Experimental alteration of coupling resistance at an electrotonic synapse.

Adjacent segments of the septate axon of the crayfish Procambarus are electrotonically coupled by junction located in the septa between them (see Pappas et al. 1970. J. Cell Biol.49:173). The coupling resistance at the septa was changed by several experimental treatments. Mechanical injury to an axon increased coupling resistance (more than 7-fold); no recovery of coupling resistance was observed, although the resting potential and resistance of the injured axon could return to near normal levels. Immersion in salines with Na propionate substituted for NaCl increased coupling resistance (mean: 6.1-fold). On return of the preparation to normal saline, coupling resistance recovered virtually completely. Immersion in low Ca(++) solutions moderately increased coupling resistance (3.5-fold or less), but return to normal saline was followed by large increases in coupling resistance (5-100-fold). 60 nM Ca(++) is near the maximum concentration that leads to increased coupling resistance on return to normal saline. Large increases in coupling resistance are associated with separation of junctional membranes (Pappas et al. 1970. Ibid.); calculations show that the separated membranes greatly increase in resistance. Increase in coupling resistance is probably an important response to injury. Mechanisms underlying changes reported here may be relevant to normal physiological processes of coupling and decoupling.

Rapid degeneration of ampullary electroreceptor organs after denervation.

Electroreceptors (ampullary organs) of the transparent catfish (Kryptopterus bicirrhus) lie in the epidermis, and contain spherical receptor cells that receive purely afferent innervation from the lateral line nerve. Section of this nerve causes rapid degenerative changes to occur in the receptors. Fine structural alterations occur in the receptor cell synapses and nerve fiber 6-12 h postoperatively. Disruption of the receptor cells begins by 18 h and most are lost by 48 h. By 72 h supporting cells and secretory cells also show marked degeneration, and by 96 h they may be totally lost. The rapid degeneration of the electroreceptor organs of Kryptopterus should make them a useful preparation for analysis of neurotrophic functions.

Morphological correlates of increased coupling resistance at an electrotonic synapse.

Close appositions between axonal membranes are present in the septum between adjacent axonal segments of the septate or lateral giant axons of the crayfish Procambarus. In sections the closely apposed membranes appear separated by a space or gap. The use of lanthanum indicates that there may be structures connecting the apposed membranes. The apparent gap is actually a network of channels continuous with the extracellular space. Adjacent axonal segments are electrotonically coupled at the septa. The coupling resistance is increased by mechanical injury of an axon, immersion in low Cl(-) solutions, and immersion in low Ca(++) solutions, followed by a return to normal physiological solution. Septa at which coupling resistance had been measured were examined in the electron microscope. The induced increases in coupling resistance are associated with separation of the junctional membranes (with the exception of the moderate increases during immersion in low Ca(++) solutions). Schwann cell processes are present between the separated axonal membranes. When nerve cords in low Cl(-) solutions are returned to normal physiological solution, coupling, i.e., electrotonic synapses. A model of an electrotonic synapse is proposed in which tween axonal membranes are again found. The association between the morphological and physiological findings provides further evidence that the junctions are the sites of electrotonic coupling, i.e., electrotonic, synapses. A model of an electrotonic synapse is proposed in which intercytoplasmic channels not open to the extracellular space are interlaced with a hexagonal network of extracellular channels between the apposed junctional membranes.

Electrical coupling between embryonic cells by way of extracellular space and specialized junctions.

The meroblastic egg of the teleost, Fundulus heteroclitus, was studied electrophysiologically from cleavage to mid-gastrula stages. The yolk is an intracellular inclusion surrounded by a membrane of high resistivity (50 k ohmcm(2)). This membrane generates a cytoplasm-negative resting potential in later stages. Cells of all stages studied are coupled electrically. In gastrulae, coupling is both by way of specialized junctions between cells and by way of intra-embryonic extracellular space, the segmentation cavity. The latter mode is present because the segmentation cavity is sealed off from the exterior by a high resistance barrier, and the outer membrane of surface cells is of high resistance (50-100 k ohmcm(2)) compared to the inner membrane. It can be inferred that clefts between surface cells are occluded by circumferential junctions. Isolated cells from late cleavage stages develop coupling in vitro, confirming the existence of coupling by way of intercellular junctions. Both modes of coupling could mediate communication between cells that is important in embryonic development.

Morphological correlates of functional differentiation of nodes of Ranvier along single fibers in the neurogenic electric organ of the knife fish Stern archus.

Electric organs in Sternarchidae are of neural origin, in contrast to electric organs in other fish, which are derived from muscle. The electric organ in Sternarchus is composed of modified axons of spinal neurons. Fibers comprising the electric organ were studied by dissection and by light- and electron microscopy of sectioned material. The spinal electrocytes descend to the electric organ where they run anteriorly for several segments, turn sharply, and run posteriorly to end blindly at approximately the level where they enter the organ. At the level of entry into the organ, and where they turn around, the axons are about 20 micro in diameter; the nodes of Ranvier have a typical appearance with a gap of approximately 1 micro in the myelin. Anteriorly and posteriorly running parts of the fibers dilate to a diameter of approximately 100 micro, and then taper again. In proximal and central regions of anteriorly and posteriorly running parts, nodal gaps measure approximately 1 micro along the axon. In distal regions of anteriorly and posteriorly running parts are three to five large nodes with gaps measuring more than 50 micro along the fiber axis. Nodes with narrow and with wide gaps are distinguishable ultrastructurally; the first type has a typical structure, whereas the second type represents a new nodal morphology. At the typical nodes a dense cytoplasmic material is associated with the axon membrane. At large nodes, the unmyelinated axon membrane is elaborated to form a closely packed layer of irregular polypoid processes without a dense cytoplasmic undercoating. Electrophysiological data indicate that typical nodes in proximal regions of anteriorly and posteriorly running segments actively generate spikes, whereas large distal nodes are inactive and act as a series capacity. Increased membrane surface area provides a morphological correlate for this capacity. This electric organ comprises a unique neural system in which axons have evolved so as to generate external signals, an adaptation involving a functionally significant structural differentiation of nodes of Ranvier along single nerve fibers.

Substituted benzyl acetates: a new class of compounds that reduce gap junctional conductance by cytoplasmic acidification.

Conductance of gap junctions in many preparations has been shown to be sensitive to cytoplasmic pH, decreasing as pH decreases below 7.5 in fish and amphibian embryos and below 7.1 in crayfish septate axon. We have found a new class of compounds, benzyl acetate derivatives, that reversibly decrease junctional conductance, gj, when applied in low concentration (approximately 1 mM). Simultaneous intracellular pH (pHi) measurements show that the ester effects are attributable to reduction in pHi. The sensitivity of gj to these compounds and the relative lack of side effects make these agents attractive for studies of the role played by gap junctions in normal tissue function. In addition, the finding of cytoplasmic acidification in response to cell exposure to esters suggests caution in interpretation of results obtained using esterified compounds for intracellular loading.

Carbon tetrachloride at hepatotoxic levels blocks reversibly gap junctions between rat hepatocytes.

Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent beta-mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

Temperature-dependence of resistance at an electrotonic synapse.

The junctional resistance at septa of the crayfish lateral giant axon is inversely related to temperature with a Q(1l), of about 3 over the range from 5 degrees to 20 degrees C. Nonjunctional axonal membrane is much less affected. Resistance changes occur rapidly with temperature changes. No correlates in ultrastructure of the synapses have been found.

Electrotonic coupling: effective sign reversal by inhibitory neurons.

Neurons in the buccal ganglia of Navanax inermis which control circumferential muscles of the pharynx showed typical electrotonic coupling when there was little synaptic activity in them. When there was much inhibitory activity, the effective sign of coupling was reversed; that is, hyperpolarization and depolarization of one cell caused depolarization and hyperpolarization of the others. A neural circuit explaining these results involes inhibitory neurons electronically coupled to and also inhibitory to the circumferential neurons that are themselves coupled. This circuit offers considerable flexibility for mediation of different activity patterns in this simple neuronal system.

Permeability and structure of junctional membranes at an electrotonic synapse.

The dye Procion Yellow M4RS crosses junctional membranes from cytoplasm to cytoplasm at electrotonic synapses between segments of the crayfish septate axon. The dye does not enter the cells from extracellular space. Thus permeability of junctional membranes is qualitatively different from that of nonjunctional membranes. Electron microscopy after fixation in the presence of lanthanum hydroxide indicates that these synapses are "gap junctions" and that there is a network of channels continuous with extracellular space between apposed junctional membranes. These channels must be interlaced with intercytoplasmic channels that are not open to extracellular space.

Gap junctional conductance is a simple and sensitive function of intracellular pH.

The pH of the cytoplasm (pHt) measured with pH-sensitive microelectrodes in cleavage-stage blastomeres of amphibian (Ambystoma) and teleost (Fundulus) embryos is about 7.7. In electrotonically coupled cell pairs, junctional conductance is rapidly and reversibly reduced by acidification of the cytoplasm. The relation between junctional conductance and pHi is the same for increasing and decreasing pH and is independent of the rate of change over a wide range. The relation is well fitted by a Hill curve with K = 50 nM (pK = 7.3) and n = 4 to 5. The closure of gap junction channels at low pHi appears to be a cooperative process involving several charged sites. The absence of hysteresis and identity of effects for fast and slow pHi changes implies that protons act directly on the channel macromolecules and not through an intermediate in the cytoplasm.

Voltage dependence of junctional conductance in early amphibian embryos.

Isolated pairs of blastomeres from early amphibian embryos (Ambystoma, Rana, Xenopus) are electrontonically coupled. Junctional conductance and permeability to the dye Lucifer Yellow are markeldy and reversibly decreased by moderate transjunctional polarization in either direction. The relationship between junctional conductance and transjunctional voltage is sufficiently steep that a physiological role in regulation of intercellular communication is plausible.

Gap junctional conductance and permeability are linearly related.

The permeability of gap junctions to tetraethylammonium ions was measured in isolated pairs of blastomeres from Rana pipiens L. and compared to the junctional conductance. In this system, the junctional conductance is voltage-dependent and decreases with moderate transjunctional voltage of either sign. The permeability to tetraethylammonium ions was determined by injecting one cell of a pair with tetraethylammonium and monitoring its changing concentration in the prejunctional and postjunctional cells with ion-selective electrodes. Junctional conductance was determined by current-clamp and voltage-clamp techniques. For different cell pairs in which the transjunctional voltage was small and the junctional conductance at its maximum value, the permeability to tetraethylammonium ions was proportional to the junctional conductance. In individual cell pairs, a reduction in the junctional conductance induced by voltage was accompanied by a proportional reduction in the permeability of the gap junction over a wide range. The diameter of the tetraethylammonium ion (8.0 to 8.5 A, unhydrated) is larger than that of the potassium ion (4.6 A, hydrated), the predominant current-carrying species. The proportionality between the permeability to tetraethylammonium ions and the junctional conductance, measured here with exceptionally fine time resolution, indicates that a common gap junctional pathway mediates both electrical and chemical fluxes between cells, and that closure of single gap junction channels by voltage is all or none.

Oculomotor neurons in fish: electrotonic coupling and multiple sites of impulse initiation.

Oculomotor neurons are electrotonically coupled in three teleosts. Electron microscopy revealed axosomatic synapses with close appositions of pre- and postynaptic membranes. Similar junctions are associated with electrotonic coupling in many other cases. Stimulation of the ipsilateral eighth nerve usually initiated impulses at sites distant from the cell bodies; stimulation of the ipsilateral ophthalmic nerve initiated impulses close to the cell bodies. Electrotonic coupling may synchronize impulses arising near the, cell bodies to generate synchronous muscle contractions. Impulses arising distant from the cell bodies may lead to contractions of graded strength.

Mutagenesis rescues spermine and Zn2+ potentiation of recombinant NMDA receptors.

Alternative splicing generates distinct forms of the NMDA receptor subunit NR1. NR1 subunits with an N-terminal insert (termed N1) form receptors in Xenopus oocytes with greatly reduced potentiation by spermine and Zn2+. Oocytes expressing NR1 receptors with N1 exhibited larger NMDA currents than oocytes expressing corresponding receptors without N1. In the present study, we used mutational analysis to investigate structural features of the N1 insert that control current amplitude and spermine and Zn2+ potentiation. Neutralization of positive charges in N1 rescued spermine and Zn2+ potentiation. Positive charges in N1 did not affect spermine or Zn2+ affinity. Neutralization of positive charges in N1 diminished the responses to the level of NR1 receptors lacking N1. The positively charged N1 may increase NMDA currents by causing a conformational change similar to that produced by spermine and Zn2+ in NR1 receptors lacking N1.

Changes in permeability caused by connexin 32 mutations underlie X-linked Charcot-Marie-Tooth disease.

The relationship between the loss of connexin 32 function and clinical manifestations of X-linked Charcot-Marie-Tooth (CMTX) disease is unknown. Here, we report that eight of nine CMTX mutations investigated form channels with measurable electrical conductance. Single-channel studies of two mutations demonstrate reduced junctional permeability caused by a decrease in either pore size (S26L) or open channel probability (M34T) that favors residency in a low-conductance substate. Permeation of second messengers such as cAMP through reflexive gap junctions between adjacent cytoplasmic loops of myelinating Schwann cells is likely to be reduced or absent in these channels. We propose that CMTX mutations impair the transduction of signals arising from normal glial-neuronal interactions and thereby cause demyelination and axonal degeneration.

Seeing is relieving: electrical synapses between visualized neurons.

Two reports of electrical synapses between identified inhibitory neurons in the neocortex suggest that many neuron types known to communicate chemically may also be electrically coupled.

Protein kinase C modulates NMDA receptor trafficking and gating.

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.

Variation in quality report viewing by providers and correlation with NICU quality metrics.

BACKGROUND: To examine variation in quality report viewing and assess correlation between provider report viewing and neonatal intensive care unit (NICU) quality. METHODS: Variation in report viewing sessions for 129 California Perinatal Quality Care Collaborative NICUs was examined. NICUs were stratified into tertiles based on their antenatal steroid (ANS) use and hospital-acquired infection (HAI) rates to compare report viewing session counts. RESULTS: The number of report viewing sessions initiated by providers varied widely over a 2-year period (median=11; mean=25.5; s.d.=45.19 sessions). Report viewing was not associated with differences in ANS use. Facilities with low HAI rates had less frequent report viewing. Facilities with high report views had significant improvements in HAI rates over time. CONCLUSIONS: Available audit and feedback reports are utilized inconsistently across California NICUs despite evidence that report viewing is associated with improvements in quality of care delivery. Further studies are needed for reports to reach their theoretical potential.

Effects of delivery room quality improvement on premature infant outcomes.

OBJECTIVE: Delivery room management interventions have been successfully implemented via collaborative quality improvement (QI) projects. However, it is unknown whether these successes translate to reductions in neonatal morbidity and mortality. STUDY DESIGN: This was a prospective pre-post intervention study of three nonrandomized hospital groups within the California Perinatal Quality Care Collaborative. A collaborative QI model (Collaborative QI) was compared with a single-site QI model (NICU QI) and a non-participant population when implementing evidence-based delivery room practices. The intervention period was between June 2011 and May 2012. Infants born with gestational age between 22 weeks 0 days and 29 weeks 6 days and birth weight ⩽1500 g were included. Outcomes were mortality and select morbidities (bronchopulmonary dysplasia (BPD), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP) and necrotizing enterocolitis (NEC)). Outcomes were compared between the baseline (January 2010 to May 2011) and post-intervention period (June 2012 to May 2013) within each comparison group. RESULTS: Ninety-five hospitals were included with 4222 infants in the baseline period and 4186 infants in the post-intervention period. The Collaborative QI group had significantly reduced odds of developing BPD post-intervention (odds ratio (OR) 0.8, 95% confidence interval (CI) 0.65 to 0.99) or composite BPD-death (OR 0.83, 95% CI 0.69 to 1.00). In both the Collaborative QI and non-participants there were also reductions in IVH, severe IVH, composite severe IVH-death, severe ROP and composite severe ROP-death. CONCLUSION: Hospitals dedicated to improving delivery room practices can impact neonatal outcomes.