Specializations of plasma membranes in Pacinian corpuscles: Implications for mechano-electric transduction

Summary Pacinian corpuscles of cat mesentery were studied with freeze-fracture and thin sectioning methods after chemical fixation.Intramembranous particles (IMPs) exhibit differences in both density and pattern of distribution between the axolemma of the smooth short axis (x-axis) region and that of the axonal spine region of the long axis (y-axis) of the axon terminal. The axolemma of thex-axis has IMPs at a density of 2687±581 per m² (mean±S.E.M.), and these particles are 9.0±1.7 nm (mean ±S.D.) in diameter. In contrast, the axolemma of they-axis has a higher density of IMPs (3607±612 per m²) which are larger (diameter, 10.0±1.7 nm). The particle distribution is not homogeneous inx-axis membranes as there are small patchy areas devoid of particles scattered throughout the entire surface. The E-face of the axolemma has a low density of IMPs ( 200 perm² in bothx- andy-axes). However, IMPs in the E-face are smaller (9nm) in thex-axis than in they-axis (10nm).The inner core lamellar cells have IMPs at a density of 3276±739 per m² and 553±169 per m² in the P- and E-faces, respectively. The particles are about 10 nm in diameter in both faces. Many gap junctions occur between lamellar cells especially near the clefts, suggesting that hemilamellae of each inner core half are kept at the same electrotonic potential.The outer core lamellar cells have IMPs at a density of 2239±403 per m² and 536±123 per m² in their P- and E-faces, respectively. The particles are approximately 10 nm in diameter in both faces. A noteworthy finding is that tight junctions are prominent at cell-to-cell appositions within individual lamellae, especially in the first and second (or sometimes third) innermost lamellae of the outer core. These tight junctions are considered to be a barrier to the leakage of fluid and/or ions between interlamellar spaces as well as between inner and outer cores.An intermediate cell layer is identified between the inner and outer cores. The connective tissue space of this cell layer corresponds to the endoneurium, indicating that intermediate layer cells are comparable to endoneurial fibroblasts. These cells exhibit a low density of IMPs (658±119 per m² in the P-face) and particles are about 9 nm in diameter.The above findings indicate that the plasmalemmata of the axon terminal of the inner and outer core cells are specialized in terms of content and distribution of IMPs. The difference in axolemmal IMPs between thex-axis and they-axis suggests that there is some separation of function in components of mechano-electric transduction between these two regions. In addition it can be assumed that the hemilamellae of each inner core half constitute an electrotonically coupled environment on each side of the axon terminal abutting thex-axis. Furthermore, the fluid of the inner core is completely segregated and probably different in composition from that of the outer core.     

Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves

Summary The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy.The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (1600 1100 m^–2, respectively), while relatively few IMPs (150 m^–2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (600–2300 m^–2) and, in most regions, E-faces displayed a relatively low IMP density (175 m^–2). Axons of > 0.4 m diameter exhibited significantly greater mean P-face IMP density than axons < 0.4 m diameter. Aggregations of E-face IMPs (350 m^–2) were occasionally observed along amyelinated axon membrane from md optic nerves.Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (1800 and 140 m^–2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (> 0.4 m) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (< 0.4 m) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (200 m^–2); however, focal aggregations (400 m^–2) of E-face particles are present.Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves. These results are consistent with persistence of action potential conduction in md axons, despite the absence of myelination in the optic nerves of the md mutant.     

Plasma membrane structure at the axon hillock, initial segment and cell body of frog dorsal root ganglion cells

Summary Analysis of the plasmalemma of frog dorsal root ganglion cells by freeze-fracture demonstrates regional differences in the distribution of intramembranous particles. Although P-face particles are distributed rather uniformly, the E-face particle concentration at the cell body (300 m^–2) is much lower than that at the axon hillock (900 m^–2), proximal initial segment (1000 m^–2), or intermediate portion of the initial segment (800 m^–2). The particle concentrations in the latter regions approach that at the node of Ranvier and, moreover, particle size analysis reveals that the E-face particles, like those at the node, include a large number that are 10 nm or more in diameter. Thin sections reveal patches of a dense undercoating on the cytoplasmic surface of the axolemma in some regions of the initial segment but not the axon hillock. It is concluded from these results that the axon hillock and the initial segment of dorsal root ganglion cells have some of the structural characteristics of the node of Ranvier.     

Effects of delayed myelination by oligodendrocytes and Schwann cells on the macromolecular structure of axonal membrane in rat spinal cord

Summary The macromolecular structure of axonal membrane from dorsal funiculi of control and irradiated spinal cord of 45-day-old rats was examined with freeze-lracture electron microscopy. In control spinal cords, virtually all myelination is mediated by oligodendrocytes, and the internodal axonal membrane of these fibres displays highly asymmetrical partitioning of intramembranous particles (IMPs). The internodal P-face particle density is 2350 IMPs per m², whereas the E-face IMP density is 150 per m². In control dorsal spinal roots, myelination is mediated by Schwann cells, and the ultrastructure of the internodal axolemma of the myelinated fibres is similar to that displayed by myelinated fibres of dorsal funiculi. On the internodal P-face of Schwann cell-myelinated fibres the IMP density is 2350 per m², whereas on the E-face the density is 175 per m². Irradiation of the lumbosacral spinal cord at 3 days of age results in a glial cell-deficient region within the spinal cord such that myelination in irradiated dorsal funiculi is delayed and subsequent myelination is mediated by both oligodendrocytes and Schwann cells. By 45 days of age, dorsal funiculi of irradiated spinal cords are well populated with fibres myelinated by oligodendrocytes and Schwann cells. However, fibres myelinated by oligodendrocytes display very thin myelin sheaths whereas Schwann cell-myelinated fibres exhibit myelin sheaths with normal thicknesses. Internodal membrane of fibres myelinated by Schwann cells and oligodendrocytes exhibit similar macromolecular structure, with 2400 IMPs per m² on P-faces and 150 IMPs per m² on E-faces. Occasional large (>1.5 m diameter) axons without glial-Schwann cell ensheathment are observed. These axons display a high density of P-face particles (2000 per m²) and a moderate density (350 per m²) of E-face IMPs on their fracture faces. These results demonstrate that CNS fibres exhibit similar axonal membrane ultrastructure irrespective of whether they are myelinated by Schwann cells or oligodendrocytes, or whether myelination is delayed. Moreover, when myelination does not occur, the axolemmal E-face IMP density, which may be related to the density of voltage-sensitive sodium channels, is not reduced.     

Noise analysis of cAMP-stimulated Na current in frog colon

The effects of oxytocin and cAMP on the electrogenic Na⁺-transport in the short-circuited epithelium of the frog colon (Rana esculenta, Rana temporaria) were investigated. Oxytocin (100 mU · ml^–1) elevated the shortcircuit current (I _sc) transiently by 70% whereas cAMP (1 mmol · l^–1) elicited a comparable sustained response. The mechanism of the natriferic action of cAMP was studied by analysing current fluctuations through apical Na⁺-channels induced by amiloride or CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamid). The noise data were used to calculate Na⁺-channel density (M) and single apical Na⁺-current (i _Na).i _Na-Values obtained with amiloride and CDPC were 1.0±0.1 pA (n=5) and 1.1±0.2 pA (n=6) respectively and unaffected by cAMP. On the other hand, cAMP caused a significant increase in M from 0.23±0.08 m^–2 (n=5) to 0.49±0.17 m^–2 (n=5) in the amiloride experiments. In our studies with CDPC we obtained smaller values for M in control (0.12±0.04 m^–2;n=6) as well as during cAMP treatment (0.19±0.06 m^–2;n=6). However, the cAMP-induced increase in M was also significant. We conclude that cAMP stimulates Na⁺-transport across the frog colon by activating silent apical Na⁺-channels. Thus, the mechanism of regulation of colonic Na-transport in frogs differs considerably from that in other vertebrates as mammals and birds.     

Membrane specialization and axo-glial association in the rat retinal nerve fibre layer: freeze-fracture observations

Summary The ultrastrucrure of non-myelinated ganglion cell axolemma within the retinal nerve fibre layer of adult rats was examined by thin section and freeze-fracture electron microscopy. Most of the axolemma within the nerve fibre layer does not exhibit any membrane specializations; intramembranous particles are partitioned with a density of 1750 m^–2 on the P-fracture face and 225 m^–2 on the E-face of the non-specialized axolemma. The nerve fibres also exhibit specialized foci of axolemma, at which the axons are abutted by the tips of blunt, radially oriented processes from Müller cells. At such sites of axo-glial association, an electron-dense undercoating is present beneath the axon membrane. Freeze-fracture analysis revealed a substantial increase in the density of E-face particles (>500 m^–2) at sites of association between the tips of blunt glial processes and the axon. These findings demonstrate that non-myelinated axolemma of the retinal nerve fibre layer can exhibit spatial heterogeneity, with patches of node-like membrane at regions of specialized association with glial cell processes. On the basis of their morphological similarity to nodes of Ranvier, we suggest that these specialized axon regions represent foci of inward ionic current.     

Differentiation of the nodal and internodal axolemma in the optic nerves of neonatal rats

Summary Axon plasma membranes (axolemma) were studied by freeze-fracture electron microscopy at stages prior to and during myelination in the optic nerves of neonatal rats. In unensheathed axons, intramembranous particles associated with the internal (P) and external (E) leaflets of the axolemma increased in number before reaching a plateau (approximately 600/m² in both leaflets) at about 9 days postnatally. In newly myelinated fibres, by contrast, the distribution of particles was asymmetrical; fewer particles (approximately 200/m²) were found on the E-face and greater numbers (approximately 1400/m²) were present on the P-face, distributions similar to those observed in mature myelinated fibres. Node-like aggregations of particles were not found in unensheathed pre-myelinated axons nor were they present in axons presumed to be ensheathed by glial cytoplasm but not yet myelinated, although nodal specializations could be easily identified in fibres with only a few turns of compact myelin. These observations show first that there is a redistribution of particles in the P- and E-faces of the internodal axolemma coincident with the onset of myelination and secondly, that nodal specializations (represented by the increased densities of E-face particles) appear after ensheathment but before the formation of compact myelin in fibres of the rat optic nerve.This work was presented in part at the Society for Neurosciences Meeting, 1980.     

Axo-glial relations in the retina-optic nerve junction of the adult rat: freeze-fracture observations on axon membrane structure

Summary The axolemmal ultrastructure of nerve fibres within the retina-optic nerve junction (ROJ) from adult rats was examined by freeze-fracture electron microscopy. In the juxtaocular (proximal) region of the ROJ, all fibres are unmyelinated. The axons generally have a membrane ultrastructure similar to that of retinal nerve fibre layer axons, with a high density of intramembranous particles (IMPs) on the P-fracture face and a low density of IMPs on the E-face. However, along some axons in this region of the ROJ, localized aggregations of E-face IMPs are observed. At levels of the ROJ closer to the optic nerve proper, the unmyelinated fibres enter a transition zone in which the axons acquire myelin sheaths. By the distal boundary of the transitional zone (optic nerve proper), virtually all fibres are myelinated. Within the transitional zone, conventional axo-glial associations and axolemmal ultrastructure is present at nodes of Ranvier. In addition, atypical axo-glial relationships and atypical nodal segments are observed in this region. At some nodes, an isolated oligodendroglial process traverses obliquely across the nodal membrane. Beneath the oligodendroglial process, the axolemma usually displays a paranodal-like ultrastructure. Finger-like oligodendroglial processes were also observed in association with non-nodal unmyelinated axon membrane. At these sites of association, the axon membrane tends to be indented and may have a paranodal-like morphology. Nodal axolemma may exhibit several atypical forms in the transition zone. At some nodes, the nodal axolemma has a low density of E-face particles. Also, nodes of extended linear length (2m) exhibit a lower-than-normal density of P-face IMPs. At heminodes, the axolemma immediately adjacent to the terminal loops lacks the usual nodal characteristics of high IMP density and high percentage of large particles. The results show that aberrant axo-glial associations accompanied by unusual ultrastructural characteristics of the axolemma are present in the ROJ of normal adult rats.     

Quantitative estimation of the area of luminal and basolateral membranes of rat parotid acinar cells: some physiological applications

Knowledge of luminal and basolateral acinar cell membrane areas of the secretory endpieces is a prerequisite for a detailed quantitative analysis of the ion transport involved in secretion of the primary saliva. In the present study, these areas were estimated in rat parotid acinar cells using standard stereological methods. A total of 480 micrographs — obtained by random sampling from eight glands from four rats — were analysed at a final magnification of 40000x. Expressed per unit cell volume, the area of the luminal acinar cell membrane was: 0.125 m² · m^–3 (SEM=0.027 m² · m^–3, n=4 animals) and the area of the basolateral membrane was: 1.54 m² · m^–3 (SEM=0.085 m² · m^–3, n=4 animals). These figures make it possible to perform a synthesis based upon different categories of experimental data, e.g. on ion fluxes, membrane potentials and single-channel conductances. Thus, we have estimated the density of open, low-conductance Cl^– channels in the luminal membrane — which are not readily accessible for direct, patch-clamp analysis — to be approximately 18 channels per m² in the stimulated state.     

Freeze-fracture study of the plasma membranes of the septal olfactory organ of Masera

Summary The olfactory border and the apical cell contacts of the organ of Masera (MO) of the mouse were investigated by freeze-fracture electron microscopy. The olfactory border is mainly composed of the terminals of receptor and supporting cells. Cells with thick microvillus-like projections, though less frequent than the other two cell types, also contribute to the border. Olfactory knobs show transitions between those displaying numerous cilia and those characterized by few or no cilia. The olfactory cilia have a typical necklace of 6–9 rows of particles. The eruption of developing cilia seems to be preceded by the formation of circular arrays of particles. The density of intramembranous particles (IMP) per m² in P- and E-faces of the ciliary membranes is 1095 ± 190 and 205 ± 65, respectively. In the microvilli of supporting cells, the density of IMP per m² is 1800 ± 270 for the P-face and 570 ± 135 for the E-face. At the base of the supporting cell microvilli, rod-shaped particles are observed. The lateral plasma membranes of these cells bear orthogonal arrays of particles. In the apical region of the MO neuroepithelium, extensive zonulae occludentes are present which seal the intercellular cleft. The zonulae occludentes between supporting and receptor cells are composed of 5–13 junctional strands, usually arranged in an elongate network. Zonulae occludentes between supporting cells are, in addition to the elongate network, also arranged in a mesh-like pattern. Gap junctions, both associated with the zonulae occludentes and independent of them, are occasionally found between supporting cells. The results obtained indicate that important similarities exist between the neuroepithelium of the MO and the olfactory epithelium proper, whereas remarkable differences exist between the MO and the vomeronasal neuroepithelium.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

Intramembranous particle distribution at the node of Ranvier and adjacent axolemma in myelinated axons of the frog brain

Summary The plasma membrane of myelinated axons in the frog brain has been examined by the freeze-fracture technique. The cytoplasmic leaflet of the axolemma contains numerous randomly distributed particles in nodal and internodal regions but relatively fewer particles in the axoglial junctional portion of the paranodal region. Particle distribution is even less uniform in the outer leaflet of the axolemma, which contains a low concentration of particles in the internodal region and a relatively high concentration at the node of Ranvier (1200 particles m^–2). The nodal particles tend to be larger than most intramembranous particles, approaching 200 Å diameter. The paranodal region of the leaflet is virtually devoid of such particles except in the narrow helical groove which faces extracellular clefts between terminating glial processes. In places this pathway widens to form lakes up to 0.3 m² area which contain large numbers of large particles resembling those at the node. The concentration of particles at the node is in the same range as the concentration of sodium channels estimated to be in this region and it is suggested on the basis of their location and concentration that these particles represent ionophores. The distribution of particles in the paranodal region suggests that the large intramembranous particles do not have free access to the axoglial junctional portion of the membrane and therefore the movement of such particles along the paranodal region of the membrane may occur primarily in the membrane of the groove spiraling through this portion of the axolemma. Such a restriction in surface area for particle movements on either side of the node of Ranvier could result in trapping of particles at the node and thus contribute to their concentration in the nodal axolemma.     

2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl]methyl)hexanamide (NPC 15437): A selective inhibitor of protein kinase C

NPC 15437 inhibited protein kinase C (PKC) activity and [³H]phorbol 12,13-dibutyrate (PDBu) binding to the enzyme in a concentration-dependent manner (IC₅₀ values, 19±2 M and 23±4 M, respectively). No inhibition of cAMP-dependent protein kinase A (PKA) or calcium/calmodulin-dependent myosin light chain kinase (MLCK) was observed. A detailed kinetic analysis of the interaction of NPC 15437 and a homogeneous preparation of PKC-alpha revealed a competitive type of inhibition with respect to activation of the enzyme by both phorbol 12-myristate 13-acetate (PMA) (K _i =5±3 M) and phosphatidylserine (PS) (K _i =12±4 M). Mixed inhibition (predominantly of the non-competitive type), with respect to activation of the enzyme by calcium, was also observed. These studies indicate that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the molecule. NPC 15437 inhibited phorbol ester-induced ear edema in mouse (IC₅₀=175 g/ear) demonstrating the ability of NPC 15437 to inhibit PKC-mediated activity in intact cells.     

Transient improvement of polymorphonuclear leukocyte function by splenectomy in β-thalassemia

Host defense mechanisms in transfusion-dependent non-splenectomized patients with -thalassemia were studied. Polymorphonuclear leukocytes (PMNLs) of non-splenectomized patients responded poorly to zymosan generated chemotactic factors. Chemotactic indices were 22.1 m ± 2.8 (mean ± S.D.) using zymosan activated serum (ZAS) as the attractant in comparison to 20.4 m ± 2.6 when fresh untreated serum was used. In contrast, chemotactic indices of normal PMNLs increased from 21.1 m to 33.6 m ± 3.1 in response to ZAS. Normal PMNL responses to a mixture of normal ZAS and thalassemic serum were inhibited; the mean chemotactic index was 18.1 m ± 5.1 with use of ZAS alone. Splenectomy temporarily reverses these alterations. Adherence to nylon wool of PMNLs suspended in fresh thalassemic serum prior to splenectomy was 3.1% ± 1.1 (mean ± S.D.); 20 days after splenectomy adherence increased to 14.0% ± 2.8 (P = 0.0001) and remained at this level for 90 days. At 120 and 150 days after splenectomy adherence decreased to 1.5% ± 0.8 and 1.0% ± 0.85 respectively. Splenectomy also transiently abrogated the failure of zymosan to generate chemotactic factors in thalassemic serum.This study was presented in part at the American Federation for Clinical Research, Central Society, Infectious Diseases, Chicago, Illinois, November 3, 1983     

Das Pferdearteriitisvirus (Equine Arteritis Virus): Seine Vermehrung in BHK 21-Zellen,die Bestimmung der Flotationsdichte und die elektronenoptische Darstellung

Zusammenfassung Pferdearteriitisvirus (EAV) Stamm Bucyrus wurde in BHK 21-Zellkulturen gezüchtet und die extra- und intrazelluläre Virusvermehrung ermittelt.Die Flotationsdichte des Virus beträgt 1,20 g/cm³.Nach Konzentrierung durch Ultrazentrifugierung und partieller Reinigung durch Dichte- und Zonenzentrifugierung im Saccharosegradienten konnten elektronenoptisch Viruspartikel eines Durchmessers von 60 ± 13 m nachgewiesen werden. Die Viria bestehen aus einer Hülle ohne Projektionen und einem Innenkörper von 35 ± 9 m.Die taxonomische Klassifikation des Virus wird diskutiert.

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Equine arteritis virus: Multiplication in BHK 21-cells, buoyant density and electron microscopical demonstration

Summary Equine Arteritis Virus (EAV) strain Bucyrus was multiplied in BHK21-cells; a growth curve considering extra- and intracellular virus was established.The buoyant density of EAV in sucrose gradients was 1.20 g/ml. After concentration of virus material by ultracentrifugation and partial purification by isopycnic and rate zonal centrifugations in sucrose gradients virus particles of 60 ± 13 m, size were demonstrated electron microscopically. The viria consist of an envelope without projections and an internal core of 35 ± 9 m in diameter.The results are discussed with respect to virus classification.

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Über Teilergebnisse der Untersuchungen wurde auf der 2. International Conference on Equine Infectious Diseases vom 15.–18. Juni 1969 in Paris berichtet. (Proc. 2nd int. Conf. Equine Infectious Diseases, Paris 1969, Karger, Basel/New York 1970.)

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Für gewissenhafte Mitarbeit danken wir Frau A. Adam, Fräulein H. Krebs und Frau R. Haller, für die Ausführung der Photoarbeiten und die Zeichnuugen Herrn E. Queißner.     

Inhibition of serotonin uptake by synaptosomes and glial cells induced by certain drugs

Uptake of [¹⁴C]serotonin by glial cells and synaptosomes in the rabbit cerebral cortex was studied. The value of K_m for serotonin uptake was the same (0.083±0.02 M) for both synaptosomes and glial cells. Cortical synaptosomes took up serotonin twice as fast as glial cells (the rates of uptake were compared as protein). Of the psychotropic drugs tested, the most active inhibitors of both synaptosomal and glial serotonin uptake were the tricyclic antidepressant imipramine and the psychostimulant cocaine which, in concentrations of 50 M, inhibited uptake of [¹⁴C]serotonin in synaptosomes by 90% and in glial cells by 75–80%.Laboratory of Neurochemical Pharmacology, Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. Department of Biochemistry, Tbilisi University. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 11, pp. 564–566, November, 1979.     

Cytodifferentiation and synaptogenesis in the neostriatum of fetal and neonatal rhesus monkeys

Summary Cytodifferentiation and synaptogenesis in the neostriatum (caudate nucleus and putamen) were analyzed by the Golgi impregnation method and electron microscopy in 14 fetuses and 8 postnatal rhesus monkeys. During the second fetal month the neostriatum consists primarily of simple, mostly bipolar, immature cells and a small number of undefined profiles ending with growth cones. The first morphologically defined synapses appear in the putamen at embryonic day 60 (E60) and in the head of the caudate nucleus at E65. Synaptic density in both structures is less than one per 1000/m² of neuropil at this stage; synapses are characterized by asymmetric junctions between axonal profiles and immature dendritic shafts, accumulation of an intermembrane web and aggregation of round clear vesicles in presynaptic profiles. During the third fetal month neuronal cell bodies and glial cells enlarge, and axonal and dendritic processes in Golgi preparations become more complex. Although the basic morphology of synapses remains unchanged, their density increases to 9/1000 m² in the putamen and 3.7/1000 m² in the caudate. During the fourth fetal month the four principal cell classes of the neostriatum emerge. Spines on the shafts of dendrites are followed closely by the appearance of axospinous synapses. Synaptic density in the putamen is still significantly higher (10.1/1000 m²) than in the caudate (5.4/1000 m²), but by the end of the fifth fetal month (E150) it is the same (80/1000 m²) in both structures. A dramatic increase in synaptic density to 125/1000 m² occurs before term (E165) with the emergence of the first asymmetric synapses as well is symmetric synapses with flat or pleomorphic vesicles that terminate predominately on dendritic shafts. Synaptic density continues to increase after birth, reaching a plateau of approximately 190/1000 m² at the end of the first postnatal month. Throughout postnatal development the proportions of symmetric and asymmetric synapses on the smooth dendritic shafts undergo systematic fluctuations which may reflect the ingrowth of various afferents as well as local cytological differentiation including the formation of cellular compartments.     

Location and size of carotid body-like organs (paraganglia) revealed in rats by the permeability of blood vessels to Evans blue dye

Summary We determined the number, distribution, size, and morphology of paraganglia near the glossopharyngeal, vagus, and sympathetic nerves of rats. The location of paraganglia was revealed by a method that takes advantage of the comparatively high permeability of their blood vessels to Evans blue dye. Rats were fixed by vascular perfusion of glutaraldehyde 2 min after receiving an intravenous injection of Evans blue dye. Paraganglia appeared as circumscribed, intensely blue structures that were readily distinguished from unstained nerves associated with them. Similarly, some groups of small intensely fluorescent (SIF) cells in autonomic and sensory ganglia were surrounded by Evans blue at a time that other portions of the ganglia contained little detectable dye. An average of 92.5 (range 41–134) paraganglia and 41 (range 17–68) blue spots in ganglia were found in the neck, thorax and abdomen of each of 10 rats. Carotid bodies had a mean length of 601 ± 123 m, width of 275 ± 65 m, and volume of 25.1 ± 11.2,m³ × 10⁶. Other paraganglia had an average length of 168 ± 108m, width of 77 ± 41 m, and volume of 0.87 ± 1.55 m³ × 10⁶. The total volume of paraganglion tissue averaged 128 m³ × 10⁶ (range 62–215 m³ × 10⁶), 59% of which was due to paraganglia other than the carotid bodies.By using fluorescence microscopy, we verified that small catecholamine-containing cells, visible because of their yellow-green fluorescence induced by formaldehyde gas, were located in regions along nerves and within ganglia that contained extravascular dye, visible because of its red fluorescence. Electron-microscopic studies confirmed that blue-stained organs (presumptive paraganglia) associated with the superior laryngeal nerve and other branches of the vagus nerve contained cells morphologically similar to glomus cells of the carotid body. Celiac ganglia contained, in addition, some cells similar to chromaffin cells of the adrenal medulla. Paraganglia (but not SIF cells in ganglia) were encapsulated by layers of perineurium, which may constitute a barrier to diffusion. Tortuous thin-walled blood vessels, some with a fenestrated endothelium, were present in all paraganglia examined and were near most groups of SIF cells in ganglia. Neural connections of the small catecholamine-containing cells varied. Most nerve terminals on cells in paraganglia resembled sensory nerve endings on glomus cells of the carotid body, although some were morphologically similar to preganglionic nerves on chromaffin cells of the adrenal medulla.     

Agonist concentration influences the pattern and time course of intracellular Ca2+ oscillations in human arterial smooth muscle cells

Oscillations in intracellular Ca²⁺ were recorded in cultured human uterine artery vascular smooth muscle cells. In the absence of external Ca²⁺, prolonged application of 3 M histamine activated a large transient increase in Ca²⁺ followed by a burst of Ca²⁺ spikes. The time course and frequency of the spikes were approximately constant until the last two to three spikes, when the inter-spike interval progressively increased. At 30 M histamine the response was different; the amplitude of the spikes decreased rapidly to zero, the rate of rise of successive transients fell and the time between spikes increased. The cessation of oscillatory activity was not associated with the depletion of intracellular Ca²⁺ stores, since increased doses of agonist or the sulphydryl reagent thimerosal could reactivate Ca²⁺ release. The changes in the pattern of intracellular Ca²⁺ spikes seen with increasing agonist concentration may reflect the involvement of different inactivation mechanisms in the termination of Ca²⁺ transients. In the presence of external Ca²⁺, histamine (3–30 M) activated regular Ca²⁺ oscillations. The frequency, but not the amplitude, of the oscillations was dependent on agonist concentration, the highest frequency of spiking was observed at 30 M histamine. In cells depolarised with 30 mM K⁺, histamine was still able to activate Ca²⁺ oscillations, but the dependence of spike frequency upon agonist concentration was abolished. Ca²⁺ oscillations could be activated in the presence of verapamil and nifedipine (10 M). These data suggest that in human uterine artery vascular smooth muscle cells histamine-induced Ca²⁺ oscillations are generated largely by a cytosolic oscillator and are modified by the influx of Ca²⁺ across the surface membrane.     

CAMP-dependent activation of ion conductances in bronchial epithelial cells

The cAMP-dependent activation of Cl^– channels was studied in a bronchial epithelial cell line (16HBE14o-) in fast and slow whole-cell, and cell-attached patch-clamp experiments. The cells are known to express high levels of cystic fibrosis transmembrane conductance regulator mRNA and protein. Isoproterenol, forskolin and histamine (all 10 mol/l) reversibly and significantly depolarized the membrane voltage (V _m) and increased the whole-cell Cl^– conductance significantly by 34.0±0.9 (n=3), 18.1±2.7 (n=50), and 25±4.5 (n=37) nS respectively. The effect of histamine was blocked by cimetidine (10 mol, n=5) but not by diphenhydramine (10 mol/l, n=4), which suggests binding of histamine to H₂ receptors. The forskolin-induced current was not inhibited significantly by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/l, n=9) nor glibenclamide (10 mol/l, n=3) and had an anion-permeability sequence of Cl^–= Br^–>I^– (n=9). In cell-attached recordings forskolin (10 mol/l) increased the conductance of the patched membrane from 65.5±13.6 pS to 150.8±33.2 pS (n=30). Although the conductance was increased significantly, clear ion-channel events occurring in parallel with the current activation were not detected in the cell attached membrane. In 4 out of 30 cell-attached recordings single-channel currents were observed. These channels, with a single-channel conductance of about 6 pS, were already active before forskolin was added. No effect of forskolin on the channel amplitude, open probability or kinetics of these channels was observed. From these data we conclude that the cAMP-induced conductance increase in 16HBE14o-cells can be correlated with the activation of very small and not resolvable (probably less than 2 pS) Cl^– channels rather than with the activation of channels with a conductance of 6–10 pS.