Subsurface cisterns and lamellar bodies in the granule cells of the guinea-pig fascia dentata

Summary Subsurface cisterns (SSC's) and less frequent lamellar bodies (LB's) were identified in the granule cells of the guinea-pig fascia dentata. Both structures, composed of flattened or collapsed agranular cisterns, are continuous with the regular rough endoplasmic reticulum and occasionally connected with each other forming LB-SSC complexes. The SSC's are apposed to glia, synaptic boutons, and nerve cell processes as well as to neighboring granule cells appearing here singly and in confronting pairs. The quantitative analysis of the various cisternal appositions compared to the distribution of the tissue components on the granule cell soma shows that the overwhelming majority of SSC's are related to glial cells.     

A freeze-fracture study of afferent and efferent synapses of hair cells in the sensory epithelium of the organ of Corti in the guinea pig

Summary Afferent and efferent synapses of hair cells in the organ of Corti of the guinea pig have been examined in freeze-fracture replicas.Afferent synapse In the inner hair cells, intramembranous particles 10 nm in diameter are aggregated on the ridge on the P-face of the presynaptic membrane directly beneath the synaptic rod. In the outer hair cells, in which the synaptic rod is located in the presynaptic cytoplasm underneath the presynaptic membrane, small aggregations of intramembranous particles 10 nm in diameter can be found on the P-face of the presynaptic membrane corresponding to the site of the presynaptic dense projection. Intramembranous particles 10 nm in diameter are also densely aggregated on the P-face of the postsynaptic membrane of the outer hair cells.Efferent synapse of the outer hair cells Large intramembranous particles 13 nm in diameter are distributed in clusters composed of four to ten particles on the P-face of the presynaptic membrane. In the P-face of the postsynaptic membrane, disc-like aggregations of intramembranous particles 9 nm in diameter are found. The subsynaptic cistern covers the cytoplasmic surface of the postsynaptic membrane of the efferent synapse; it may cover more than one postsynaptic membrane when several efferent synapses are in close proximity to one another.     

Subsurface tubular system in the outer sensory cells of the rat cochlea

Summary The system of subsurface tubules in the outer sensory cells of the rat cochlea consists of tubules lying immediately beneath the cell membrane. The tubules extend from the region below the cuticular plate, follow without interruption along the contours of the cell membrane, and terminate as a complicated, branched system under the nucleus. Several mitochondria are found on the cytoplasmic side of the tubules. Microtubules are observed in the space between the subsurface tubules and the mitochondria, in contact with the membranes of the subsurface system and, rarely, in contact with small, stalked vesicles.Current theories on the role of the subsurface system are discussed and a new one, considering possible involvement of the system in transport of the synaptic transmitter, is proposed.     

An ultrastructural study of the development of afferent and efferent synapses on outer hair cells of the guinea pig organ of Corti

The guinea pig organ of Corti was studied using transmission electron microscopy, the second turn of the cochlea being examined at various ages between 20 days before birth and 30 days postnatal. Outer hair cells were examined at each of these ages. At all ages studied, the efferent (presynaptic) terminals are large and are packed with synaptic vesicles, whereas the afferent (postsynaptic) terminals are generally smaller, with a relatively small number of vesicles. During development, the subsynaptic cistern changes from a fragmented, diffuse profile extending over 50-70% of the length of the efferent contact zones, to a continuous, compact structure spanning neighbouring synapses. Synaptic vesicles in the efferent terminals are predominantly rounded in early development, flattened vesicles appearing postnatally. The synaptic bodies at afferent synapses do not change noticeably during development. Quantitative analysis revealed that the area of efferent terminals and the length of their active zone increase with increasing age, the same parameters decreasing in afferent terminals. Synaptic vesicles in the efferent terminals decrease in diameter, but remain constant in afferent terminals, with increasing age. The number of hair cell membrane invaginations decreases as development proceeds.     

Subsurface cisterns and lamellar bodies: Particular forms of the endoplasmic reticulum in the neurons

Summary Structures identified as subsurface cisterns (SSC's) and lamellar bodies (LB's) have been observed in the neurons, but not in the glial cells, of the rat and cat substantia nigra. The SSC's are most often opposite what appears to be glial cells, but they are also subsynaptic in position. A single, large (0.4–1.5 ), unfenestrated, usually flattened cistern closely underlies the inner aspect of the plasma membrane of the perikaryon and proximal parts of the neuronal processes at a regular interval ranging about 100–130 Å. They are sheet-like or discoid in configuration and consists of a pentalaminar structure which usually widens at its lateral edges where its membranes are continuous with each other or with rough ER profiles. Filaments, about 70 Å thick, bridge the cleft between the SSC and the overlying plasmalemma. One or more ER cisterns devoid of ribosomes except on their outermost membrane may be stacked up parallel to an SSC and immediately subjecent to it. A dense filamentous network intervenes between the SSC and its closely applied ER cisterns. At higher magnification, it is seen to consist of a finely textured material which is apparently composed of loosely packed tiny particles. These constituent subunits in turn may represent transverse sections of very fine filaments rather than granules. A mitochondrion frequently occurs in the immediate vicinity of an SSC and may be closely applied to its deep surface. Stacks of unfenestrated, parallel, regularly spaced (about 300–400 Å) cisterns, designated lamellar bodies, appear deeper in the karyoplasm. They are most often flattened and appear as pentalaminar structures. These cisterns, as well as the dense filamentous network intervening between them, are structurally similar to those closely applied to SSC's. They are also devoid of ribosomes except on their outermost surfaces. Whorls of similar cisterns are also occasionally observed. Another particular feature of the rough ER consists of the close apposition of two cisterns without any ribosome attached to the inner membranes of the latter structure. It evokes a simplified type of LB's. It is of particular interest to point out that all these cisterns, i.e. the SSC's, their closely applied cistern(s) and those forming the LB's, are connected to the RER membranes, so that a continuous channel occurs between the nuclear membrane and the SSC which closely underlies the plasma membrane. Our observations show that the SSC's and the LB's are structurally related forms of the ER. A parallel may be drawn between the SSC and the lateral element(s) of a dyad (triad). The structural complex consisting of an SSC, the overlying plasmalemma and the cross-bridges linking them, indeed, bears some resemblance to a dyad. It is suggested that membranes which are closely applied may interact, resulting in alterations in their respective properties. These patches of the neuronal plasma membrane associated with SSC's may, therefore, have special properties because of this relationship, resulting in a non-uniform spread of an action potential on the neuronal surface. The possible significance of SSC's in relation to neuronal electrophysiology, as well as of the latter structures and LB's in relation to cell metabolism, is to be discussed.This work was supported by grant MA-3448 from the Medical Research Council of Canada. The skilful technical assistance of Mrs. Marjolaine Turcotte is gratefully acknowledged.     

Orthogonal arrays of intramembrane particles in the supporting cells of the guinea-pig vestibular sensory epithelium

Membrane specializations of the contact region between afferent nerve endings and supporting cells of the sensory epithelia of guinea-pig vestibular endorgans were examined by thin-section and freeze-fracture electron microscopy. The calyx-type nerve endings (C-endings) are separated from supporting cells (SC) by a 25-30 nm space. At irregular intervals along the upper lateral surface of supporting cells, the intercellular space narrows markedly to form special close contacts between the C-ending and SC plasma membranes. Freeze-fracture replicas reveal membrane specializations--orthogonal arrays of particulate units--in the region where the close intercellular contacts were found in sections. Orthogonal arrays consisting of from 5 to 20 units were observed on the cytoplasmic (P) fracture face of the lateral SC plasma membrane. These particulate units from a 12 x 12-nm square, and each unit is composed of four 6-nm subunits. Possible roles of the orthogonal arrays are discussed.     

Defining the cellular environment in the organ of Corti following extensive hair cell loss: a basis for future sensory cell replacement in the Cochlea

Background

Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss.

Methodology/Principal Findings

Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca''s, suggesting genetic background influences the rate of re-organisation.

Conclusions/Significance

The lack of dedifferentiation amongst supporting cells and their replacement by cells from the outer side of the organ of Corti are factors that may need to be considered in any attempt to promote endogenous hair cell regeneration. The variability of the cellular environment along an individual cochlea arising from patch-like generation of flat epithelium, and the possible variability between individuals resulting from genetic influences on the rate at which remodelling occurs may pose challenges to devising the appropriate regenerative therapy for a deaf patient.     

Fine structure of a sensory organ in the arista of Drosophila melanogaster and some other dipterans

Summary The arista, a characteristic appendage of dipteran antennae, consists of 2 short segments at the base and a long distal shaft. A small sensory ganglion, from which arises the aristal nerve, is located proximally in the shaft. The fine structure of the aristal sensory organ was studied in detail in the fruitfly (Drosophila) and for comparison in the housefly (Musca) and the blowfly (Calliphora). In Drosophila, the aristal sense organ consists of 3 identical sensilla that terminate in the hemolymph space of the aristal shaft, and not in an external cuticular apparatus. Each sensillum comprises 2 bipolar neurons and 2 sheath cells; a third sheath cell envelops the somata of all six neurons of the ganglion. The neurons have long slender dendrites with the usual subdivision into an inner and an outer segment. One of the outer segments is highly lamellated and bears small particles (BOSS-structures) on the outside of its cell membrane; the other outer segment is unbranched and has a small diameter. The fine structure of the first dendrite is strongly reminiscent of thermoreceptors known from the antennae of other insects. These thermoreceptors are often coupled with hygroreceptors; however, we can only speculate whether the second dendrite of the aristal organ also has this function. Our present results argue against mechanoreceptive functions, as formerly postulated. The aristal sense organs in Musca and Calliphora are similar to those in Drosophila, but contain more sensilla (12 in Musca, 18 in Calliphora).     

Outer hair cell somatic electromotility in vivo and power transfer to the organ of Corti

The active amplification of sound-induced vibrations in the cochlea, known to be crucial for auditory sensitivity and frequency selectivity, is not well understood. The outer hair cell (OHC) somatic electromotility is a potential mechanism for such amplification. Its effectiveness in vivo is putatively limited by the electrical low-pass filtering of the cell's transmembrane potential. However, the transmembrane potential is an incomplete metric. We propose and estimate two metrics to evaluate the effectiveness of OHC electromotility in vivo. One metric is the OHC electromechanical ratio defined as the amplitude of the ratio of OHC displacement to the change in its transmembrane potential. The in vivo electromechanical ratio is derived from the recently measured in vivo displacements of the reticular lamina and the basilar membrane at the 19 kHz characteristic place in guinea pigs and using a model. The ratio, after accounting for the differences in OHC vibration in situ due to the impedances from the adjacent structures, is in agreement with the literature values of the in vitro electromechanical ratio measured by others. The second and more insightful metric is the OHC somatic power. Our analysis demonstrates that the organ of Corti is nearly optimized to receive maximum somatic power in vivo and that the estimated somatic power could account for the active amplification.     

Non-sensory cells in the deafened organ of Corti: approaches for repair

After moderate cochlear trauma, hair cells degenerate and their places are taken by phalangeal scars formed by differentiated supporting cells. A short time after trauma, these supporting cells can respond to an induced expression of genes which signal hair cell differentiation during normal development and transdifferentiate into new hair cells. However, these non-sensory cells often lose their differentiated features after severe insults or prolonged hearing loss and become a simple, flat epithelium. The flat organ of Corti can serve as a substrate for gene- and stem cell-based therapies. Despite its prevalence, the flat epithelium is not well characterized. Recent data show that cells of the flat epithelium can divide and maintain the structural confluence of the membranous labyrinth. The mitotic potential of these cells should facilitate production of cells for therapies based on recapitulation of development or insertion of stem cells.     

Localization of cholinergic and purinergic receptors on outer hair cells isolated from the guinea-pig cochlea.

Acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are shown to act in opposing fashion on guinea-pig cochlear outer hair cells (OHCS) via receptors localized within different fluid compartments of the organ of Corti. The cholinergic (efferent) receptors localized at the basal (perilymphatic) region of these cells activated a rapidly desensitizing hyperpolarizing K+ current. In contrast, purinergic (ATP) receptors were localized at the apical (endolymphatic) surface of OHCS and activated a depolarizing nonselective cation current which exhibited inward rectification and lacked desensitization. Localization of the receptors was determined by using whole-cell patch-clamp, by recording onset latencies and response amplitudes to pulses of either ACh or ATP pressure-applied at selected sites along the length of isolated OHCS. Under voltage-clamp at -60 mV, the largest ACh-induced (outward) currents were recorded when ACh was directed at the basal region of the cells. Conversely, the maximum (inward) ATP currents were obtained when ATP was directed toward the apical surface of these cells. Onset latencies increased rapidly from a minimum of approximately 10 ms for either ACh or ATP as the drug pipette was moved away from these optimal sites. The ATP response was antagonized by amiloride in a dose-dependent manner with a KD of approximately 400 microM. The localization of P2-type purinoceptors to the endolymphatic surface of OHCS suggests that ATP mediates a humoral modulation of the mechano-electrical transduction process.     

New tunes from Corti's organ: the outer hair cell boogie rules

The amplification of acoustic stimuli is a feature of hair cells that evolved early on in vertebrates. Though standard stereocilia mechanisms to promote such amplification may persist in the mammal, an additional mechanism evolved to enhance high frequency sensation. Only in mammals, a special cell type, the outer hair cell, arose that possesses a remarkably fast somatic mechanical response, which probably endows the passive cochlea with a boost in sensitivity by a factor of 100 (40dB), at least. Experiments conducted over the past few years have shed light on many aspects of outer hair cell electromotility, including the molecular identification of the motor, the effects of a knockout, and underlying mechanisms of action. A review of this remarkable progress is attempted.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Ciliary-structure precursor bodies as stable constituents in the sensory cells of the vomero-nasal organ of reptiles and mammals

Summary The sensory cells of the vomero-nasal organ in reptiles and mammals do not develop cilia. In several species they contain centrioles together with cilium-structure precursor bodies measuring 400–700 Å in diameter. These structures resemble axonemal precursor bodies which are known to occur in developing ciliated cells. They are enclosed in a fibrogranular matrix. The precursor bodies are resistant to pepsin digestion in Araldite sections. In Tupaia precursor bodies may join periodically in a row. In the vomero-nasal receptor cells the precursor bodies can be considered stabilized with a corresponding reduction of cilia. The periodically arranged precursor bodies could represent a special storage form.