Functional organization of the peripheral efferent vestibular system in the frog

The functional organization of the efferent vestibular system (EVS) was studied in the isolated frog labyrinth. To ascertain whether, besides the efferent branching fibres that innervate several end-organs, the EVS is also endowed with efferent non-branching axons which might control a given population of sensory units in each end-organ, the 8th nerve and one of its branchlets were electrically stimulated while recordings of the spontaneous activity arising from the different sensors were made by impaling single afferent axons in all the 8th nerve branchlets. The results demonstrated that the vast majority of the sensory units whose activity was modified by stimulating the whole 8th nerve was also affected by stimulating an 8th nerve branchlet. These findings therefore rule out the possibility that the EVS is endowed with projective fibres and strengthen the view that the EVS is a highly divergent system with collaterals arising from single parent axons that innervate several end-organs. These experiments have also shown that the percentage of sensory units which are actually controlled by the EVS varies amongst the different labyrinthine organs. It is maximal in the sacculus (ca. 90%), somewhat lower in canal organs (ca. 80%) and the utriculus (ca. 70%) and considerably lower in the lagena (ca. 50%). This EVS arrangement therefore might allow information arising from some organs to be modified more extensively than that from others.     

Peripheral organization of the vestibular efferent system in the frog: an electrophysiological study

The distribution and the properties of efferent fibers in vestibular nerve were studied in the isolated frog labyrinth. Electrical stimulation of the central stump of any vestibular nerve branchlet elicited compound action potentials in all the other eighth nerve branchlets, indicating the existence of neural links between the various vestibular organs. The same experimental paradigm, when repeated in frogs with chronic section of the eighth nerve roots, demonstrated that these pathways are efferent collaterals extending to all vestibular organs. There are more collaterals linking the 3 semicircular canals than the otolith organs and the otoliths with the canal organs. Efferent connections in the eighth nerve were preserved in full after ablation of the ipsilateral hemi-cerebellum, suggesting that the efferent pathways probably originate in the brainstem. Intracellular recordings from single afferent fibers of both canal and otolith organs revealed that efferent fiber activation could elicit either inhibition or facilitation of the receptor discharge. It was concluded that the frog efferent vestibular system is endowed with non-selective control channels which allow single neurons to influence the receptor activity of different labyrinthine organs.     

Pharmacology of the vestibular hair cell-afferent fiber synapse in the frog

The isolated, intact, membranous labyrinth of the frog (Rana temporaria) has been investigated electrophysiologically in vitro to determine the nature of the transmitter substance at the synapse between the vestibular hair cells and afferent fibers. Spontaneous synaptic activity can be monitored with intra-axonal recordings from the afferents. Increased K+ in the bath results in an increase in frequency of presynaptic release, as indicated by an increased frequency of spontaneous synaptic potentials. Adding Mg2+ and lowering Ca2+ results in a decrease in synaptic potential frequency (often to zero) with no change in their mean amplitude, indicating pre-synaptic blockade. Extracellular recordings from individual vestibular afferents indicate that bath-applied glutamate and related acidic amino acids consistently increase the firing rates of these afferents in a dose-dependent manner with no evidence of desensitization. In the presence of presynaptic blockade (high Mg2+/low Ca2+), bath application of glutamate and its agonists results in a reversible depolarization of vestibular afferents, suggesting a postsynaptic action of these substances. 2-Amino-5-phosphonovaleric acid, kynurenic acid, and other acidic amino acid antagonists reversibly decrease the amplitudes of spontaneously occurring synaptic potentials without affecting their frequency, indicating subsynaptic blockade. These antagonists also block the postsynaptic depolarizations due to glutamate and its agonists. GABA and its agonists and antagonists have no consistent effect upon afferent activity. These findings suggest that glutamate, aspartate, or a related compound is the transmitter at this synapse. However, the antagonists used, or the receptors themselves, are not selective enough to discriminate adequately between the agonists. Therefore, which of these glutamate agonists are actually involved in synaptic transmission remains to be determined.     

Responses of afferent and efferent neurons to visual inputs in the vestibular nerve of the frog

In the frog immobilized by intralymphatic injection of D-tubocurarine, stimulation of the visual apparatus with either electrical shocks applied to the optic chiasma or light pulses elicited, in many cases, an increase and a decrease of firing of efferent and afferent vestibular neurons, respectively, recorded from the horizontal semicircular canal nerve. Optokinetic stimulation was completely inefficient in modulating the efferent and afferent discharge. These results show that stimulation of the visual system can modify vestibular apparatus fuctioning at the most peripheral level. However, it is likely that the effects observed were due to an arousal phenomenon or/and to a motor corollary discharge.     

New ultrastructural features in teh vestibular labyrinth of the toadfish, Opsanus tau

Electron microscopy of the toadfish vestibular labyrithn demonstrated the usual cytoarchitectural efferent-hair cell organization with type II hair cells having efferent synaptic terminals directly onto the hair cell bodies. Additional, unexpected findings were: (1) direct efferent-afferent synapses; and (2) two types of synaptic vesicles either found both in the same efferent terminals or in two separate classes of efferent terminals.     

Activation of the efferent system in the isolated frog labyrinth: Effects on the afferent EPSPs and spike discharge recorded from single fibers of the posterior nerve

Intra-axonal recordings were obtained from single afferent fibres of the posterior nerve in the isolated labyrinth of the frog (Rana esculenta). EPSPs spike discharge were recorded both at rest and during rotary stimulation of the canal. Electrical stimulation of either the distal end of the cut posterior nerve or of the central stumps of the anterior-horizontal nerves elicited a frequency-dependent inhibitory effect on the afferent discharge arising from the posterior canal. Denervation experiments revealed that inhibition is mediated by efferent fibres exhibiting a high degree of branching in the proximal part of the eight nerve. The inhibitory effect was selectively cancelled by (1)d-tubocurarine 10-⁶ M; (2) atropine 5 x 10-⁵ M;(3) acetylcholine or carbachol 10⁻⁴ M; (4) eserine 10⁻⁵ M. Inhibition is thus most likely to be sustained by the release of acetylcholine from the efferent nerve terminals. Experiments in which the ionic composition of the external medium was modified suggest that the transmitter acts mainly by opening the chloride ion channels of the hair cell membrane. In some units the same stimulation pattern evoked a consistent increase in both EPSP and spike discharge, instead of inhibition. Such facilitation was unaffected by drugs or ionic modifications which block the efferent synapse, but disappeared after denervation. Inhibition and facilitation, therefore, act as two control mechanisms which are able to modify substantially, at the first stage of processing, the sensory information which is sent to the vestibular second order neurones.     

Origin of centrifugal fibers to the labyrinth in the frog (Rana esculenta). A study with the fluorescent retrograde neuronal tracer ‘Fast blue’

After injecting a solution of a fluorescent retrograde neuronal tracer (Fast blue, Diamidino compound 253/50) into the perilymphatic space of the frog labyrinth (Rana esculenta), labeled cells were found in the ventral and dorsal nuclei of the VIIIth nerve and in the nucleus reticularis medius. We consider these labeled cells to be the origin of the efferent innervation of the frog labyrinth. No evidence was found for the existence of a direct cerebello-labyrinthine connection.     

Presynaptic evidence for zinc release at the mossy fiber synapse of rat hippocampus

Vesicular zinc (Zn(2+)) is found in a subset of glutamatergic nerve terminals throughout the mammalian forebrain and is colocalized with glutamate. Despite well-documented neuromodulatory roles, exocytosis of endogenous Zn(2+) from presynaptic terminals has never been directly demonstrated, because existing studies have measured elevated Zn(2+) concentrations by examining the perfusate. Thus, the specific origin of synaptic Zn(2+) remains a controversial subject. Here, we describe synaptic Zn(2+) trafficking between cellular compartments at hippocampal mossy fiber synapses by using the fluorescent indicator Zinpyr-1 to label the hippocampal mossy fiber boutons. We determined endogenous Zn(2+) exocytosis by direct observation of vesicular Zn(2+) as decreasing fluorescence intensity from presynaptic axonal boutons in the stratum lucidum of CA3 during neural activities induced by the stimulation of membrane depolarization. This presynaptic fluorescence gradually returned to a level near baseline after the withdrawal of moderate stimulation, indicating an endogenous mechanism to replenish vesicular Zn(2+). The exocytosis of the synaptic Zn(2+) was also dependent on extracellular Ca(2+) and was sensitive to Zn(2+)-specific chelators. Vesicular Zn(2+) loading was sensitive to the vacuolar-type H(+)-ATPase inhibitor concanamycin A, and our experiments indicated that blockade of vesicular reloading with concanamycin A led to a depletion of that synaptic Zn(2+). Furthermore, synaptic Zn(2+) translocated to the postsynaptic cell body upon release to produce increases in the concentration of weakly bound Zn(2+) within the postsynaptic cytosol, demonstrating a feature unique to ionic substances released during neurotransmission. Our data provide important evidence for Zn(2+) as a substance that undergoes release in a manner similar to common neurotransmitters.     

Mode of synaptic transmission between vestibular afferents and neurons of the vestibular nucleus in the frog

Excitatory postsynaptic potentials (EPSPs) were evoked in vestibular neurons of the isolated frog brainstem by stimulation of the ipsilateral vestibular nerve or by direct intra-axonal activation of single vestibular fibers. Composite as well as single-fiber EPSPs usually displayed two components. A Ca2+-deficient, Mn2+-containing solution abolished the delayed chemical components of the EPSPs, but did not affect the short latency responses suggesting a dual (electrical-chemical) mode of transmission between some vestibular afferents and vestibular neurons.     

Prominent expression of Narp in central vestibular pathways: selective effect of labyrinth ablation

Recent in vitro studies demonstrated that Narp, a secreted immediate early gene (IEG) product, induces AMPA receptor clustering. Accordingly, Narp has been implicated in mediating activity-dependent changes in synaptic efficacy. To help define the role of Narp in vivo, we conducted immunohistochemical studies of Narp in rat brain. Unexpectedly, we found robust Narp expression in several discrete areas linked to the vestibular system: the anterodorsal nucleus (ADN) of the thalamus, which relays head orientation information to the cortex, the lateral vestibulospinal (Deiters') nucleus and Purkinje cells in the flocculonodular lobe of the cerebellum. Although strong Narp expression in Deiters' nucleus and the cerebellum was present consistently, Narp expression in the ADN displayed a high degree of variability among animals. To check if this variability in ADN Narp expression reflects its dependence on fluctuating levels of vestibular input, we monitored Narp immunostaining following bilateral labyrinth ablation. This procedure significantly suppressed Narp immunostaining in the ADN, indicating that it is stimulated by naturally occurring vestibular input. In contrast, labyrinth ablation did not affect Narp staining in Deiters' nucleus or the flocculonodular lobe of the cerebellum, presumably because these areas are driven by inputs from multiple systems. As previous studies implicate Narp in synaptic plasticity, these findings suggest that this IEG may mediate ongoing adjustments in synaptic strength or connectivity in several pathways linked to the vestibular system.     

Development of synaptic organization in the tangential vestibular nucleus: a quantitative electron microscope study

The objective of the present study is to quantify the developmental changes in the total synaptic pattern of one part, the soma, of one particular cell type, the principal cells of the tangential vestibular nucleus. The term "synaptic space" is defined and quantified. Intermediate stages in the development of synaptic organization are compared. The findings show that the synaptic space available to the full complement of afferents is constant throughout development, while specific terminals in the afferent population change their synaptic space allotments. The synaptic-junction covering is invariant for small terminals at intermediate stages of development with set proportions between "active" and "non-active" zones of the synaptic surfaces. However, the spoon endings and the postsynaptic target cells are covered by synaptic junctions in variable amounts. The findings are important to the fields of neuroembryology and neural plasticity, for the system provides a useful basis to measure the influence of factors in the local environment and the role of formation of synaptic connections in the competition for synaptic space. This study will assist investigators to probe the mechanisms operating in the selection of competing afferents for the limited amount of surface area available under the changing conditions of maturation and aging in the central nervous system.     

Distribution of choline acetyltransferase immunoreactivity in the vestibular nuclei of normal and unilateral vestibular neurectomized cats

Post‐lesion recovery of vestibular functions is a suitable model for studying adult central nervous system plasticity. The vestibular nuclei complex (VN) plays a major role in the recovery process and neurochemical reorganizations have been described at this brainstem level. The cholinergic system should be involved because administration of cholinergic agonists and antagonists modify the recovery time course. This study was aimed at analysing the postlesion changes in choline acetyltransferase immunoreactivity (ChAT‐Ir) in the VN of cats killed 1 week, 3 weeks or 1 year following unilateral vestibular neurectomy. ChAT‐positive neurons and varicosities were immunohistochemically labelled and quantified (cell count and surface measurement, respectively) by means of an image analysing system. The spatial distribution of ChAT‐Ir within the VN of control cats showed darkly stained neurons and varicosities mainly located in the caudal parts of the medial (MVN) and inferior (IVN) VN, the nucleus prepositus hypoglossi (PH) and, to a lesser extent, in the medial part of the superior vestibular nucleus (SVN). Lesion‐induced changes consisted in a significant increase in both the number of ChAT‐positive neurons (IVN, SVN) and the surface of ChAT‐positive varicosities (IVN, SVN, PH). They were observed bilaterally in the acute (1 year and 3 weeks) and compensated (1 year) cats for the SVN and PH, while they persisted only in the IVN on the lesioned side in the compensated cats. These findings demonstrate vestibular lesion‐induced reorganization of the cholinergic system in the IVN, SVN and PH which could contribute to postural and oculomotor function recovery.     

Identification of multiple groups of efferent vestibular neurons in the adult pigeon using horseradish peroxidase and DAPI

This study was designed to investigate the efferent innervation of the pigeon labyrinth.Horseradish peroxidase (HRP) was injected and confined within the endolymphatic space of one labyrinth in 9 adult pigeons. The brain was perfused by transcardiac carotid catheterization and the HRP reacted by the tetramethylbenzidine (TMB) blue reaction process. Five different groups of HRP-labeled vestibular efferent neurons were identified. Three groups were located within the confines of the ipsilateral vestibular nuclear complex (in the lateral, tangential and descending nucleus) and two additional groups, each bilateral, were located in the reticular formation. In 9 additional pigeons, 4,6-diamidino-2-phenylindole (DAPI) was also injected and confined within the endolymphatic space of one labyrinth. DAPI-labeled cells were noted in 3 of the 5 locations (tangential nucleus, and both reticular groups) which in the other animals contained HRP-labeled cells.These findings raise the possibility of different physiological roles for the efferent vestibular groups in the ipsilateral vestibular nuclear complex and bilateral reticular formation.     

Ontogeny of electrophysiological properties and dendritic pattern in second-order chick vestibular neurons

The pattern of development of several subpopulations of second-order vestibular neurons was investigated by using intracellular recordings from chicken brain slices to define the timing of morphological and electrophysiological changes occurring at 3 critical ages. Two embryonic stages, embryonic day 13 (E13) and E15–16, and also newborn chicks were selected according to previous anatomical findings showing the differentiation of primary vestibular afferents and their synapses within a distinctive brainstem vestibular nucleus, the tangential nucleus. The responses of these cells to depolarizing and hyperpolarizing current pulses and their postsynaptic responses to vestibular nerve stimulation were recorded, while simultaneously biocytin was injected for subsequent morphogenetic analysis. From this study, developmental schedules of membrane properties, synaptic responses, and dendritic differentiation were established for the 2 cell populations of the tangential nucleus and other neurons located in the surrounding vestibular nuclei. Compared with all other second-order vestibular neurons, the principal cells of the tangential nucleus exhibited a distinctive schedule. Mainly, this includes their pattern of firing on depolarization, the shape and duration of the vestibular-evoked excitatory postsynaptic potential, and the time of onset of dendritic outgrowth. In regard to these observations, E15–16 appears to be a turning point in principal cell ontogeny, whereas these features occur earlier in development for other second-order vestibular neurons. These findings, which indicate that the principal cells may have distinct membrane properties at specific ages, are discussed in light of their unique vestibular innervation and the possible consequences for vestibular signal processing. J. Comp. Neurol. 384:621–633, 1997. © 1997 Wiley-Liss, Inc.     

Ascending and descending projections of the lateral vestibular nucleus in the frog Rana esculenta

The lectin Phaseolus vulgaris leucoagglutinin was injected into the frog lateral vestibular nucleus (LVN) to study its antero- and retrograde projections. The following new observations were made. 1) In the diencephalon, vestibular efferents innervate the thalamus in a manner similar to that of mammalian species. The projections show a preference for the anterior, central, and ventromedial thalamic nuclei. 2) In the mesencephalon, vestibular fibers terminate in the tegmental nuclei and the nucleus of medial longitudinal fascicle. 3) In the rhombencephalon, commissural and internuclear projections interconnect the vestibular nuclei. Some of the termination areas in the reticular formation can be homologized with the mammalian inferior olive and the nucleus prepositus hypoglossi. Another part of the vestibuloreticular projection may transmit vestibular impulses toward the vegetative centers of the brainstem. A relatively weak projection is detected in the spinal nucleus of the trigeminal nerve, dorsal column nuclei, and nucleus of the solitary tract. 4) In the spinal cord, vestibular terminals are most numerous in the ipsilateral ventral horn and in the triangular area of the dorsal horn. 5) The coincidence of retrogradely labeled cells with vestibular receptive areas suggests reciprocal interconnections between these structures and the LVN. 6) In seven places, the LVN projections overlap the receptive areas of proprioceptive fibers, suggesting a convergence of sensory modalities involved in the sense of balance.     

Endocannabinoid transport tightly controls 2-arachidonoyl glycerol actions in the hippocampus: effects of low temperature and the transport inhibitor AM404

The control of endocannabinoid actions on cortical neurons by a putative carrier-mediated uptake is still poorly understood at the level of synaptic transmission. We investigated the effect of an endocannabinoid, 2-arachidonoyl glycerol (2-AG), on inhibitory postsynaptic currents (IPSCs) in hippocampal slices under physiological conditions, and when uptake was altered by a selective blocker or lower temperature. Bath application of 2-AG (20 micro m) caused a 40% reduction in the amplitude of IPSCs evoked in the perisomatic region of CA1 pyramidal neurons at room temperature; this effect could be blocked by a specific CB(1) receptor antagonist, AM251. By contrast, a smaller (20%) but significant suppression of inhibitory transmission was found by 2-AG at 33-35 degrees C. This reduced blocking effect at physiological temperature could be brought back to 40% by coapplying the endocannabinoid uptake blocker, AM404 (10 or 20 micro m) with 2-AG. In parallel experiments, we measured [(3)H]2-AG uptake at different temperatures in primary cultures prepared from cortical neurons. These data confirmed a striking inhibition of [(3)H]2-AG uptake at room temperature compared with values observed at 37 degrees C. Uptake could be significantly modified by anandamide, 2-AG and AM404, suggesting a common transporter for the two endocannabinoids. These findings together demonstrate the presence of an effective endocannabinoid uptake in cortical neurons, which could considerably alter the spatial and temporal constraints of endocannabinoid signalling at physiological temperature, and which may critically change the interpretation of findings at room temperature.     

Lack of localization of 5-HT6 receptors on cholinergic neurons: implication of multiple neurotransmitter systems in 5-HT6 receptor-mediated acetylcholine release

The involvement of the cholinergic system in learning and memory together with the cognitive enhancing properties of 5-HT6 receptor antagonists led us to study the relationship between 5-HT6 receptors and cholinergic neurotransmission. A selective cholinergic lesion, induced by injection of the immunotoxin 192-IgG-Saporin into the nucleus basalis magnocellularis, failed to alter the density of 5-HT6 receptor mRNA or protein expression in the deafferentated frontal cortex, suggesting that 5-HT6 receptors are not located on cholinergic neurons. The 5-HT6 receptor antagonist SB-357134 (0.001-1 microM) induced a concentration-dependant K+-evoked [3H]acetylcholine (ACh) release in vitro in rat cortical and striatal slices, which was blocked by tetrodotoxin. SB-357134, up to 1 microM, stimulated glutamate release in cortical and striatal slices. In the cortex, riluzole (1 microM) blocked the SB-357134-induced K+-stimulated [3H]ACh release, and simultaneous administration of MK-801 (1 microM) and SB-357134 (0.05 microM) elicited an increase in K+-evoked ACh release. In the striatum, SB-357134, 1 microM, decreased dopamine release, and the increase in K+-evoked [3H]ACh release induced by 5-HT6 receptor blockade was reversed by the D1 receptor antagonist, SCH23390 (1 microM). In both the frontal cortex and striatum, bicuculline, 1 microM, showed no effect on SB-357134-evoked [3H]ACh. These results are discussed in terms of neurochemical mechanisms involved in 5-HT6 receptor functions.     

Excitatory actions of GABA in developing chick vestibular afferents: Effects on resting electrical activity

The aim of this study was to characterize the effect of γ-aminobutyric acid (GABA) in the resting multiunit activity of the vestibular afferents during development using the isolated inner ear of embryonic and postnatal chickens (E15-E21 and P5). GABA (10⁻³ to 10⁻⁵ M; n = 133) and muscimol (10⁻³M) elicited an increase in the frequency of the basal discharge of the vestibular afferents. We found that GABA action was dose-dependent and inversely related to animal age. Thus, the largest effect was observed in embryonic ages such as E15 and E17 and decreases in E21 and P5. The GABA_A receptor antagonists, bicuculline (10⁻⁵M; n = 10) and picrotoxin (10⁻⁴M; n = 10), significantly decreased the excitatory action of GABA and muscimol (10⁻³M). Additionally, CNQX 10⁻⁶M, MCPG 10⁻⁵M and 7ClKyn 10⁻⁵M (n = 5) were co-applied by bath substitution (n = 5). Both the basal discharge and the GABA action significantly decreased in these experimental conditions. The chloride channel blocker 9-AC 0.5 mM produced an important reduction in the effect of GABA 10⁻³ (n = 5) and 10⁻⁴M (n = 5). Thus, our results suggest an excitatory role of GABA in the resting activity of the vestibular afferents that can be explained by changes in the gradient of concentration of Cl⁻ during development. We show for the first time that the magnitude of this GABA effect decreases at later stages of embryonic and early postnatal development. Taking into account the results with glutamatergic antagonists, we conclude that GABA has a presynaptic action but is not the neurotransmitter in the vestibular afferent synapses, although it could act as a facilitator of the spontaneous activity and may regulate glutamate release. Synapse 67:374–381, 2013. © 2013 Wiley Periodicals, Inc.