Effect of calcium on the dissociation of the mature rat heart into individual and paired myocytes: electrical properties of cell pairs.

The dissociation of adult rat heart into individual, functionally intact, calcium-tolerant myocytes requires precise manipulation of extracellular calcium levels. Dissociation of intercellular connections is achieved by lowering extracellular calcium to micromolar levels for a short period. By imposing a very small increment in free calcium activity (from 14 to 17 microM) during this period, we achieve a significant yield of functionally intact pairs of myocytes still joined at the intercalated disc. We obtain fewer intact cells, but many of these are paired end to end. These findings permit us to describe some structural characteristics of intercellular connections between cardiac cells and to report unambiguous measurements of electrotonic coupling and dye transfer between rat cardiac cell pairs. We find that the strength of electrical coupling between cells isolated as pairs with intact junctional contacts is much greater than that measured between cell pairs that have formed new junctional contacts.

Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes.

Physiological properties of isolated pairs of rat hepatocytes were examined within 5 h after dissociation. These cells become round when separated, but cell pairs still display membrane specializations. Most notably, canaliculi are often present at appositional membranes which are flanked by abundant gap and tight junctions. These cell pairs are strongly dye-coupled; Lucifer Yellow CH injected into one cell rapidly diffuses to the other. Pairs of hepatocytes are closely coupled electrically. Conductance of the junctional membrane is not voltage sensitive: voltage clamp studies demonstrate that gj is constant in response to long (5 s) transjunctional voltage steps of either polarity (to greater than +/- 40 mV from rest). Junctional conductance (gj) between hepatocyte pairs is reduced by exposure to octanol (0.1 mM) and by intracellular acidification. Normal intracellular pH (pHi), measured with a liquid ion exchange microelectrode, was generally 7.1-7.4, and superfusion with saline equilibrated with 100% CO2 reduced pHi to 6.0-6.5. In the pHi range 7.5-6.6, gj was constant. Below pH 6.6, gj steeply decreased and at 6.1 coupling was undetectable. pHi recovered when cells were rinsed with normal saline; in most cases gj recovered in parallel so that gj values were similar for pHs obtained during acidification or recovery. The low apparent pK and very steep pHi-gj relation of the liver gap junction contrast with higher pKs and more gradually rising curves in other tissues. If H+ ions act directly on the junctional molecules, the channels that are presumably homologous in different tissues must differ with respect to reactive sites or their environment.

Cell junctions in early embryos of squid (Loligo pealei).

Squid embryos examined by freeze-fracture and thin-section electron microscopy exhibit identifiable gap junctions during mid-cleavage stages (stages 7-8), and junctional complexes composed of adherent appositions, elaborate septate junctions and gap junctions at slightly later stages (stages 12-13). During germinal layer establishment (stages 12-13) cytoplasmic bridges frequently link the embryonic cells. The presence of gap junctions in cleavage-stage embryos provides the morphological substrate for a demonstrated pathway of direct cell-cell communication that is modifiable by experimental treatments and may be physiologically regulatable. The existence of septate junctions and adherent contacts at later stages suggests that some functional specialization, perhaps the establishment of a strongly joined framework of cells at the surface of the embryo, accompanies the formation of germinal layers.

Freeze-fracture morphology of the vestibular hair cell--primary afferent synapse in the chick.

The intramembrane specializations at vestibular hair cell-primary afferent synapses have been identified and characterized in complementary freeze-fracture replicas from prehatch and hatchling chick cristae and maculae. Hair cell protoplasmic (P) faces at sites where presynaptic bodies are present exhibit small, tightly packed arrays of 9 nm particles. Hair cell external (E) faces have corresponding arrays of pits. Multiple arrays are often observed in contiguity. Opposite the presynaptic bodies, postsynaptic afferent boutons and calyces exhibit a more extensive array of scattered, irregular E-face particles. Corresponding P-fracture faces of afferent boutons and calyces display little topographical specialization opposite these E-face arrays, which are presumed to be the intramembrane correlate of the postsynaptic density. Examination of complementary replicas has allowed identification of the intramembrane synaptic specializations for all membrane faces at the synaptic apposition.

Fine structure of cochlear innervation in the cat.

Examination of adult and juvenile cat cochleas by electron microscopy and semi-serial sections permitted identification of the cytological features characteristic of the afferent and efferent nerve fiber populations identified in Golgi impregnations of the cochlea. This study demonstrated the distribution of synaptic contacts made by these fiber populations. As in the Golgi findings, radial and outer spiral afferent fibers were identified in well separated zones of the inner spiral bundle. The trunks of the outer spiral fibers, containing many microtubules and few neurofilaments, at first coursed spirally below the inner hair cells on the proximal face of the inner pillar, turned abruptly between adjacent pillar cells and entered the tunnel without branching. Radial afferents, containing many neurofilaments and a few microtubules, coursed through the inner spiral bundle, maintaining a radial or oblique orientation and proceeded directly toward the inner hair cells. Efferent fibers in the region of the inner spiral bundle were distinguishable by size, by orientation, and, to a lesser extent, by cytology. Small (1 micron) efferent fibers, containing few neurofilaments, an occasional microtubule, and mitochondria, occurred in the inner and tunnel spiral bundles and formed large varicosities, which contacted radial afferents. A separate population of much thicker efferents, containing many neurofilaments, mitochondria and dense-cored vesicles, but no microtubules, did not enter the inner spiral bundle but coursed directly to the level of the tunnel spiral bundle on the proximal face of the inner pillar cells. These fibers crossed the tunnel at the level of the tunnel spiral bundle and, upon reaching the outer hair cells, formed large synaptic contacts on outer hair cells and on outer spiral fibers as well. Some of these efferent fibers also synapse on afferent fibers while crossing the tunnel. The findings agree with previous observations with the Golgi method showing that entirely separate populations of spiral ganglion cells innervate the inner and outer hair cells. Likewise, there are efferent fibers innervating only inner or outer hair cells, but the probability of efferent fibers to both inner and outer hair cells cannot be ruled out.

A study of cochlear innervation in the young cat with the Golgi method.

Individual afferent and efferent nerve fibers were identified and traced in Golgi-impregnated cochleas of cats from newborn to one month old. Afferent radial fibers project radially without varicosities to terminate at the base of one or two inner hair cells. Outer spiral fibers have both radial and spiral orientations within the organ of Corti, do not form varicosities while crossing the base of the tunnel, and spiral for long distances in the outer spiral bundles. They contact many outer hair cells of more than one row both en passant and by small terminal branchlets. Two separate groups of efferent fibers are identifiable. Thin efferent fibers with many large varicosities spiral for long distances in the inner and tunnel spiral bundles; varicosities in the inner spiral bundle may contact radial afferent fibers or hair cells, depending on age. Thick radial efferent fibers course radially through the tunnel spiral bundle and across the upper part of the tunnel, often in fascicles. They contact a few outer hair cell bases by large terminals. The spiral expanse of the terminals is limited. These fibers are most common in the more basal turns of the organ. The present results confirm the anatomical separation of radial and spiral afferent fiber systems and identify two separate efferent populations beyond the neonatal period in the cat. The major features of afferent innervation discernible in Golgi-impregnated cochleas are present at birth, although some simplification of afferent fibers probably occurs during the first postnatal week. In contrast, the efferent fiber pattern undergoes important changes during the first few weeks after birth. In mature animals, the fine spiral efferents probably contact only afferent fibers, whereas the thick radial efferents may contact both outer hair cells and spiral afferent fibers. The possibility that some individual efferents branch to both inner and outer hair cell regions in the older cats cannot be ruled out.

Synaptogenesis in the vestibular sensory epithelium of the chick embryo.

The formation of synapses between sensory cells and the terminals of afferent axons has been examined in the embryonic chick labyrinth. Neurites initially cross the otocyst basal lamina and ramify among the undifferentiated epithelial cells by stage 25 Hamburger and Hamilton. At the same time granular vesicles, with diameters averaging 130nm, appear in the basal cytoplasm of a few of the epithelial cells. These vesicles often exist in groups at sites contact with; neurites. By stages 27-28, non membrane-bound densities are frequently found in association with groups of granular vesicles at the plasma membrane. Smaller, clear synaptic vesicles are also a prominent component of these arrangements in presumptive hair cells. Presynaptic ribbons opposite postsynaptic densites are identifiable at about stage 28, and their number increases during subsequent embryonic stages. Specialized appositions, including adherent, postsynaptic and possibly gap junctional contacts, join epithelial cells and nerve terminals throughout this period. The distribution of these junctions is variable, and is not necessarily correlated with the sites of formation of presynaptic ribbons. By stage 32, well-developed chemical synapses consisting of presynaptic ribbons witah vesicle halos and postsynaptic densities are common features of hair cell-afferent nerve terminal contact regions. In addition, possible sites of gap junctional contact between adjacent intra-epithelial nerve endings found at stage 32 presage those found in the cristae and maculae of pre-hatch (stage 45) embryos and adults.

Hormonally induced cell shape changes in cultured rat ovarian granulosa cells.

Cultured rat ovarian granulosa cells undergo a dramatic morphological change when exposed to follicle-stimulating hormone (FSH). Exposure to FSH causes the flattened epithelioid granulosa cells to assume a nearly spherical shape while retaining cytoplasmic processes which contact the substrate as well as adjacent cells. This effect of FSH is preceded by a dose-dependent increase in intracellular cAMP, is potentiated by cyclic nucleotide phosphodiesterase inhibitors, and is mimicked by dibutyryl cAMP. Prostaglandins E1 or E2 and cholera enterotoxin also cause the cells to change shape. A subpopulation of the cells responds to luteinizing hormone. These morphological changes, which are blocked by 2,4-dinitrophenol, resemble those produced by treating cultures with cytochalasin B. Electron microscopy shows that the unstimulated, flattened cells contain bundles of microfilaments particularly in the cortical and basal regions. After FSH stimulation, microfilament bundles are not found in the rounded granulosa cell bodies but they are present in the thin cytoplasmic processes. These data suggest that the morphological change results from a cAMP-mediated, energy-dependent mechanism that may involve the alteration of microfilaments in these cells.

Pigmentation of the stria vascularis. The contribution of neural crest melanocytes.

Because all stages of melanogenesis (premelanosome, melanosome and melanin granules) were found in intermediate cells of the rat stria vascularis, they can be classified as melanocytes. The marginal and basal cells of these rat specimens contained no pigment. Early in development, the melanocytes or future intermediate cells are located beneath the strial basal lamina. They penetrate this lamina to insert themselves between marginal cells. Both the melanocytes and marginal cells are believed to participate actively in the tissue rearrangements which ultimately bring the two cell types into intimate structural relationship. The ingrowing melanocytes and the mature, pigmented, intermediate cells which they form are frequently associated with blood vessels. Pretreatment with Dopa to enhance the melanin content of early melanocytes did not enable us to identify their route of migration into the stria.

Glycogen in the cochlea during development.

A recently developed technique for demonstrating glycogen by electron microscopy was used to study its location at various stages of cochlear development. It appears first in dispersed form in most of the cells of the otocyst at the stage of the 14-day rat embryo. Glycogen clums were found in Reissner's membrane at 17 days and in the future pillar cells shortly afterwards. Near the time of birth, glycogen appeared in the stria vascularis. By a few days after birth, heavy deposits were evident in the stria but glycogen had faded from the pillar cells and Reissner's membrane. Ten days after birth glycogen is no longer apparent in any of the cells of the cochlear duct except the outer hair cells.