Non-linear Membrane Properties in Entorhinal Cortical Stellate Cells Reduce Modulation of Input-Output Responses by Voltage Fluctuations

The presence of voltage fluctuations arising from synaptic activity is a critical component in models of gain control, neuronal output gating, and spike rate coding. The degree to which individual neuronal input-output functions are modulated by voltage fluctuations, however, is not well established across different cortical areas. Additionally, the extent and mechanisms of input-output modulation through fluctuations have been explored largely in simplified models of spike generation, and with limited consideration for the role of non-linear and voltage-dependent membrane properties. To address these issues, we studied fluctuation-based modulation of input-output responses in medial entorhinal cortical (MEC) stellate cells of rats, which express strong sub-threshold non-linear membrane properties. Using in vitro recordings, dynamic clamp and modeling, we show that the modulation of input-output responses by random voltage fluctuations in stellate cells is significantly limited. In stellate cells, a voltage-dependent increase in membrane resistance at sub-threshold voltages mediated by Na⁺ conductance activation limits the ability of fluctuations to elicit spikes. Similarly, in exponential leaky integrate-and-fire models using a shallow voltage-dependence for the exponential term that matches stellate cell membrane properties, a low degree of fluctuation-based modulation of input-output responses can be attained. These results demonstrate that fluctuation-based modulation of input-output responses is not a universal feature of neurons and can be significantly limited by subthreshold voltage-gated conductances.     

The role of intrinsic neuronal properties in the encoding of auditory information in the cochlear nuclei.

It is now possible to relate the intrinsic electrical properties of particular cells in the cochlear nuclei of mammals with their biological function. In the layered dorsal cochlear nucleus, information concerning the location of a sound source seems to be contained in the spatial pattern of activation of a population of neurons. In the unlayered, ventral cochlear nucleus, however, neurons carry information in their temporal firing patterns. The voltage-sensitive conductances that make responses to synaptic current brief enable bushy cells to convey signals from the auditory nerve to the superior olivary complex with a temporal precision of at least 120 microseconds.     

Background calcium permeable channels in glomerulosa cells from adrenal gland

The cell-attached recording mode of the patch-clamp technique was used to study Ca2+ permeable background currents of glomerulosa cells from rat and bovine adrenal gland. With a pipette filled with 110 mM BaCl2 or 90 mM CaCl2, three different types of unitary currents were detected. The B1 channel demonstrates a nonlinear I-V curve. The conductances are 4 and 7 pS at -40 and -70 mV, respectively. The curve of the opening probability vs. membrane potential is bell shaped with its maximum at -70 mV. The B2 channel has a conductance of 6 pS, while the B3 channel shows a nonlinear I-V relationship with conductances close to 17 and 10 pS at HPs of -60 and -20 mV. The three types of currents are insensitive to dihydropyridines. We suggest that these background currents could be responsible for the basal calcium influx and aldosterone secretion previously observed in nonstimulated glomerulosa cells.     

Extracellular currents alter gap junction intercellular communication in synovial fibroblasts

We studied the effect of extremely low frequency (ELF) currents on gap junction intercellular communication (GJIC) mediated by connexin43 protein. Confluent monolayers of synovial fibroblasts (HIG-82) and neuroblastoma cells (5Y) were exposed in bath solution to 0-75 mA/m(2) (0-56 mV/m), 60 Hz. Single channel conductance, cell membrane current-voltage (I-V) curves, and Ca(2+) influx were measured using the nystatin single and double patch methods. The conductances of the closed and open states of the gap junction channel in HIG-82 cells were each significantly reduced (by 0.76 and 0.39 pA, respectively) in cells exposed to 20 mA/m(2). Current densities as low as 10 mA/m(2) significantly increased Ca(2+) influx in HIG-82 cells. No effects were seen in 5Y cells. The I-V curves of the plasma membranes of both types of cells were independent of 60 Hz electric fields and current densities, 0-75 mA/m(2), indicating that the effect of the 60 Hz fields on GJIC in HIG-82 cells was not mediated by a change in membrane potential. We conclude that ELF electric fields can alter GJIC in synovial cells via a mechanism that does not depend on changes in membrane potential, but may depend on Ca(2+) influx. The results open the possibility that GJIC mediated responses in synovial cells, such as for example, their secretory responses to proinflammatory cytokines, could be antagonized by the application of ELF electric fields.     

Ionic conductances of squid giant fiber lobe neurons

The cell bodies of the neurons in the giant fiber lobe (GFL) of the squid stellate ganglion give rise to axons that fuse and thereby form the third-order giant axon, whose initial portion functions as the postsynaptic element of the squid giant synapse. We have developed a preparation of dissociated, cultured cells from this lobe and have studied the voltage-dependent conductances using patch-clamp techniques. This system offers a unique opportunity for comparing the properties and regional differentiation of ionic channels in somatic and axonal membranes within the same cell. Some of these cells contain a small inward Na current which resembles that found in axon with respect to tetrodotoxin sensitivity, voltage dependence, and inactivation. More prominent is a macroscopic inward current, carried by Ca2+, which is likely to be the result of at least two kinetically distinct types of channels. These Ca channels differ in their closing kinetics, voltage range and time course of activation, and the extent to which their conductance inactivates. The dominant current in these GFL neurons is outward and is carried by K+. It can be accounted for by a single type of voltage-dependent channel. This conductance resembles the K conductance of the axon, except that it partially inactivates during relatively short depolarizations. Ensemble fluctuation analysis of K currents obtained from excised outside-out patches is consistent with a single type of K channel and yields estimates for the single channel conductance of approximately 13 pS, independently of membrane potential. A preliminary analysis of single channel data supports the conclusion that there is a single type of voltage-dependent, inactivating K channel in the GFL neurons.     

A quantitative profile of the synapses on the stellate cell body and axon in the cochlear nucleus of the chinchilla

Summary. One of the most numerous neurons in the cochlear nucleus is the type I stellate cell. Previous attempts to understand the structural basis for its signal coding assumed that integration of synaptic potentials arising from axodendritic synapses should account for the generation of its response properties. However, the present study documents the importance of excitatory and inhibitory types of synapses on the soma and axon. Retrograde transport of cholera toxin B subunit, injected in the inferior colliculus of chinchillas, was used to label exclusively type I stellate cells in the anteroventral cochlear nucleus. The relative distribution of terminal types by vesicle morphology was pleomorphic < large spherical < flattened < smaller spherical. The somatic perimeter covered by endings ranged from almost none to nearly half. More flattened-vesicle terminals contacted somata in the high-frequency than in the low-frequency region. Eight of twenty axons received endings that contained large spherical vesicles and made asymmetric junctions; half of these extensively apposed the initial segment, forming a collar of presumed excitatory input. Thus, type I stellate cells are a heterogeneous group. Inhibitory synapses probably compose the majority of terminals. Some cells receive mostly inhibitory synapses near the presumed site of the spike generator, but others also have a prominent excitatory input. These findings call for a new look at the mechanisms for signal coding in stellate cells in the auditory system in particular and raise issues concerning the stochastic nature of information processing in sensory systems in general.     

Isolation, culture, and preliminary characterization of ellipsoids (sheathed capillaries of Schweigger-Seidel) of the pig spleen. II. An enzyme histochemical study of in vitro cultivated ellipsoids

Whole isolated ellipsoids (sheathed capillaries of Schweiger-Seidel) of the pig spleen were explanted in Medium 199 containing 20% fetal calf serum or horse serum respectively. Cultures were kept in a gas phase of 5% carbon dioxide in air at 37 degrees C. After about 4 days in culture the outgrowth of two morphologically different cell types was apparent. Small cells of fusiform or stellate morphology displayed high activity of acid phosphatase. N-acetyl-beta-glucosaminidase and beta-glucuronidase activity were also detectable. Furthermore these cells were highly reactive for unspecific esterase and gamma-glutamyl transpeptidase activity. Endogenous peroxidase activity was present in the cytoplasm and in the perinuclear space. Stellate cells therefore are thought of as ellipsoid macrophages. Additional observations reported are the expression of Fc-receptors on stellate cells. They triggered the phagocytosis of opsonized test particles. The second cell type showed fibroblastic morphology. The large well spread cells did exhibit low activities of acid phosphatase and N-acetyl-beta-glucosaminidase. The other enzyme activities examined were not detectable. The nature of these cells is not well understood at present. Most likely they are constituents of the framework of the ellipsoids. No transitions between stellate cells and fibroblastic cells were found.     

TRPC5 activation kinetics are modulated by the scaffolding protein ezrin/radixin/moesin-binding phosphoprotein-50 (EBP50)

TRPC1-7 proteins are members of a family of mammalian non-specific cation channels that mediate receptor-operated, phospholipase Cbeta/Cgamma dependent Ca(2+) influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C-terminal "VTTRL" motif that binds to PDZ-domains of the scaffolding protein, EBP50 (NHERF1; Tang et al., J Biol Chem 275:37559-37564). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. We have cloned rat TRPC5 (rTRPC5), functionally expressed it in HEK293 cell, and studied channel regulation with patch-clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) were localized to the plasma membrane. rTRPC5 did not display any significant basal activity in unstimulated HEK293 cells. In cells co-expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPgammaS or histamine developed in two phases: a slowly developing, small inward current was followed by a rapidly developing, transient, large inward current. Each phase had a characteristic non-linear current-voltage (I-V) relationship. Deletion of the VTTRL motif had no detectable effect on the biophysical properties of the channel. Co-expression of EBP50 with rTRPC5 caused a significant delay in the time-to-peak of the histamine-evoked, transient large inward current. EBP50 did not modify the activation kinetics of the VTTRL-deletion mutant. We conclude that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics.     

Mapping the distribution of outer hair cell voltage-dependent conductances by electrical amputation.

The mammalian outer hair cell (OHC) functions not only as sensory receptor, but also as mechanical effector; this unique union is believed to enhance our ability to discriminate among acoustic frequencies, especially in the kilohertz range. An electrical technique designed to isolate restricted portions of the plasma membrane was used to map the distribution of voltage-dependent conductances along the cylindrical extent of the cell. We show that three voltage-dependent currents, outward K, I(K,n), and I(Ca) are localized to the basal, synaptic pole of the OHC. Previously we showed that the lateral membrane of the OHC harbors a dense population of voltage sensor-motor elements responsible for OHC motility. This segregation of membrane molecules may have important implications for auditory function. The distribution of OHC conductances will influence the cable properties of the cell, thereby potentially controlling the voltage magnitudes experienced by the motility voltage sensors in the lateral membrane, and thus the output of the "cochlear amplifier."     

Internalization of retinol-binding protein in parenchymal and stellate cells of rat liver

We have studied uptake of retinol-binding protein (RBP) by rat liver cells. First, we compared the in vivo uptake in different liver cells of 125I-labeled RBP with that of other well-known ligands. We found that the ligands studied were recognized differently by the various cell types in the liver, and that RBP was most efficiently taken up by parenchymal and stellate cells. We then studied the in vivo uptake of RBP in liver cells by immunocytochemistry at the electron microscopic level using ultrathin cryosections. Ten min after injection, RBP was localized to parenchymal cells and stellate cells. In these cells, RBP was detected on the cell surface and in vesicles near the cell surface. RBP was observed mainly in association with the membrane in these vesicles. Two hours after injection, RBP was localized not only on the cell surface and in vesicles close to the cell surface, but also in larger vesicles located deeper in the cytoplasm of these cells. RBP in larger vesicles was observed at a distance from the vesicular membrane. Finally, we compared the distribution of endocytosed RBP in liver parenchymal cells with that of asialo-orosomucoid, a ligand known to be internalized by receptor-mediated endocytosis. We detected both ligands on the cell surface and in small vesicles located close to the cell surface and in larger vesicles located deeper in the cytoplasm. Asialo-orosomucoid and RBP were seldom observed in the same small vesicles, but the larger vesicles contained both ligands. These data suggest that RBP is internalized in parenchymal and stellate cells of the liver by receptor-mediated endocytosis.     

Mast cell ionic channels: significance for stimulus-secretion coupling.

The activation of mast cells (MC) due to immunological stimulation causes an immediate and dramatic inflammatory response. We review current evidence indicating that the membrane permeabilities for calcium, chloride, sodium, and potassium have a significant role in the activation of these cells, and in some cases, specific ionic channels have been identified. Moreover, a number of intracellular mechanisms controlling these channels are pointed out, including different classes of G proteins, intracellular calcium, cAMP, and products of phosphoinositol breakdown. However, the interplay between factors controlling membrane conductances for different ions is not currently understood. The diversity of ionic effects on MC activation is depicted, illustrating that the ionic mechanisms of MC activation are specific for different MC types. Since nerve/mast cell interaction is a key element in the burgeoning field of neuroimmunology, we discuss the role of ionic channels as targets of neurotransmitter action in MC activation.     

Simultaneous functional expression of swelling and forskolin-activated chloride currents in primary cultures of rabbit distal convoluted tubule

Ionic Cl⁻ currents induced by cell swelling and forskolin were studied in primary cultures of rabbit distal convoluted tubule (DCTb) by the whole-cell patch clamp technique. We identified a Cl⁻ conductance activated by cell swelling with an hyperosmotic pipette solution. The initial current exhibited an outwardly rectifying I-V relationship, whereas steady state current showed strong decay at depolarized membrane potentials. The ion selectivity was I⁻ > Br⁻ > Cl⁻ > > glutamate. The forskolin-activated Cl⁻ conductance demonstrated a linear I-V relationship and its ion selectivity was Br⁻ > Cl⁻ > I⁻ > glutamate. This last conductance could be related to the CFTR (cystic fibrosis transmembrane conductance regulator) previously identified in these cells. NPPB inhibited both Cl⁻ currents, and DIDS inhibited only the swelling-activated Cl⁻ current. Forskolin had no effect on the activation of the swelling-activated Cl⁻ current. In DCTb cells which exhibited swelling-activated Cl⁻ currents subsequently inhibited by DIDS, forskolin could activate CFTR related Cl⁻ currents. In the continuous presence of I⁻ which inhibited CFTR conductance, forskolin did not modify the swelling-activated current. The results suggest that both Cl⁻ conductances could be co-expressed in the same DCTb cell and that CFTR did not modulate the swelling-activated conductance.     

Cholesterol influences voltage-gated calcium channels and BK-type potassium channels in auditory hair cells

The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.     

From global to local: exploring the relationship between parameters and behaviors in models of electrical excitability

Models of electrical activity in excitable cells involve nonlinear interactions between many ionic currents. Changing parameters in these models can produce a variety of activity patterns with sometimes unexpected effects. Further more, introducing new currents will have different effects depending on the initial parameter set. In this study we combined global sampling of parameter space and local analysis of representative parameter sets in a pituitary cell model to understand the effects of adding K ⁺ conductances, which mediate some effects of hormone action on these cells. Global sampling ensured that the effects of introducing K ⁺ conductances were captured across a wide variety of contexts of model parameters. For each type of K ⁺ conductance we determined the types of behavioral transition that it evoked. Some transitions were counterintuitive, and may have been missed without the use of global sampling. In general, the wide range of transitions that occurred when the same current was applied to the model cell at different locations in parameter space highlight the challenge of making accurate model predictions in light of cell-to-cell heterogeneity. Finally, we used bifurcation analysis and fast/slow analysis to investigate why specific transitions occur in representative individual models. This approach relies on the use of a graphics processing unit (GPU) to quickly map parameter space to model behavior and identify parameter sets for further analysis. Acceleration with modern low-cost GPUs is particularly well suited to exploring the moderate-sized (5-20) parameter spaces of excitable cell and signaling models.     

Morphological alterations in newly born dentate gyrus granule cells that emerge after status epilepticus contribute to make them less excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.     

Voltage oscillations and ionic conductances in hair cells isolated from the alligator cochlea

Summary Tall hair cells were isolated by enzymatic and mechanical dissociation from selected regions of the apical half of the alligator (A. mississippiensis) cochlea. Single cells were subjected to voltage-clamp and current-clamp using the tight-seal whole-cell recording technique. Most hair cells isolated from the apex of the cochlea produced slowly regenerative depolarizations or Na action potentials during current injection, whereas hair cells isolated from more basal regions usually produced voltage oscillations (ringing) in response to depolarizing current injection, an indication of electrical resonance. Resonant frequencies ranged from 50 to 157 Hz in different cells. The higher-frequency cells tended to have larger and more rapidly activating outward currents than did the lower-frequency cells. An inward Ca current and an outward Ca-activated K current were present in all hair cells. In addition, an inwardly rectifying current and a small, transient outward current were often seen. Thus, we conclude that an electrical tuning mechanism is present in alligator hair cells. The role of the ionic conductances in shaping hair cell responses to current injection, and the possible contributions of these electrical responses to cochlear function are discussed.     

A Monte Carlo simulation of plasmid replication during the bacterial division cycle

Plasmids have cell cycle replication patterns that need to be considered in models of their replication dynamics. To compare current theories for control of plasmid replication with experimental data for timing of plasmid replication with the cell cycle, a Monte Carlo simulation of plasmid replication and partition was developed. High-copy plasmid replication was simulated by incorporating equations previously developed from the known molecular biology of ColE1-type plasmids into the cell-cycle simulation. Two types of molecular mechanisms for low-copy plasmid replication were tested: accumulation of an initiator protein in proportion to cell mass and binding of the plasmid origin to the cell membrane. The low-copy plasmids were partitioned actively, with a specific mechanism to mediate the transfer from mother to daughter cells, whereas the high-copy plasmids were partitioned passively with cell mass.The simulation results and experimental data demonstrate cell-cycle-specific replication for the low-copy F plasmid and cell-cycle-independent replication for the high-copy pBR322, ColBM, and R6K plasmids. The simulation results indicate that synchronous replication at multiple plasmid origins is critical for the cell-cycle-specific pattern observed in rapidly growing cells. Variability in the synchrony of initiation of multiple plasmid origins give rise to a cell-cycle-independent pattern and is offered as a plausible explanation for the controversy surrounding the replication pattern of the low-copy plasmids. A comparison of experimental data and simulation results for the low-copy F plasmid at several growth rates indicates that either initiation mechanism would be sufficient to explain the timing of replication with the cell cycle. The simulation results also demonstrate that, although cell-cycle-specific and cell-cycle independent replication patterns give rise to very different gene-expression patterns during short induction periods in age-selected populations, long-term expression of genes encoded on low-copy and high-copy plasmids in exponentially growing cells have nearly the same patterns. These results may be important for the future use of low-copy plasmids as expression vectors and validate the use of simpler models for high-copy plasmids that do not consider cell-cycle phenomena. (c) 1996 John Wiley & Sons, Inc.