Otolith endorgan input to the Mauthner neuron in the goldfish

The Mauthner (M-) cell of the goldfish, Carassius auratus, triggers the rapid escape response of the fish in response to various stimuli, including visual and auditory. The large size and accessibility of the M-cell make it an ideal model system for the study of synaptic transmission, membrane properties, and sensory-motor gating. Although physiological recordings have suggested that afferents from all three of the inner ear endorgans (the saccule, lagena, and utricle) synapse directly on the ipsilateral M-cell, the specific contacts and anatomical distributions of these inputs along the M-cell lateral dendrite remain unknown. We traced specific branches of the auditory (VIIIth) nerve from the three otolith organs of the fish inner ear to the M-cell. The goldfish sacculus gives rise to the vast majority of inputs that contact a large portion of the M-cell lateral dendrite, and these inputs vary greatly in size. In contrast to the ubiquitous distribution of saccular inputs, those from the lagena are segregated to distal regions of the M-cell and synapse on the distal dorsal branch of the lateral dendrite. Similarly, inputs from the utricle are also segregated to distal regions, synapsing on the ventral branch of the lateral dendrite. These results demonstrate that nerves from all three endorgans contact the M-cell, with input-specific segregation of synapses along the M-cell lateral dendrite.     

Quantitative analyses of postembryonic hair cell addition in the otolithic endorgans of the inner ear of the european hake,merluccius merluccius (gadiformes,teleostei)

Bony fishes add sensory hair cells to the saccule and lagena of the ear for at least several years after hatching. However, it is not known whether hair cell proliferation occurs for the whole lifetime of an animal, whether proliferation occurs in all endorgans of the ear, or whether the rate of proliferation is the same in all of the endorgans. To obtain answers to these questions, the extent of postembryonic hair-cell proliferation was determined in the saccule, lagena, and utricle of the ear in the European hake, Merluccius merlaccius, for fish ranging from 7 to 75 cm in total length (6 months to 9 years of age). Results demonstrated that hair-cell addition continued throughout this period in all three otic endorgans, although endorgan size was proportionally greatest in smaller animals. Of the three endorgans, cell addition was greatest in the saccule. Moreover, far more cells were added to the caudal end of the saccule than to the rostral end. Each saccule of the largest hake had over 900,000 hair cells. It is estimated that each saccule adds approximately 110,000 new hair cells each year (or 302 cells/day) over the life span of the fish studied. A significant number of small ciliary bundles, thought to represent newly proliferated hair cells, was found throughout each endorgan, and the number of such bundles declined as the rate of hair cell proliferation decreased. The results demonstrate that extensive proliferation occurs in all three otolithic endorgans of the ears in a fish and that such proliferation continues for virtually the whole life of the animal. The functional significance of this addition is not known. © 1994 Wiley-Liss, Inc.     

The inner ear of the lungfish Protopterus

The sensory end organs of the inner ear of the lungfish, Protopterus, were examined using scanning and transmission electron microscopy. The utricle has a structure and hair cell orientation pattern that are typical for vertebrates, although the hair cells are unusually large. There are the typical three semicircular canals extending from the utricle, with the typical hair cell orientations, but the lateral canal sensory crista looks like the "hemicrista" of some amphibians and amniotes, lacking a saddle-shaped flare on one wall of the ampulla. Unlike most vertebrates that have the saccule and lagena as two separate pouches ventral to the utricle, the lungfish has a single large ventral pouch that contains a single large pasty otoconial mass. This mass covers two hair cell patches, each like a striola with prominent hair cell ciliary bundles, that are presumed to represent saccular and lagenar maculae. However, these two major sensory patches are not completely separate maculae because they lie within a less densely populated field of smaller hair cells, which forms an extrastriolar region that surrounds and fills the region between the two striolae of higher hair cell density. The more caudal lagenar striola is a vertically elongated stripe with hair cell orientation vectors facing antiparallel on either side of a midline drawn vertically along the macula, resembling the macula lagena of some bony fishes but not of tetrapods. The more rostral saccular striola is a curving band with hair cell orientation vectors facing away from its midline, but because this macula curves in three dimensions, the vectors at the rostral end of this striola are oriented mediolaterally, whereas the vectors on the caudal half of this striola are oriented dorsoventrally. The presence of a macula neglecta was confirmed near the posterior canal as a tiny single patch of a few dozen hair cells with all the cell orientations directed caudally. The ciliary bundles on the cells in the striolar-like regions of all of three otolithic organs average over 80 cilia, a number far greater than for any other fish studied to date. The features of the single sacculolagenar pouch with separate striolar-like regions, the cellular orientation in the otolith organs, and the large cells and ciliary bundles in Protopterus also were observed in specimens of the other extant lungfish genera, Lepidosiren and Neoceratodus.     

Organization of the efferent vestibular nuclei and nerves of the toadfish, Opsanus tau

The efferent vestibular nuclei and nerves were studied in the toadfish, Opsanus tau, with morphological and electrophysiological techniques. The origin and course of the efferent vestibular nerves was extensively documented. One major morphological observation was that the efferent nerves comprise a peripheral network that is anatomically distinct, and separable by dissection from the primary afferents innervated by each end organ. These anatomically distinct nerves are likely to be a major asset in physiological studies of efferent vestibular function. The retrograde transport of horseradish peroxidase (HRP) from each of the nerves innervating the vestibular and lateral line organs was used to delineate the subgroups of efferent neurons projecting to these end organs. The efferent vestibular nuclei are located in the posterior medulla in and around the median longitudinal fasciculi (MLF). We divided the nuclei cytoarchitecturally into lateral, medial, and dorsal subdivisions. The lateral cells had bilateral dendritic trees while the dorsal cells had ipsilateral, unilateral dendritic trees. There was a higher proportion of lateral cells that innervated the canal organs and the utricle while the dorsal cells tended to innervate the other organs. The total number of cells obtained by summing those from separate nerve label was twice the total cell count present in the nuclei. Indirectly, this indicates that some cells project to more than one end organ. Efferent neurons were penetrated with glass microelectrodes, and their end organs and patterns of connectivity with other end organs were investigated by stimulating various vestibular nerves. Posterior semicircular canal efferent cells are electrically coupled to each other and could be activated electrically or chemically by stimulating other ipsilateral or contralateral vestibular nerves. It is suggested that electrical coupling might be responsible for the uniform behavior of these cells under certain conditions. Morphological and physiological experiments suggested that the semicircular canals are innervated by their own, exclusive populations of efferent neurons while other end organs may share efferent innervation. Single cells were injected intracellularly with HRP and their morphology was studied and characterized by light microscopy. Intracellular label confirmed the morphological features demonstrated by retrograde transport of HRP and also revealed that some cells had central axon collaterals that terminated within the MLF. These morphological and physiological results provide a basis for understanding the behavior of efferent vestibular neurons in the alert animal.     

Spatial and temporal response properties of secondary neurons that receive convergent input in vestibular nuclei of alert cats

Responses to natural stimulation were studied in electrically identified secondary vestibular neurons of awake cats. A class of neurons was identified whose response dynamics and responses to rotations in several vertical and horizontal planes indicated that they received semicircular canal input. Each canal neuron had clearly defined planes of maximal and null sensitivity to rotation. The orientation of these planes indicated that 44% of the neurons received input from one pair of canals, 40% from two, and 16% from all 3 canal pairs. Many cells also had oculomotor-related discharges and/or responded weakly to neck rotation.     

Gamma-aminobutyric acid is present in a spatially discrete subpopulation of hair cells in the crista ampullaris of the toadfish Opsanus tau

Although gamma-aminobutyric acid (GABA) and glutamate are known to be present in the vestibular sensory epithelia of a variety of species, the functional relationship between these two transmitters is not clear. The present study addresses the three-dimensional spatial distribution of GABA and glutamate immunoreactivity in the vestibular labyrinth of the oyster toadfish by using whole end organs labeled by immunofluorescence with monoclonal anti-GABA and/or antiglutamate antibodies and visualized as whole mounts by multiphoton confocal microscopy. We find glutamate-immunoreactive hair cells present throughout the sensory epithelium. In contrast, prominent GABA immunoreactivity is restricted to a small population of hair cells located in the central region of the crista. Double immunofluorescence reveals two distinct staining patterns in GABA-labeled hair cells. Most ( approximately 80%) GABA-labeled cells show trace levels of glutamate, appropriate for the metabolic/synthetic role of cytoplasmic glutamate. The remainder of the GABA-stained cells contain substantial levels of both GABA and glutamate, suggesting transmitter colocalization. In the toadfish utricle, glutamatergic hair cells are present throughout the macula. GABA-immunoreactive hair cells follow the arc of the striola, and most GABA-labeled receptor cells coexpress glutamate. The localization of GABA was explored in other species as well. In the pigeon, GABAergic hair cells are present throughout the crista ampullaris. Our findings demonstrate that multiple, neurochemically distinct types of hair cells are present in vestibular sensory epithelia. These observations, together with the excitatory activity generally associated with 8th nerve afferent fibers, strongly suggest that GABA serves an important, specific, and complex role in determining primary afferent response dynamics.     

Analysis of magnetic elements in otoliths of the macula lagena in homing pigeons with inductively coupled plasma mass spectrometry

Objective

The macula lagena in birds is located at the apical end of the cochlea and contains many tiny otoliths. The macula lagena is innervated and has neural projections to the brainstem, but its physiological function is still unclear. It remains disputable that it is because otoliths in the lagena are rich in elements Fe and Zn that birds can obtain geomagnetic information for homing. To clarify this issue, we carried out a study to determine whether or not otoliths in the lagena of homing pigeons are richer in magnetic elements than those in the saccule and the utricle.

Methods

The contents of ferromagnetic elements (Fe, Co, Ni) and other metal elements in lagenal otoliths of adult homing pigeons were precisely analyzed with inductively coupled plasma mass spectrometry (ICP-MS) of high sensitivity, and then they were compared with those in saccular and utricular otoliths (all the contents were normalized to Ca).

Results

In adult homing pigeons, the contents of ferromagnetic elements (Fe, Co, Ni) in lagenal otoliths were less than 0.7% (normalized to Ca element) and were the same order in magnitude as those in saccular and utricular otoliths. The content of Fe in lagenal otoliths was not significantly different from that in utricular otoliths and was even lower than that in saccular otoliths. The content of Co in lagenal otoliths was lower than that in saccular otoliths and higher than that in utricular otoliths. The content of Ni in lagenal otoliths was not significantly different from that in saccular otoliths and was higher than that in utricular otoliths. The contents of other metal elements Na, Mg, K, Al, Mn and Pb in lagenal otoliths were not significantly different from those in utricular and saccular otoliths. The contents of metal elements Zn, Ba and Cu in lagenal otoliths were lower than those in saccular otoliths.

Conclusion

The contents of magnetic elements in lagenal otoliths of homing pigeons are not much higher than those in utricular and saccular otoliths, which does not support the hypothesis that birds depend on high contents of Fe and Zn in lagenal otoliths for sensation of geomagnetic information. Similarities in morphology, element ingredient and element content between lagenal otoliths and utricular otoliths suggest that the two types of otolithic organs may play similar roles in sensing gravitational and acceleration signals.     

Fos expression in otolith-related brainstem neurons of postnatal rats following off-vertical axis rotation

To determine the critical time of responsiveness of developing otolith organ-related brainstem neurons and their distribution, Fos protein expression in response to off-vertical axis rotations (OVAR) was mapped in conscious Sprague Dawley rats from P5 to adulthood. OVAR was used to activate sequentially all utricular hair cells per 360 degrees revolution. We detected the coding of horizontal head positions in otolith organ-related neurons within the vestibular nucleus as early as P7. In the vestibular nuclear complex and its subgroups, the density of Fos-immunoreactive (Fos-ir) neurons increased steadily with age and reached the adult level by P21. In both labyrinthectomized rats subjected to OVAR and normal rats kept stationary, labeled neurons were found sporadically in the aforementioned brain regions in each age group, confirming that Fos labeling observed in neurons of normal experimental rats subjected to OVAR was due to otolith organ stimulation. Whereas OVAR-induced Fos-ir neurons were also first observed in vestibular-related brain areas, such as the prepositus hypoglossal nucleus, gigantocellular reticular nucleus, and locus coeruleus, of normal experimental rats at P7, those in the inferior olive were observed only from P14 onward. This indicates the unique maturation time of inferior olivary neurons in gravity-related spatial coding. In general, age-dependent increase in OVAR-induced Fos-ir neurons was observed in brain areas that received otolith inputs. The locus coeruleus was exceptional in that prominent OVAR-induced Fos-ir neuronal number did not change with maturation, and this was well above the low but significant number of Fos-ir neurons in control preparations. Taken together, our results suggest that neuronal subpopulations within the developing network of the horizontal otolith system provide an anatomical basis for the postnatal development of otolith organ-related sensorimotor functions. J. Comp. Neurol. 470:282-296, 2004.     

Morphologic hippocampal asymmetry in male and female rats

Our findings indicate that the hippocampus of the rat varies in structure in the right and left hemispheres and in males and females. A morphologic study of the thickness of the dorsal hippocampus was carried out on celloidin-embedded histologic sections from male, Long-Evans rats of the following ages: 6, 10, 14, 26, 41, 55, 77, 90, 108, 185, 300, 400, and 650 days. Between 15 and 25 rats per age group were used, a total of 225 male rats. In addition, the hippocampi were measured on 47 Long-Evans female, 90-day-old rats: 11 normal and 36 animals ovariectomized one day after birth. We found that in males the right hippocampus was significantly thicker than the left for some but not all age groups. In the female at 90 days of age, the left hippocampus was thicker than the right. This left-right pattern in the female hippocampus did not reverse, as it did in the cerebral cortex after ovariectomy at birth.     

Selective vulnerability of hippocampal CA3 neurons to hypoxia after mild concussion in the rat

Immunohistochemical staining for microtubule-associated protein 2 (MAP2) and synaptophysin was used to investigate the effect of hypoxia on hippocampal neurons after mild concussion in the rat. Male Sprague-Dawley rats were divided into four groups: Group 1 (n = 3) was subjected to a mild impact-acceleration closed head injury group 2 (n = 3) was subjected to 30 min of moderate hypoxia, group 3 (n = 5) was subjected to head trauma followed by 30 min of moderate hypoxiaand group 4 (n = 3) comprised sham-operated controls. All rats were fixed by transcardial perfusion 24 h after insult. No damage was observed in CA1 or CA2 neurons in any of the rats. However, rats in group 3 showed selective damage of hippocampal CA3 neurons manifested by a pycnosis and a marked decrease in MAP2 immunoreactivity. Presynaptic terminals visualized by synaptophysin immunostaining showed no differences among groups. The loss of immunoreactivity for the post-synaptic somal and dendritic protein marker MAP2 from the CA3 subfield 24 h after combined insults indicates an increased vulnerability of pyramidal cells in this brain area. [Neurol Res 1995; 17: 455-460]     

Differential vulnerability of male and female rats to the timing of various perinatal insults

The results of five experiments showed that exposure to diazepam, hypoxia and monosodium glutamate during the prenatal or early postnatal period of rapid brain development may result in different behavioral consequences depending on the timing of the exposure rather than the nature of the agent. Moreover, male and female offspring may be affected differently by the same agent at different periods of development. Prenatal insults of various kinds impair the later performance of males but not the females in a complex learning task, while postnatal insults seem to affect detrimentally this same behavior in both males and females. The effects of perinatal insults on maze learning and open field activity do not lend themselves to explanation by "feminization" or "masculinization" of behavior caused by interference with prenatal gonadal hormones.     

Maturation of canal‐related brainstem neurons in the detection of horizontal angular acceleration in rats

We examined the functional maturation of canal‐related brainstem neurons in Sprague‐Dawley rats at postnatal day (P)1 to adult. Conscious animals were subjected to cycles of angular acceleration and deceleration so as to selectively activate hair cells of the horizontal semicircular canals. Brainstem neurons were monitored for c‐fos expression by immuno‐hybridization histochemistry as an indicator of neuronal activation. Fos‐immunoreactive canal‐related neurons were identifiable from P4 onwards in the vestibular nucleus and downstream vestibular relay stations, prepositus hypoglossal nucleus, and inferior olive. In the vestibular nucleus and prepositus hypoglossal nucleus, the number of canal‐related neurons increased progressively with age, reaching the adult level by P21. Those in the inferior olive increased in number from P4 to P14 but decreased significantly afterwards until adulthood. The topography was not clear in the vestibular nucleus and prepositus hypoglossal nucleus. Canal‐related neurons in P4–7 rats were spread throughout the rostrocaudal length of each subnucleus but clusters of canal‐related neurons tended to form within specific subnuclei by P21. These were concentrated in the caudal halves of medial and spinal vestibular nuclei and the rostral parts of superior vestibular nucleus and prepositus hypoglossal nucleus. In the inferior olive, the topography was evident early in the course of development. Canal‐related neurons were exclusively located in four subnuclei: dorsal medial cell column, dorsal cap, subnucleus A, and subnucleus C, but not in other subnuclei. Taken together, our data revealed the developmental profile of neuronal subpopulations within the horizontal canal system, thus providing an internal neural representation for postnatal coding of horizontal head rotations in spatial perception. J. Comp. Neurol. 518:1742–1763, 2010. © 2009 Wiley‐Liss, Inc.     

Quantitative study of the coexpression of Fos and N-methyl-D aspartate (NMDA) receptor subunits in otolith-related vestibular nuclear neurons of rats

The expression of NMDA receptor subunits (NR1 and NR2A/B) was demonstrated immunocytochemically in otolith-related neurons within the vestibular nuclear complex and its subnuclei of conscious Sprague-Dawley adult rats. All experimental animals were subjected to constant velocity off-vertical axis rotation (OVAR). The rotating gravity vector during OVAR sequentially activates hair cells on all sectors of the utricular maculae; neurons so activated within the vestibular nuclei were denoted by the expression of Fos protein. Control animals, i.e., labyrinthectomized rats subjected to OVAR and normal rats that remained stationary, showed only a few sporadically scattered labeled neurons. In the brainstem of normal rats subjected to OVAR, a high density of Fos-immunoreactive (Fos-ir) neurons was found in the vestibular nuclear complex (namely, spinal vestibular nucleus, SpVe; medial vestibular nucleus, Mve; superior vestibular nucleus, SuVe) and subnuclei (namely, group x and group y), whereas a lower density was found in the lateral vestibular nucleus (LVe). A double-immunofluorescence study indicated that both NR1 and NR2A/B subunits were highly expressed in Fos-ir neurons within the vestibular nuclei. Fos/NR1 or Fos/NR2A/B double-labeled neurons constitute over three-quarters of the total number of Fos-ir neurons in SpVe, MVe, LVe, SuVe, and groups x and y. Our findings suggest that NMDA-type ionotropic glutamate receptors play a key role in the OVAR-induced neuronal activation of the vestibular nuclei, thus providing a morphological basis for further study of glutamatergic central otolith neurons and their involvement in sensorimotor regulation and autonomic functions of rats.     

Duration of neurofibrillary changes in the hippocampal pyramidal neurons

The total number of neurons with and without neurofibrillary changes in sectors CA1 to CA4, subiculum, and dentate gyrus of 16 subjects with Alzheimer disease (AD) was estimated. The duration of neurofibrillary changes was calculated on the basis of regressions between the duration of AD and neuronal numbers. In the CA1 and subiculum, it takes 3.4 and 5.4 years, respectively, for an intact neuron affected by neurofibrillary pathology to become a ghost tangle.     

Immunolocalization of BK channels in hippocampal pyramidal neurons

Neurons are highly specialized cells in which the integration and processing of electrical signals critically depends on the precise localization of ion channels. For large-conductance Ca(2+)- activated K(+) (BK) channels, targeting to presynaptic membranes in hippocampal pyramidal cells was reported; however, functional evidence also suggests a somatodendritic localization. Therefore we re-examined the subcellular distribution of BK channels in mouse hippocampus using a panel of independent antibodies in a combined approach of conventional immunocytochemistry on cultured neurons, pre- and postembedding electron microscopy and immunoprecipitation. In cultured murine hippocampal neurons, the colocalization of BK channels with both pre- and postsynaptic marker proteins was observed. Electron microscopy confirmed targeting of BK channels to axonal as well as dendritic membranes of glutamatergic synapses in hippocampus. A postsynaptic localization of BK channels was also supported by the finding that the channel coimmunoprecipitated with PSD95, a protein solely expressed in the postsynaptic compartment. These results thus demonstrate that BK channels reside in both post- and presynaptic compartments of hippocampal pyramidal neurons.     

Neonatal exposure to monosodium L-glutamate induces loss of neurons and cytoarchitectural alterations in hippocampal CA1 pyramidal neurons of adult rats

Glutamatergic post-synaptic receptors are closely related to the known excitotoxic effects of high doses of L-glutamate. Several behavioral abnormalities, glial reaction, and an increase of expression of the NMDA receptor sub-units have been observed in the rat hippocampus after early monosodium glutamate exposure. Thus, a quantitative morphological study was carried out to determine the effects of early exposure to monosodium glutamate on post-synaptic structures that mediate glutamate excitatory neurotransmission in the hippocampal CA1 field. Four milligrams per gram body weight of monosodium glutamate was subcutaneously injected into neonatal Wistar rats, at 1, 3, 5, and 7 days. Cell loss and several cytoarchitectonic parameters were evaluated in pyramidal cells from the hippocampal CA1 field in the treated rats at 60 days of age. An untreated group of rats were used as controls. Cell number in the hippocampus of experimental rats was 11.5% less than that in control animals. In addition, both dendritic arborization and dendritic spine density were adversely affected, and thin and mushroom-shaped spines became proportionally more numerous, while the opposite occurred to stubby spines. These results strongly suggest the occurrence of cell death and also show some cytoarchitectural modifications in the surviving neurons. These could lead to functional alterations in the hippocampal integrative activity, due to an early cytoexcitotoxic effect of monosodium glutamate.     

Vestibular efferent fibers to ampulla of anterior, lateral and posterior semicircular canals in cats

Origins of vestibular efferent fibers to ampulla of semicircular canals in cats were examined using retrograde transport of horseradish peroxidase. The anterior canal was innervated from bilateral parvocellular reticular nucleus (PCRN), contralateral gigantocellular reticular nucleus and ipsilateral lateral reticular nucleus (LRN); the lateral canal, from ipsilateral PCRN and LRN as well as ipsilateral lateral vestibular nucleus; and the posterior canal, from bilateral PCRN and ipsilateral medial and lateral vestibular nuclei.