Distribution and Structure of Synapses on Medial Vestibular Nuclear Neurons Targeted by Cerebellar Flocculus Purkinje Cells and Vestibular Nerve in Mice: Light and Electron Microscopy Studies.

Adaptations of vestibulo-ocular and optokinetic response eye movements have been studied as an experimental model of cerebellum-dependent motor learning. Several previous physiological and pharmacological studies have consistently suggested that the cerebellar flocculus (FL) Purkinje cells (P-cells) and the medial vestibular nucleus (MVN) neurons targeted by FL (FL-targeted MVN neurons) may respectively maintain the memory traces of short- and long-term adaptation. To study the basic structures of the FL-MVN synapses by light microscopy (LM) and electron microscopy (EM), we injected green florescence protein (GFP)-expressing lentivirus into FL to anterogradely label the FL P-cell axons in C57BL/6J mice. The FL P-cell axonal boutons were distributed in the magnocellular MVN and in the border region of parvocellular MVN and prepositus hypoglossi (PrH). In the magnocellular MVN, the FL-P cell axons mainly terminated on somata and proximal dendrites. On the other hand, in the parvocellular MVN/PrH, the FL P-cell axonal synaptic boutons mainly terminated on the relatively small-diameter (< 1 µm) distal dendrites of MVN neurons, forming symmetrical synapses. The majority of such parvocellular MVN/PrH neurons were determined to be glutamatergic by immunocytochemistry and in-situ hybridization of GFP expressing transgenic mice. To further examine the spatial relationship between the synapses of FL P-cells and those of vestibular nerve on the neurons of the parvocellular MVN/PrH, we added injections of biotinylated dextran amine into the semicircular canal and anterogradely labeled vestibular nerve axons in some mice. The MVN dendrites receiving the FL P-cell axonal synaptic boutons often closely apposed vestibular nerve synaptic boutons in both LM and EM studies. Such a partial overlap of synaptic boutons of FL P-cell axons with those of vestibular nerve axons in the distal dendrites of MVN neurons suggests that inhibitory synapses of FL P-cells may influence the function of neighboring excitatory synapses of vestibular nerve in the parvocellular MVN/PrH neurons.

Sex differences in morphometric aspects of the peripheral nerves and related diseases.

BACKGROUND: The elucidation of the relationship between the morphology of the peripheral nerves and the diseases would be valuable in developing new medical treatments on the assumption that characteristics of the peripheral nerves in females are different from those in males. METHODS: We used 13 kinds of the peripheral nerve. The materials were obtained from 10 Japanese female and male cadavers. We performed a morphometric analysis of nerve fibers. We estimated the total number of myelinated axons, and calculated the average transverse area and average circularity ratio of myelinated axons in the peripheral nerves. RESULTS: There was no statistically significant difference in the total number, average transverse area, or average circularity ratio of myelinated axons between the female and male specimens except for the total number of myelinated axons in the vestibular nerve and the average circularity ratio of myelinated axons in the vagus nerve. CONCLUSIONS: The lower number of myelinated axons in the female vestibular nerve may be one of the reasons why vestibular disorders have a female preponderance. Moreover, the higher average circularity ratio of myelinated axons in the male vagus nerve may be one reason why vagus nerve activity to modulate pain has a male preponderance.

Macroscopic description of the external and middle ear of paca (Cuniculus paca Linnaeus, 1766) / Descrição macroscópica da orelha externa e media da paca (Cuniculus paca Linnaeus, 1766)

Paca (Cuniculus paca), one of the largest rodents of the Brazilian fauna, has inherent characteristics of its species which can conribute as a new option for animal experimantation. As there is a growing demand for suitable experimental models in audiologic and otologic surgical research, the gross anatomy and ultrastructural ear of this rodent have been analyzed and described in detail. Fifteen adult pacas from the Wild Animals Sector herd of Faculdade de Ciências Agrárias e Veterinárias, Unesp-Jaboticabal, were used in this study. After anesthesia and euthanasia, we evaluated the entire composition of the external ear, registering and ddescribing the details; the temporal region was often dissected for a better view and detailing of the tympanic bulla which was removed and opened to expose the ear structures analyzed mascroscopically and ultrastructurally. The ear pinna has a triangular and concave shape with irregular ridges and sharp apex. The external auditory canal is winding in its path to the tympanic mebrane. The tympanic bulla is is on the back-bottom of the skull. The middle ear is formed by a cavity region filled with bone and membranous structures bounded by the tympanic membrane and the oval and round windows. The tympanic membrane is flat and seals the ear canal. The anatomy of the paca ear is similar to the guinea pig and from the viewpoint of experimental model has major advantages compared with the mouse ear.

A paca (Cuniculus paca), um dos maiores roedores da fauna brasileira, possui características inerentes à sua espécie que podem contribuir como uma nova opção de animal experimental; assim, considerando-se que há crescente busca por modelos experimentais apropriados para pesquisas audiológicas e otológica cirúrgicas foram analisados e descritos em detalhes a anatomia macroscópica e ultraestrutural da orelha desse roedor. Para o estudo, utilizaram-se 15 animais adultos provenientes do plantel do Setor de Animais Silvestres da Faculdade de Ciências Agrárias e Veterinárias, Unesp-Jaboticabal, Jaboticabal/SP. Após anestesia e eutanásia, avaliou-se toda a composição da orelha externa, registrando-se e descrevendo-se os detalhes, também se dissecou a região temporal para melhor visibilização e detalhamento da bula timpânica e estas foram removidas e abertas a fim de expor as estruturas da orelha, as quais foram analisadas, macroscopicamente e ultraestruturalmente. O pavilhão auricular apresenta forma triangular e côncava com cristas irregulares e ápice pontiagudo; o conduto auditivo externo é sinuoso em seu trajeto até a membrana timpânica; a bula timpânica encontra-se na parte posterior-inferior do crânio; a orelha média é formada por uma região cavitária preenchida por estruturas ósseas e membranosas. É delimitada pela membrana timpânica e as janelas redonda e oval, sendo a membrana timpânica de forma plana e que veda todo o conduto auditivo. A anatomia da orelha da paca é semelhante à da cobaia e do ponto de vista de modelo experimental apresenta grandes vantagens em comparação com a orelha do rato.

Implementation of linear sensory signaling via multiple coordinated mechanisms at central vestibular nerve synapses.

Signal transfer in neural circuits is dynamically modified by the recent history of neuronal activity. Short-term plasticity endows synapses with nonlinear transmission properties, yet synapses in sensory and motor circuits are capable of signaling linearly over a wide range of presynaptic firing rates. How do such synapses achieve rate-invariant transmission despite history-dependent nonlinearities? Here, ultrastructural, biophysical, and computational analyses demonstrate that concerted molecular, anatomical, and physiological refinements are required for central vestibular nerve synapses to linearly transmit rate-coded sensory signals. Vestibular synapses operate in a physiological regime of steady-state depression imposed by tonic firing. Rate-invariant transmission relies on brief presynaptic action potentials that delimit calcium influx, large pools of rapidly mobilized vesicles, multiple low-probability release sites, robust postsynaptic receptor sensitivity, and efficient transmitter clearance. Broadband linear synaptic filtering of head motion signals is thus achieved by coordinately tuned synaptic machinery that maintains physiological operation within inherent cell biological limitations.

Ion channels in mammalian vestibular afferents may set regularity of firing.

Rodent vestibular afferent neurons offer several advantages as a model system for investigating the significance and origins of regularity in neuronal firing interval. Their regularity has a bimodal distribution that defines regular and irregular afferent classes. Factors likely to be involved in setting firing regularity include the morphology and physiology of the afferents' contacts with hair cells, which may influence the averaging of synaptic noise and the afferents' intrinsic electrical properties. In vitro patch clamp studies on the cell bodies of primary vestibular afferents reveal a rich diversity of ion channels, with indications of at least two neuronal populations. Here we suggest that firing patterns of isolated vestibular ganglion somata reflect intrinsic ion channel properties, which in vivo combine with hair cell synaptic drive to produce regular and irregular firing.

[Electron microscopy analysis of the structural elements of the vestibular input to nodulus Purkinje's cells in rats exposed to a 9-day space flight].

Electron microscopy was used to study structural elements of the vestibular afferent input to the cerebellar nodulus Purkinje's cells--terminals of mossy fibers and granular cells in the granular layer and parallel fibers and Purkinje's cell dendrites in the molecular layer in rats decapitated in 2-3 hours and 9 days after the 9-day space flight aboard NASA shuttle Columbia (STS 40, SLS-1 mission). Analysis of the revealed ultrastructural changes on the base of morphofunctional correlations leads to the following conclusions: 1) space flight induced a prolonged reduction in vestibular input to most of the mossy fiber terminals and nodulus Purkinje's cells; 2) within the initial hours of recovery the vestibular input to a part of mossy fiber terminals and granular cells was increasing due to elevation of the sensitivity of vestibular receptors in microgravity; 3) regain of the vestibular input to Purkinje's cells after space flight is hampered by structural, as a result of microgravity effects, and also functional, developing shortly after space flight, impediments, and 4) in 9 d after landing the vestibular input to Purkinje's cells was almost normal. The observed reduction in the vestibular input to the nodulus Purkinje's cells during and after the spaceflight microgravity is presumably the key to the mechanism altering the velocity storage in mammals in microgravity and on return from space flight.

The extraocular motor nuclei: organization and functional neuroanatomy.

The organization of the motoneuron subgroups in the brainstem controlling each extraocular eye muscle is highly stable through the vertebrate species. The subgroups are topographically organized in the oculomotor nucleus (III) and are usually considered to form the final common pathway for eye muscle control. Eye muscles contain a unique type of slow non-twitch, fatigue-resistant muscle fiber, the multiply innervated muscle fibers (MIFs). The recent identification the MIF motoneurons shows that they too have topographic organization, but very different from the classical singly innervated muscle fiber (SIF) motoneurons. The MIF motoneurons lie around the periphery of the oculomotor nucleus (III), trochlear nucleus (IV), and abducens nucleus (VI), slightly separated from the SIF subgroups. The location of four different types of neurons in VI are described and illustrated: (1) SIF motoneurons, (2) MIF motoneurons, (3) internuclear neurons, and (4) the paramedian tract neurons which project to the flocculus. Afferents to the motoneurons arise from the vestibular nuclei, the oculomotor and abducens internuclear neurons, the mesencephalic and pontine burst neurons, the interstitial nucleus of Cajal, nucleus prepositus hypoglossi, the supraoculomotor area and the central mesencephalic reticular formation and the pretectum. The MIF and SIF motoneurons have different histochemical properties and different afferent inputs. The hypothesis that SIFs participate in moving the eye and MIFs determine the alignment seems possible but is not compatible with the concept of a final common pathway.

Connections between the facial, vestibular and cochlear nerve bundles within the internal auditory canal.

The vestibular, cochlear and facial nerves have a common course in the internal auditory canal (IAC). In this study we investigated the average number of nerve fibres, the average cross-sectional areas of the nerves and nerve fibres, and the apparent connections between the facial, cochlear and vestibular nerve bundles within the IAC, using light and scanning electron microscopy. The anatomical localization of the nerves within the IAC was not straightforward. The general course showed that the nerves rotated anticlockwise in the right ear from the inner ear end towards the brainstem end and vice versa for the left ear. The average number of fibres forming vestibular, cochlear, and facial nerves was not constant during their courses within the IAC. The superior and the inferior vestibular nerves showed an increase in the number of nerve fibres from the inner ear end towards the brainstem end of the IAC, whereas the facial and the cochlear nerves showed a reduction in the number of fibres. This suggests that some of the superior and inferior vestibular nerve bundles may receive fibres from the facial and/or cochlear nerves. Scanning electron microscopic evaluations showed superior vestibular-facial and inferior vestibular-cochlear connections within the IAC, but no facial-cochlear connections were observed. Connections between the nerves of the IAC can explain the unexpected vestibular disturbances in facial paralysis or persistence of tinnitus after cochlear neurectomy in intractable tinnitus cases. The present study offers morphometric and scanning electron microscopic data on the fibre connections of the nerves of the IAC.

Demyelination of vestibular nerve axons in unilateral Ménière's disease.

We conducted a study to determine whether vestibular nerves in patients with unilateral Ménière's disease whose symptoms are refractory to medical management exhibit neuropathologic changes. We also endeavored to determine whether retrocochlear abnormalities are primary or secondary factors in the disease process. To these ends, we obtained vestibular nerve segments from five patients during retrosigmoid (posterior fossa) neurectomy, immediately fixed them, and processed them for light and electron microscopy. We found that all five segments exhibited moderate to severe demyelination with axonal sparing. Moreover, we noted that reactive astrocytes produced an extensive proliferation of fibrous processes and that the microglia assumed a phagocytic role. We conclude that the possible etiologies of demyelination include viral and/or immune-mediated factors similar to those seen in other demyelinating diseases, such as multiple sclerosis and Guillain-Barré syndrome. Our findings suggest that some forms of Ménière's disease that are refractory to traditional medical management might be the result of retrocochlear pathology that affects the neuroglial portion of the vestibular nerve.

Subcellular immunolocalization of NMDA receptor subunit NR1, 2A, 2B in the rat vestibular periphery.

The immunohistochemical localization of the NMDA glutamate receptor subunits NR1, NR2A, and NR2B was investigated in the rat vestibular periphery at the light and electron microscopy level using specific antipeptide antibodies. The afferent calyceal terminals and nerve fibers innervating type I vestibular hair cells were strongly NR1, NR2A, and NR2B immunoreactive. Under electron microscopy, the basolateral type I hair cell membrane was NR1 immunoreactive. The type II hair cell and its afferent boutons were NR1, NR2A, and NR2B non-immunoreactive. Nearly all of Scarpa's ganglion neurons were NR1 immunoreactive, but there was a subset of NR2A non-immunoreactive neurons. Additionally, the larger sized Scarpa's ganglia neurons were NR2B immunoreactive, while the smaller neurons were non-immunoreactive. These findings are strong evidence for functional NMDA receptor mediation or modulation of afferent excitatory neurotransmission from type I but not type II vestibular hair cells to the primary afferent nerve. The receptor subtype(s) may be a combination of NR1/NR2A, NR1/NR2B, and/or NR1/NR2A/NR2B.

Role of substance P in the peripheral vestibular and auditory system.

The central role of substance P (SP) has attracted growing interest in the past two decades. One of the important physiological functions of SP and other tachykinins is that of a neurotransmitter in primary afferent neurons. Recent immunocytochemical, biochemical and electrophysiological investigations on various neurotransmitters support the hypothesis that SP has a similar function in the vestibular and auditory systems of all mammals including humans. The purpose of this review is to give an overview of the distribution and concomitant physiological functions of this peptide in these sensory systems.

Vestibular compensation after ganglionectomy: ultrastructural study of the tangential vestibular nucleus and behavioral study of the hatchling chick.

The tangential nucleus is a major part of the avian vestibular nuclear complex, and its principal cells are structurally distinctive neurons participating in the vestibuloocular and vestibulocollic reflexes. After unilateral peripheral vestibular lesion, a behavioral recovery of function defined as vestibular compensation is observed. Because sprouting and hypertrophy of synapses have been reported in other regions of immature animals after central nervous system injury, we investigated whether this also occurs in the vestibular nuclei during compensation. To test this hypothesis, unilateral vestibular ganglionectomy was performed on 4-6-day-old hatchlings and vestibular function was tested during the next 2 months. Degeneration and evidence for regeneration of synapses were studied in the tangential nucleus at 1, 3, 7, and 56 days after surgery. Spoon endings, large vestibular terminals on the principal somata, degenerated 1-3 days after surgery. However, the small synaptic terminals showed no significant change in the percentage or number covering the soma or in mean terminal lengths in the deafferented or contralateral tangential nucleus. Furthermore, there was no evidence of neuron death in the tangential nucleus. Vestibular compensation occurred in three stages: 0-3 days, when vestibular synapses degenerated and severe behavioral deficits were seen; 4-9 days, when primary vestibular fibers degenerated centrally and marked improvement in both the static and the dynamic symptoms were observed; and 10-56 days, when changes in neuronal morphology were not detected but the dynamic symptoms gradually improved. Accordingly, after unilateral vestibular ganglionectomy, vestibular compensation proceeded without ultrastructural evidence of sprouting or hypertrophy of axosomatic synapses in the hatchling tangential nucleus. This rapid behavioral recovery of function distinguishes the vestibular system from other sensory systems, which, in general, exhibit much less robust recovery after injury to their peripheral receptors.

Morphological alterations in the inner ear of the arylsulfatase A-deficient mouse.

Metachromatic leukodystrophy of humans is an inherited sulfatide lipidosis due to deficiency of arylsulfatase A (ASA). As an animal model, ASA(-/-) mice have been generated. A previous study showed that the mice lose most of their spiral (acoustic) ganglion cells and develop deafness by the end of the first year of life. The present report describes the sulfatide histochemistry and ultrastructure of the inner ears of ASA(-/-) mice at 0.5-26 months of age. Lysosomal accumulation of sulfatides was observed in various cell types such as Schwann cells that maintain the myelin sheaths around the spiral and vestibular ganglion cells, periaxonal Schwann cells, macrophages, and spiral and vestibular ganglion cell perikarya. In the spiral ganglion, the only surviving neurons were those which are primarily non-myelinated (type 2 cells). However, the myelinated spiral neurons and their processes were rarely encountered within the process of dying, suggesting that this was a rather rapid process. Since the myelin sheaths around dying perikarya and axons appeared structurally normal, the primary cause of the neuronal cell death seems to reside in the neuron. In contrast to the spiral ganglion, the vestibular ganglion as a whole survived throughout the period of observation. The organ of Corti and the vestibular apparatus appeared preserved at the light microscopic level, despite massive sulfatide storage in the vestibular hair cells.

Herpes simplex virus in the vestibular ganglion and the geniculate ganglion-role of loose myelin.

This study presents the first direct evidence for herpes simplex virus type 1 (HSV-1) infection in the neurons of the vestibular ganglion. Although many investigators have reported electron microscopic evidence of HSV-1 infection in sensory ganglia, HSV-1 infection in the vestibular ganglion has not been described. Vestibular ganglion neurons have a unique structure, with a loose myelin sheath instead of the satellite cell sheath that is seen in other ganglia. This loose myelin is slightly different from compact myelin which is known as too tight for HSV-1 to penetrate. The role of loose myelin in terms of HSV-1 infection is completely unknown. Therefore, in an attempt to evaluate the role of loose myelin in HSV-1 infection, we looked for HSV-1 particles, or any effects mediated by HSV-1, in the vestibular ganglion as compared with the geniculate ganglion. At the light microscopic level, some neurons with vacuolar changes were observed, mainly in the distal portion of the vestibular ganglion where the communicating branch from the geniculate ganglion enters. At the electron microscopic level, vacuoles, dilated rough endoplasmic reticulum and Golgi vesicles occupied by virus were observed in both ganglia neurons. In contrast, viral infections in Schwann and satellite cells were observed only in the geniculate ganglion, but not in the vestibular ganglion. These results suggest that loose myelin is an important barrier to HSV-1 infection, and it must play an important role in the prevention of viral spread from infected neurons to other cells.

Neuronal organization of the utricular macula concerned with innervation of single vestibular neurons in the cat.

We investigated whether cross-striolar inhibition, which may increase sensitivity to linear acceleration, contributed to utricular (UT) afferent innervation of single vestibular neurons (VNs). Excitatory and inhibitory postsynaptic potentials (EPSPs, IPSPs, respectively) were recorded from VNs after focal stimulation of the UT macula (M). From a total of 83 VNs, 25 (30%) neurons received inputs from both sides of the UTM, and the response patterns were opposite, i.e. cross-striolar inhibition was observed. In roughly 2/3 of these neurons, stimulation of the medial side of the UTM evoked EPSPs, while stimulation of the lateral side evoked IPSPs. In the remaining 1/3 neurons, the response patterns were opposite. Thirty-two (39%) of the 83 neurons received the identical pattern of inputs from both sides of the UTM: EPSPs in 26 neurons and IPSPs in six neurons. Twenty-six (31%) of the 83 neurons received inputs from either the medial or the lateral side of the UTM. These findings suggest that cross-striolar inhibition existed in the UT system, although it was not a dominant circuit that increased the sensitivity as in the saccular system [15].

Immunoelectron microscopy of AMPA receptor subunits reveals three types of putative glutamatergic synapse in the rat vestibular end organs.

To characterize the synapses between hair cells and afferent nerve endings in the rat vestibular end organs, the ultrastructural localization of AMPA receptor subunits (GluR1-4) was examined by postembedding immunogold cytochemistry. Immunoreactivities for GluR2/3 and GluR4 were associated with the synapses between type I hair cells and the surrounding chaliceal nerve endings and with the bouton type nerve endings contacting type II hair cells. There was no detectable immunoreactivity for GluR1. A third type of immunoreactive synapse was found between the outer face of chalices and type II hair cells. While the linear densities of gold particles (particles per micrometer postsynaptic specialization) of bouton type endings and chaliceal nerve endings were the same, the former type of ending showed larger postsynaptic specializations and, hence, a higher number of receptor molecules. These data indicate that there are three types of putative glutamatergic synapse in the vestibular end organ.

[Immunocytochemical study of gamma-aminobutyric acid-ergic innervation in the end-organs of human vestibule].

OBJECTIVE: To investigate gamma-aminobutyric acid-ergic (GABAergic) innervation in the end-organs of human vestibule. METHODS: A modified pre-embedding immunostaining technique of immunoelectron microscopy were applied to accomplish this study with a polyclonal antibody to gamma-aminobutyric acid. RESULTS: GABA-immunoreactive products were confined to the nerve terminals, which were rich in synaptic vesicles and the non-myelinated fibers. The GABA-immunoreactive nerve fibers synapse with afferent calices surrounding the type I hair cells. CONCLUSION: This study shows that GABAergic fibers of human vestibular end-organs belong to the vestibular efferent system.

A morphological and morphometric study of the peripheral process of the human vestibular nerve following posterior cranial fossa neurectomy.

Three vestibular nerve specimens removed at transmeatal neurectomy were studied in order to understand better retrograde degeneration and regeneration after vestibular neurectomy in the posterior cranial fossa. In two cases this procedure followed retrolabyrinthine retrosigmoid posterior fossa vestibular neurectomy. The subjects, three patients with Menière's disease, were compared with one another and two autopsy controls with no known otological problem. The specimens were obtained at the distal end of the internal auditory canal and transversely sectioned. Many collapsed Schwann cell basement membranes were observed. The ratio of small-diameter nerve fibres increased significantly after neurectomy. Onion bulb formation around myelinated nerve fibres with small diameters and Schwann cell proliferation around the soma of vestibular ganglion cells reflected remyelination. We conclude that peripheral processes of vestibular nerve fibres can undergo retrograde degeneration and subsequent regeneration after transection of the central process.

[Traverse connectivity of innervation in the vestibular sensory epithelium].

OBJECTIVE: To further understand the traverse connections of nerve components in the vestibular sensory epithelium. METHODS: The nerve innervations in the vestibular sensory epithelia and infraepithelia of ten healthy guinea pigs (16 ears) were observed by transmission electron microscopy. RESULTS: Type I sensory cells were intricately connected with type II cells. Afferent and efferent fibers as well as their endings had also complicated connections with nerve trunks and branches. The traverse connections appeared among nerve components in the vestibular sensory epithelium were extremely complicated, forming multilevel connections. They stemmed from and end in the crista ampullaris and the maculae. CONCLUSION: These complicated and multilevel connections are the basis of nerve activity in the vestibular apparatus.

Myelin-sheath abnormalities in the vestibular nerves of chronically diabetic rats.

Electron microscopic examination of the saccular and utricular nerves of rats with streptozocin-induced diabetes mellitus revealed differences from age-matched control animals, mainly related to myelin sheaths. Nerves from diabetic rats had a high incidence of a variety of osmiophilic inclusion bodies, often associated with disrupted myelin-sheath lamellae, lysosomal bodies, and periaxonal expansions of Schwann cell cytoplasm. The occurrence of osmiophilic inclusion bodies within nerve profiles was correlated with the severity of diabetes as measured by blood glucose levels (p < 0.001, linear-regression ANOVA). Duration of diabetes was not related to incidence of inclusion bodies. No evidence of demyelination/remyelination or neuronal degeneration was observed. An unusual Schwann cell reaction was noted, involving lysosomal digestion of large, pinched-off portions of myelinated nerve fibers of some diabetic rats. Both this Schwann cell reaction and the high incidence of inclusion bodies may be due to increased nonenzymatic glycosylation of myelin proteins in the diabetic animals.