Planum Semilunatum Research Articles

Migraine, vertigo and migrainous vertigo: Links between vestibular and pain mechanisms

This review develops the hypothesis that co-morbid balance disorders and migraine can be understood as additive effects of processing afferent vestibular and pain information in pre-parabrachial and pre-thalamic pathways, that have consequences on cortical mechanisms influencing perception, interoception and affect. There are remarkable parallel neurochemical phenotypes for inner ear and trigeminal ganglion cells and these afferent channels appear to converge in shared central pathways for vestibular and nociceptive information processing. These pathways share expression of receptors targeted by anti-migraine drugs. New evidence is also presented regarding the distribution of serotonin receptors in the planum semilunatum of the primate cristae ampullaris, which may indicate involvement of inner ear ionic homeostatic mechanisms in audiovestibular symptoms that can accompany migraine.

Na+ currents in vestibular type I and type II hair cells of the embryo and adult chicken.

In birds, type I and type II hair cells differentiate before birth. Here we describe that chick hair cells, from the semicircular canals, begin expressing a voltage-dependent Na current (INa) from embryonic day 14 (E14) and continue to express the current up to hatching (E21). During this period, INa was present in most (31/43) type I hair cells irrespective of their position in the crista, in most type II hair cells located far from the planum semilunatum (48/63), but only occasionally in type II hair cells close to the planum semilunatum (2/35). INa activated close to -60 mV, showed fast time- and voltage-dependent activation and inactivation, and was completely, and reversibly, blocked by submicromolar concentrations of tetrodotoxin (Kd = 17 nM). One peculiar property of INa concerns its steady-state inactivation, which is complete at -60 mV (half-inactivating voltage = -96 mV). INa was found in type I and type II hair cells from the adult chicken as well, where it had similar, although possibly not identical, properties and regional distribution. Current-clamp experiments showed that INa could contribute to the voltage response provided that the cell membrane was depolarized from holding potentials more negative than -80 mV. When recruited, INa produced a significant acceleration of the cell membrane depolarization, which occasionally elicited a large rapid depolarization followed by a rapid repolarization (action-potential-like response). Possible physiological roles for INa in the embryo and adult chicken are discussed.

Regional analysis of whole cell currents from hair cells of the turtle posterior crista.

The turtle posterior crista is made up of two hemicristae, each consisting of a central zone containing type I and type II hair cells and a surrounding peripheral zone containing only type II hair cells and extending from the planum semilunatum to the nonsensory torus. Afferents from various regions of a hemicrista differ in their discharge properties. To see if afferent diversity is related to the basolateral currents of the hair cells innervated, we selectively harvested type I and II hair cells from the central zone and type II hair cells from two parts of the peripheral zone, one near the planum and the other near the torus. Voltage-dependent currents were studied with the whole cell, ruptured-patch method and characterized in voltage-clamp mode. We found regional differences in both outwardly and inwardly rectifying voltage-sensitive currents. As in birds and mammals, type I hair cells have a distinctive outwardly rectifying current (I(K,L)), which begins activating at more hyperpolarized voltages than do the outward currents of type II hair cells. Activation of I(K,L) is slow and sigmoidal. Maximal outward conductances are large. Outward currents in type II cells vary in their activation kinetics. Cells with fast kinetics are associated with small conductances and with partial inactivation during 200-ms depolarizing voltage steps. Almost all type II cells in the peripheral zone and many in the central zone have fast kinetics. Some type II cells in the central zone have large outward currents with slow kinetics and little inactivation. Although these currents resemble I(K,L), they can be distinguished from the latter both electrophysiologically and pharmacologically. There are two varieties of inwardly rectifying currents in type II hair cells: activation of I(K1) is rapid and monoexponential, whereas that of I(h) is slow and sigmoidal. Many type II cells either have both inward currents or only have I(K1); very few cells only have I(h). Inward currents are less conspicuous in type I cells. Type II cells near the torus have smaller outwardly rectifying currents and larger inwardly rectifying currents than those near the planum, but the differences are too small to account for variations in discharge properties of bouton afferents innervating the two regions of the peripheral zone. The large outward conductances seen in central cells, by lowering impedances, may contribute to the low rotational gains of some central-zone afferents.

Molecular probes of the vestibular nerve: I. Peripheral termination patterns of calretinin, calbindin and peripherin containing fibers

Vestibular afferents have different physiological properties that can be at least partially correlated with the morphology that the peripheral ending makes with type I and type II hair cells. If the location of the ending in the sensory epithelium is included, the correlations are further improved. It is also known that vestibular afferents can be immunohistochemically stained for a variety of different substances. We have concentrated on three of these markers, calretinin, calbindin and peripherin, because the sources of afferents to the vestibular nuclear complex that contain these substances are restricted, in two cases to the primary afferents. We demonstrate that calretinin is found only in the calyx-only afferents that are located at the apex of the cristae ampullaris and along the striola of the maculae. The area containing stained calyces is equal to or slightly smaller than the central zone of the cristae as defined by the Goldberg group [J. Neurophysiol. 60 (1988) 167]. Calbindin is also found in calyces at the apex of the cristae and along the striola of the otoliths. Examination of adjacent sections of all endorgans indicates that calbindin staining overlaps with calretinin, but is always several hair cells wider on each side. Peripherin also stains fibers in the neuroepithelium. The greatest density of staining is in the peripheral zone of the cristae, i.e. at the base and toward the planum semilunatum. We suggest that these substances are useful markers for specific sets of vestibular afferents.

Electron microscopic studies of ion- and H2O-transporting epithelial cells in the horizontal ampulla of the pigeon.

Earlier morphological studies of the epithelial structure in the semicircular canals of mammals have focused on the sensory cells of the crista ampullaris. This report draws attention to the fact that there exist at least seven further cell types in the horizontal ampulla walls of pigeon with various functions; the role of ion- and H2O-transporting epithelial cells is dealt with here in detail. While the dark cells appear to play a decisive role in the regulation of ionic composition, the cells in the planum semilunatum may transport H2O and assist in the regulation of endolymph volume. In addition, protein-secreting structures are located in the apical region of the cells of the planum semilunatum. The question whether the proteins are dispersed in the endolymph or contribute to cupula formation remains unclear. The morphology and possible functions of these two cell types are discussed on the basis of electron microscopic results.

Responses to efferent activation and excitatory response-intensity relations of turtle posterior-crista afferents.

Multivariate statistical formulas were used to infer the morphological type and longitudinal position of extracellularly recorded afferents. Efferent fibers were stimulated electrically in the nerve branch interconnecting the anterior and posterior VIIIth nerves. Responses of bouton (B) units depended on their inferred position: BP units (near the planum semilunatum) showed small excitatory responses; BT units (near the torus) were inhibited; BM units (in an intermediate position) had a mixed response, including an initial inhibition and a delayed excitation. Calyx-bearing (CD-high) units with an appreciable background discharge showed large per-train excitatory responses followed by smaller post-train responses that could outlast the shock train by 100 s. Excitatory responses were smaller in calyx-bearing (CD-low) units having little or no background activity than in CD-high units. Excitatory response-intensity functions, derived from the discharge during 2-s angular-velocity ramps varying in intensity, were fit by empirical functions that gave estimates of the maximal response (r(MAX)), a threshold velocity (v(T)), and the velocity producing a half-maximal response (v(1/2)). Linear gain is equal to r(MAX)/v(S), v(S) = v(1/2) - v(T). v(S) provides a measure of the velocity range over which the response is nearly linear. For B units, r(MAX) declines by as much as twofold over the 2-s ramp, whereas for CD units, r(MAX) increases by 15% during the same time period. At the end of the ramp, r(MAX) is on average twice as high in CD as in B units. Thresholds are negligible in most spontaneously active units, including almost all B and CD-high units. Silent CD-low units typically have thresholds of 10-100 deg/s. BT units have very high linear gains and v(S) < 10 deg/s. Linear gains are considerably lower in BP units and v(S) > 150 deg/s. CD-high units have intermediate gains and near 100 deg/s v(S) values. CD-low units have low gains and v(S) values ranging from 150 to more than 300 deg/s. The results suggest that BT units are designed to measure the small head movements involved in postural control, whereas BP and CD units are more appropriate for monitoring large volitional head movements. The former units are silenced by efferent activation, whereas the latter units are excited. This suggests that the efferent system switches the turtle posterior crista from a "postural" to a "volitional" mode.

Morphological identification of physiologically characterized afferents innervating the turtle posterior crista.

The turtle posterior crista consists of two hemicristae. Each hemicrista extends from the planum semilunatum to the nonsensory torus and includes a central zone (CZ) surrounded by a peripheral zone (PZ). Type I and type II hair cells are found in the CZ and are innervated by calyx, dimorphic and bouton afferents. Only type II hair cells and bouton fibers are found in the PZ. Units were intraaxonally labeled in a half-head preparation. Bouton (B) units could be near the planum (BP), near the torus (BT), or in midportions of a hemicrista, including the PZ and CZ. Discharge properties of B units vary with longitudinal position in a hemicrista but not with morphological features of their peripheral terminations. BP units are regularly discharging and have small gains and small phase leads re angular head velocity. BT units are irregular and have large gains and large phase leads. BM units have intermediate properties. Calyx (C) and dimorphic (D) units have similar discharge properties and were placed into a single calyx-bearing (CD) category. While having an irregular discharge resembling BT units, CD units have gains and phases similar to those of BM units. Rather than any single discharge property, it is the relation between discharge regularity and either gain or phase that makes CD units distinctive. Multivariate statistical formulas were developed to infer a unit's morphological class (B or CD) and longitudinal position solely from its discharge properties. To verify the use of the formulas, discharge properties were compared for units recorded intraaxonally or extracellularly in the half-head or extracellularly in intact animals. Most B units have background rates of 10-30 spikes/s. The CD category was separated into CD-high and CD-low units with background rates above or below 5 spikes/s, respectively. CD-low units have lower gains and phases and are located nearer the planum than CD-high units. In their response dynamics over a frequency range from 0.01-3 Hz, BP units conform to an overdamped torsion-pendulum model. Other units show departures from the model, including high-frequency gain increases and phase leads. The longitudinal gradient in the physiology of turtle B units resembles a similar gradient in the anamniote crista. In many respects, turtle CD units have discharge properties resembling those of calyx-bearing units in the mammalian central zone.

Three-dimensional analysis of vestibular efferent neurons innervating semicircular canals of the gerbil.

Anterograde labeling techniques were used to examine peripheral innervation patterns of vestibular efferent neurons in the crista ampullares of the gerbil. Vestibular efferent neurons were labeled by extracellular injections of biocytin or biotinylated dextran amine into the contralateral or ipsilateral dorsal subgroup of efferent cell bodies (group e) located dorsolateral to the facial nerve genu. Anterogradely labeled efferent terminal field varicosities consist mainly of boutons en passant with fewer of the terminal type. The bouton swellings are located predominately in apposition to the basolateral borders of the afferent calyces and type II hair cells, but several boutons were identified close to the hair cell apical border on both types. Three-dimensional reconstruction and morphological analysis of the terminal fields from these cells located in the sensory neuroepithelium of the anterior, horizontal, and posterior cristae were performed. We show that efferent neurons densely innervate each end organ in widespread terminal fields. Subepithelial bifurcations of parent axons were minimal, with extensive collateralization occurring after the axons penetrated the basement membrane of the neuroepithelium. Axonal branching ranged between the 6th and 27th orders and terminal field collecting area far exceeds that of the peripheral terminals of primary afferent neurons. The terminal fields of the efferent neurons display three morphologically heterogeneous types: central, peripheral, and planum. All cell types possess terminal fields displaying a high degree of anisotropy with orientations typically parallel to or within +/-45 degrees of the longitudinal axis if the crista. Terminal fields of the central and planum zones predominately project medially toward the transverse axis from the more laterally located penetration of the basement membrane by the parent axon. Peripheral zone terminal fields extend predominately toward the planum semilunatum. The innervation areas of efferent terminal fields display a trend from smallest to largest for the central, peripheral, and planum types, respectively. Neurons that innervate the central zone of the crista do not extend into the peripheral or planum regions. Conversely, those neurons with terminal fields in the peripheral or planum regions do not innervate the central zone of the sensory neuroepithelium. The central zone of the crista is innervated preferentially by efferent neurons with cell bodies located in the ipsilateral group e. The peripheral and planum zones of the crista are innervated preferentially by efferent neurons with cell bodies located in the contralateral group e. A model incorporating our anatomic observations is presented describing an ipsilateral closed-loop feedback between ipsilateral efferent neurons and the periphery and an open-loop feed-forward innervation from contralateral efferent neurons. A possible role for the vestibular efferent neurons in the modulation of semicircular canal afferent response dynamics is proposed.

Electrophysiological properties of vestibular sensory and supporting cells in the labyrinth slice before and during regeneration.

The whole cell patch-clamp technique in combination with the slice preparation was used to investigate the electrophysiological properties of pigeon semicircular canal sensory and supporting cells. These properties were also characterized in regenerating neuroepithelia of pigeons preinjected with streptomycin to kill the hair cells. Type II hair cells from each of the three semicircular canals showed similar, topographically related patterns of passive and active membrane properties. Hair cells located in the peripheral regions (zone I, near the planum semilunatum) had less negative resting potentials [0-current voltage in current-clamp mode (Vz) = -62.8 +/- 8.7 mV, mean +/- SD; n = 13] and smaller membrane capacitances (Cm = 5.0 +/- 0.9 pF, n = 14) than cells of the intermediate (zone II; Vz = -79.3 +/- 7.5 mV, n = 3; Cm = 5.9 +/- 1.2 pF, n = 4) and central (zone III; Vz = -68.0 +/- 9.6 mV, n = 17; Cm = 7.1 +/- 1.5 pF, n = 18) regions. In peripheral hair cells, ionic currents were dominated by a rapidly activating/inactivating outward K+ current, presumably an A-type K+ current (IKA). Little or no inwardly rectifying current was present in these cells. Conversely, ionic currents of central hair cells were dominated by a slowly activating/inactivating outward K+ current resembling a delayed rectifier K+ current (IKD). Moreover, an inward rectifying current at voltages negative to -80 mV was present in all central cells. This current was composed of two components: a slowly activating, noninactivating component (Ih), described in photoreceptors and saccular hair cells, and a faster-activating, partially inactivating component (IK1) also described in saccular hair cells in some species. Ih and IK1 were sometimes independently expressed by hair cells. Hair cells located in the intermediate region (zone II) had ionic currents more similar to those of central hair cells than peripheral hair cells. Outward currents in intermediate hair cells activated only slightly more quickly than those of the cells of the central region, but much more slowly than those of the peripheral cells. Additionally, intermediate hair cells, like central hair cells, always expressed an inward rectifying current. The regional distribution of outward rectifying potassium conductances resulted in macroscopic currents differing in peak-to-steady state ratio. We quantified this by measuring the peak (Gp) and steady-state (Gs) slope conductance in the linear region of the current-voltage relationship (-40 to 0 mV) for the hair cells located in the different zones. Gp/Gs average values (4.1 +/- 2.1, n = 15) from currents in peripheral hair cells were higher than those from intermediate hair cells (2.3 +/- 0.8, n = 4) and central hair cells(1.9 +/- 0.8, n = 21). The statistically significant differences (P < 0.001) in Gp/Gs ratios could be accounted for by KA channels being preferentially expressed in peripheral hair cells. Hair cell electrophysiological properties in animals pretreated with streptomycin were investigated at approximately 3 wk and approximately 9-10 wk post injection sequence (PIS). At 3 wk PIS, hair cells (all zones combined) had a statistically significantly (P < 0.001) lower Cm (4.6 +/- 1.1 pF, n = 24) and a statistically significantly (P < 0.01) lower Gp(48.4 +/- 20.8 nS, n = 26) than control animals (Cm = 6.2 +/- 1.6 pF, n = 36; Gp = 66 +/- 38.9 nS, n = 40). Regional differences in values of Vz, as well as the distribution of outward and inward rectifying currents, seen in control animals, were still obvious. But, differences in the relative contribution of the expression of the different ionic current components changed. This result could be explained by a relative decrease in IKA compared with IKD during that interval of regeneration, which was particularly evident in peripheral hair cells. (ABSTRACT TRUNCATED)

Localization of the GM1 ganglioside in the vestibular system using cholera toxin

Cholera toxin is an ubiquitous activator of intracellular adenylate cyclase and is divided in two major components: A and B. The B-component consists of several subunits that specifically bind to the external cell membrane. The receptor for the toxin, the GM1 ganglioside, is concentrated in nervous tissues. The B subunit of the cholera toxin, conjugated to different molecules (i.e. choleragenoid) is therefore a sensitive anatomical tracer and has been used to detect the presence of GM1 in mammalian tissues. Using choleragenoid, unlabeled and labeled with FITC, we have determined the distribution of the GM1 ganglioside in the vestibular system of the chinchilla.Vestibular tissues were fixed in 4% paraformaldehyde in phosphate buffer, decalcified in 10% EDTA and prepared as either whole-mount, surface-preparations, or for radial cryosections. Positive control tissue consisted of binding to normal brain tissues. Negative controls consisted of several treatments: masking of the GM1 receptors with unlabeled choleragenoid, tissue extraction of GM1 using ethanol, and preabsorbing the choleragenoid with bovine GM1. In addition, to exclude staining of glycoproteins that may have a carbohydrate structure similar to GM1, tissues were digested with trypsin prior to choleragenoid exposure.In the vestibular system, a strongly positive reaction was observed in: the sensory stereocilia and supporting cells of the maculae and cristae, epithelial cells of the planum semilunatum, and polygonal cells of the semicircular canal. Positive but less strong reactivity was observed in the sensory cell body of maculae and cristae, nerve fibers, epithelial cells of utricle and ampulla walls and flattened epithelial cells of the semicircular canals. No reactivity was present in the supporting connective tissue cells and fibrils, blood vessels, gelatinous cupula of the cristae ampullaris and statoconial membranes. Brain tissue showed strong choleragenoid reactivity. The negative controls showed no or greatly reduced reactivity to choleragenoid. Trypsin digestion did not decrease reactivity to choleragenoid.

Planum semilunatum of the rat: new light and electron microscopy observations.

Planum semilunatum (PSL) cells of the rat ampullae were studied by light, transmission, and scanning electron microscopy. The observed ultrastructure of rat PSL cells was similar to that described in other species, but is in disagreement with earlier reports of rat PSL cells, most probably because of previous divergent definitions of the PSL. Regions of PSL cells were easily distinguished from other nonsensory epithelia at the lateral ends of the crista ampullaris. The PSL region consisted of irregular-shaped columnar to cuboidal pentagonal or hexagonal cells that interdigitated with one another by lateral membrane infoldings. In the PSL region the subepithelial reticular layer appeared thickened and formed wartlike impressions in the basal surface of the PSL cells. These morphological characteristics of the subepithelial reticular layer were unique to the PSL region in the ampulla and may reflect special adaptations of the PSL region to mechanical stress. Furthermore, the thick subepithelial reticular layer may have implications for transport across the PSL region.

Quantitative immunoelectron microscopic localization of Na, K-ATPase α-subunit in the epithelial cells of rat vestibular apparatus

Na, K-ATPase was quantitatively localized in the epithelial cells of rat vestibular apparatus such as macula utriculi, macula sacculi and crista ampullaris. Immunogold localization method was carried out at the saturation level of antibody using an affinity purified antibody against the α-subunit of rat kidney Na, K-ATPase. Numerous gold particles were found on the basolateral membrane of the dark cells, a small number of gold particles were found on the basolateral membrane of the transitional epithelium cells and hair cells, but the luminal surface membranes of the hair cells, transitional epithelium cells, planum semilunatum cells and dark cells were rarely labeled by gold particles. Significance of the abundant localization of Na, K-ATPase on the basolateral surface of the dark cells in the production of endolymph was discussed.

Immunohistochemical findings of IgG recognized in the planum semilunatum of the guinea pig.

The planum semilunatum is located on the lateral side of the crista ampullaris of the semicircular canal. With regard to the functions of the planum semilunatum, some researchers assume the existence of secretory activity based on the microstructural features observed in isotopic experiments. In the present study, in order to confirm the existence of an immunological mechanism in the ampulla of the semicircular canal, the distribution of immunoglobulins in the semi-circular canal of guinea pigs was investigated immunohistochemically. Positivity of IgG was observed in the constituent cells of the planum semilunatum or beneath the basement membrane of the cells. It is concluded that ample IgG, which is an important constituent of the body fluids, is collected within this region. The planum semilunatum containing such ample amounts of IgG might be involved in the immunological mechanism operating in the ampullar of the semicircular canal.

Expression of nerve growth factor (NGF) receptors in the developing inner ear of chick and rat.

The expression of nerve growth factor receptors (NGFRs) was studied in the developing inner ear with in situ hybridization in chick embryos and with immunocytochemistry in rat embryos to determine sites of possible functions of NGF or NGF-like molecules in inner ear development. NGFR expression in the chick otocyst and acoustic ganglion is compared with epithelial differentiation and the onset of afferent innervation as determined with fluorescent carbocyanine tracers. In the inner ear of the chick embryo, NGFR mRNA expression shows an alternating pattern in mesenchymal and epithelial tissues. NGFR mRNA is heavily expressed in the mesenchyme surrounding the otocyst (E2-3), ceases at E3-5, and reappears in a thin layer of mesenchymal cells surrounding the membraneous epithelia (E5-13). In the otocyst epithelium, NGFR mRNA expression develops in one anterior and one posterior focus at E3-4.5. NGFR mRNA is expressed in the primordia of the ampullary cristae (E5-7) and possibly the anlage of the utricle; label transiently concentrates in the planum semilunatum of the cristae ampullares and in superior portions of the semicircular canals at E9, but is not seen in differentiating hair cells. In the acoustic ganglion, NGFR mRNA expression begins at E4; at the same time, the first peripheral acoustic nerve processes penetrate the otic epithelium (E4-4.5). The acoustic ganglia remain weakly NGFR mRNA-labeled in the posthatch animal. In the rat embryo, NGFR immunoreactivity is present in the auditory placode at E9, in the periotic mesenchyme at E9-10, and in the medial half of the otocyst at E10-11. At E12, epithelial NGFR expression becomes restricted anteriorly and posteriorly in a pattern similar to that of the chick otocyst and ceases at E13. NGFR immunoreactivity appears transiently in pillar cells of the cochlea in the third week of gestation. NGFR and NGFR mRNA is expressed after E11 in the acoustic ganglia. While NGFR transcripts are expressed in the cochlear ganglion cell bodies, NGFR protein becomes restricted to neuronal processes by the third week of gestation. The vestibular, but not the cochlear (spiral) ganglia remain NGFR-labeled in the adult rat. Onset of NGFR mRNA expression in the acoustic ganglion during the period of afferent fiber ingrowth into the otocyst epithelium is consistent with the hypothesis that NGF-like molecules may have a neurotrophic function for acoustic ganglion cells. Transient expression of NGFRs in secretory cells of the vestibular endorgan and pillar cells in the organ of Corti implicate a role for neurotrophins in the differentiation of these epithelial cell types.

Distribution of glycoconjugates in ion transport cells of gerbil inner ear.

Ion transport cells in gerbil inner ear were differentiated histochemically by staining glycoconjugates (GCs) with a battery of horseradish peroxidase-conjugated lectins. Strong staining with PSA and LCA showed a high content of N-linked oligosaccharides in transport cell GCs. Reactivity with PHA-L and PHA-E identified GC with triantennary and with bisected biantennary N-linked oligosaccharides, respectively, in these cells. High affinity for DSA and PWM demonstrated abundant N-acetyl lactosamine in N-linked side chains. Ion transporting epithelial cells reacting with lectins specific for N-linked oligosaccharides included strial marginal cells and outer sulcus cells of the cochlea and dark cells, transitional cells, and planum semilunatum cells of the vestibular system. In general, all of the inner ear transport epithelial cells revealed a similar lectin binding profile, with the one exception that SBA reacted strongly with ion transporting cells in the vestibular system but only weakly with those in the cochlea. Fibrocytes specialized for ion transport located in distinct areas in the suprastrial and inferior regions of the spiral ligament also stained with lectins that demonstrate N-glycosylation. However, transport fibrocytes differed from transport epithelial cells in two ways. First, they reacted e with HPA, DBA, VVA, and SJA specific for O-linkages and second, they failed to react with UEA I. The staining pattern for N-glycosylated GC resembled that for Na+, K(+)-ATPase in inner ear, suggesting a relationship between these constituents.

Immunohistochemical identification of proteoglycans in gelatinous membranes of cat and gerbil inner ear

Proteoglycans have been identified in gelatinous membranes of adult cat and gerbil inner ears using highly specific histochemical techniques. The tectorial and otoconial membranes and cupula of both species stained strongly with high iron diamine which is specific for sulfate esters and with monoclonal antibody against keratan sulfate proteoglycan (KSPG). The cat tectorial membrane also showed strong immunoreactivity with monoclonal antibody against chondroitin sulfate proteoglycan (CSPG) but the gerbil tectorial membrane reacted only weakly with this antibody. Otoconial membranes and the cupula of both species showed little if any immunostaining with antibodies against CSPG. Supporting cells in the vestibular neurosensory epithelium and planum semilunatum cells in the ampullae of the cat stained strongly with anti-KSPG, demonstrating the origin of KSPG in the cat. These cell types failed to stain in the gerbil, however, suggesting a different mechanism of secretion or a slower rate of turnover of membraneous KSPG in the gerbil. Interdental cells of both species failed to react with either antibody, leaving the origin of tectorial membrane proteoglycans in question. The approach used here provides a highly sensitive and reliable means of assessing the contribution of specific proteoglycans to inner ear structure and function.

Immunolocalization of Na+, K+-ATPase and carbonic anhydrase in the gerbil's vestibular system

The distribution of two ion transport enzymes in the vestibular system was investigated immunocytochemically. Immunostaining demonstrated abundant Na+, K+-ATPase in the basolateral plasmalemma of all dark cells and of cuboidal (transitional) cells bordering maculae and planum semilunatum cells bordering cristae. Na+, K+-ATPase was also present in nerve terminals impinging on vestibular hair cells and around nerve fibers and ganglion cells. Na+, K+-ATPase containing cells with fine intertwining processes were found within the perilymphatic stroma beneath maculae and cristae. These cells and interpersed nerves form a distinct, highly cellular plate that lies under neurosensory epithelium selectively. The catalytic α subunit of Na+, K+-ATPase in vestibular epithelia differs antigenically from the α subunit in nerves and from the α subunit in salivary gland and renal epithelium. Carbonic anhydrase (CA) isozyme II was localized in the apex of all supporting cells in neurosensory epithelia. In contrast, CA II immunostaining varied in vestibular dark cells showing heterogeneity in ion transport activity among these cells. Immunostaining evidenced CA II also in perilymphatic stromal cells which were presumably fibroblastic in nature and which correspond in location with the Na+, K+-ATPase positive cells under the vestibular neurosensory epithelium.

Ultrastructural localization of carbonic anhydrase in the vestibular end organs of the guinea pig

Carbonic anhydrase activity was demonstrated cytochemically on an ultrastructural level in the vestibular end organs of the guinea pig. Reaction product was found in the dark cells, transitional cells, cells of the planum semilunatum and supporting cells. In the dark cells, reaction product was observed in the cytoplasm as well as in the basal infoldings. Reaction product was also observed in the basal infoldings of the transitional cells and the cells of the planum semilunatum. The globular structures inside the supporting cells, transitional cells and the cells of the planum semilunatum were also surrounded by the reaction product. These findings suggest that carbonic anhydrase may have different functions, such as water and ion transport, respiration, nutrition and calcium carbonate deposition in the vestibular end organs.

Morphological Study of the Cells in the Planum Semilunatum

The fine structures of the epithelial cells in the planum semilunatum were investigated using scanning and transmission electron microscopy. The chicks and guinea pigs were used for this study. In both guinea pig and chick, the body of the planum semilunatum cell was cylindrical. Its length decreased towards the utricular and canal side, resulting in cuboid-shaped cells. In the guinea pig, the flat surfaced hexagonal cells with few microvilli were arranged like flagstones over the whole area of the planum semilunatum, while in the chick the cells with many prominent microvilli were found on both sides of the crista. The cytoplasm contained well developed endoplasmic reticula and abundant granules. Interdigitations between the neighbouring cells were well developed. Basement membrane was slightly folded but no basal infolding was observed. The morphological characteristics of the planum semilunatum cell highly suggest that these cells have some secreting function.

Cytochemical study of K+-p-nitrophenyl phosphatase (K+-NPPase) in planum semilunatum and dark cells of the squirrel monkey.

We examined the ultrastructure of the planum semilunatum, and also cytochemically demonstrated K+-dependent p-nitrophenyl phosphatase (K+-NPPase) activity in planum semilunatum and ampullary dark cells of the squirrel monkey. K+-NPPase activity was found in the basal part of the planum semilunatum. The reaction products were limited to the cytoplasmic side of the plasma membranes. A more intense reaction was found in the basal infoldings and the lateral interdigitations of the dark cells. On the other hand, the apical cell surface of both cell types showed no K+-NPPase activity. The present results show that both the planum semilunatum and the dark cells may both be involved in maintaining endolymph homeostasis.