissected cadavers to identify the ramification of the suprascapular artery, and directly injected 2.5% patent blue dye to observe the distribution of its cutaneous perforators. Results indicate that a suprascapular flap is not practicable. The branching pattern of the suprascapular artery from the subclavian artery varies widely, making dissection of the vessels highly problematic. Moreover, no constant presence of cutaneous perforators from the artery could be identified in either the suprascapular fossa or the shoulder.

Fate of newly formed periglomerular cells in the olfactory bulb.

It is well established that olfactory receptor cells are replaced during life. Periglomerular (PG) cells of the olfactory bulb have recently been demonstrated to be produced following proliferation and migration of periventricular neuronal precursor cells even in adulthood. The purpose of the present study was to examine the fate of newly formed PG cells in adult rodents. Using 5-bromodeoxyuridine (BrdU), we carried out a quantitative immunohistochemical analysis of BrdU-positive cells in the bulbar glomerular layer at different survival periods. Each number of BrdU-positive PG cells per 100 olfactory glomeruli was 34.1 +/- 3.3 (1 week), 57.2 +/- 2.7 (2 weeks), 28.0 +/- 4.7 (4 weeks) and 25.9 +/- 1.6 (8 weeks). These results indicate that bulbar PG cells, similar to olfactory receptor cells, are mostly replaced during life, and that the olfactory system is composed of disposable neuronal networks centrally as well as peripherally.

A new video/computer method to measure the amount of overall movement in experimental animals (two-dimensional object-difference method).

Evaluation of the amount of overall animal movement is important for investigations of motor control mechanisms in the central nervous system. We describe a new method to quantify overall free movements of an animal without any markers using a video camera and a personal computer equipped with a video-capture board. The operating principle is that the amount of overall movement of an object can be expressed by the difference in total area occupied by the object in two consecutive picture frames. The software for this application operates in real-time. Using this method and with proper setting for the cage and recording view, we can estimate three-dimensional movements of animals. The major advantages are low cost, easy operation and high sensitivity. The experimental results indicate that this method can be applied to various fields of motion analysis.