Martha C. Nowycky

Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones.

1. Calcium currents in cultured dorsal root ganglion (d.r.g.) cells were studied with the whole‐cell patch‐clamp technique. Using experimental conditions that suppressed Na+ and K+ currents, and 3‐10 mM‐external Ca2+ or Ba2+, we distinguished three distinct types of calcium currents (L, T and N) on the basis of voltage‐dependent kinetics and pharmacology. 2. Component L activates at relatively positive test potentials (t.p. greater than ‐10 mV) and shows little inactivation during a 200 ms depolarization. It is completely reprimed at a holding potential (h.p.) of ‐60 mV, and can be isolated by using a more depolarized h.p. (‐40 mV) to inactivate the other two types of calcium currents. 3. Component T can be seen in isolation with weak test pulses. It begins activating at potentials more positive than ‐70 mV and inactivates quickly and completely during a maintained depolarization (time constant, tau approximately 20‐50 ms). The current amplitude and the rate of decay increase with stronger depolarizations until both reach a maximum at approximately ‐40 mV. Inactivation is complete at h.p. greater than ‐60 mV and is progressively removed between ‐60 and ‐95 mV. 4. Component N activates at relatively strong depolarizations (t.p. greater than ‐20 mV) and decays with time constants ranging from 50 to 110 ms. Inactivation is removed over a very broad range of holding potentials (h.p. between ‐40 and ‐110 mV). 5. With 10 mM‐EGTA in the pipette solution, substitution of Ba2+ for Ca2+ as the charge carrier does not alter the rates of activation or relaxation of any component. However, T‐type channels are approximately equally permeable to Ca2+ and Ba2+, while L‐type and N‐type channels are both much more permeable to Ba2+. 6. Component N cannot be explained by current‐dependent inactivation of L current resulting from recruitment of extra L‐type channels at negative holding potentials: raising the external Ba2+ concentration to 110 mM greatly increases the amplitude of L current evoked from h.p. = ‐30 mV but produces little inactivation. 7. Cadmium ions (20‐50 microM) virtually eliminate both N and L currents (greater than 90% block) but leave T relatively unaffected (less than 50% block). 200 microM‐Cd2+ blocks all three components. 8. Nickel ions (100 microM) strongly reduce T current but leave N and L current little changed. 9. The dihydropyridine antagonist nifedipine (10 microM) inhibits L current (approximately 60% block) at a holding potential that inactivates half the L‐type channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Single-channel recordings of three types of calcium channels in chick sensory neurones.

1. T‐, and L‐type Ca2+ channels were studied in cell‐attached patch recordings from the cell bodies of chick dorsal root ganglion neurones. All experiments were performed with isotonic BaCl2 (110 mM) in the recording pipette and with isotonic potassium aspartate in the bathing solution to zero the cell membrane potential. 2. L‐type channels are distinguished by a unitary slope conductance of 25 pS, activation over the range of membrane potentials between 0 and +40 mV, little inactivation over the course of a 136 ms depolarization, and availability for opening even at depolarized holding potentials (h.p. greater than ‐40 mV). L channels show a predominant mode of gating (mode 1) characterized by brief openings (approximately 1 ms), occasionally interspersed with another pattern of gating characterized by much longer openings (mode 2). 3. The dihydropyridine (DHP) Ca2+ agonist Bay K 8644 promotes mode 2 activity and shifts the voltage dependence of L‐type channel activation towards more negative potentials. It leaves the unitary current‐voltage relation unchanged. 4. Nifedipine, a DHP Ca2+ antagonist, strongly inhibits L‐type channel activity through an increase in the proportion of blank sweeps. 5. T‐type Ca2+ channels are distinguished by a much smaller unitary slope conductance (8 pS) and by activation and inactivation over relatively negative ranges of potential. Inactivation is complete by the end of 136 ms pulses to test potentials beyond ‐20 mV. 6. N‐type Ca2+ channels are distinguished by an intermediate unitary slope conductance (13 pS), and by activation over a range of potentials between those of T‐ and L‐type channels. Inactivation of N‐type channels takes place over an exceptionally broad range of holding potentials (‐80 to ‐20 mV). 7. Cell‐attached patch data on the voltage dependence of activation and inactivation of T‐ and N‐type channels are in excellent agreement with results from whole‐cell recordings (Fox, Nowycky & Tsien, 1987) if allowances are made for variations in external surface potential. 8. Patches containing one or two channels of a single type were used for analysis of gating kinetics. The predominant pattern of activity for each of the channel types is an exponential distribution of relatively brief (approximately 1 ms) openings, and a bi‐exponential distribution of short and long closings. 9. Patches containing all possible combinations of channel types were observed. However, preliminary evidence suggests that channels are distributed unevenly over the cell body; clustering of N‐type channels is particularly prominent.(ABSTRACT TRUNCATED AT 400 WORDS)

Time courses of calcium and calcium-bound buffers following calcium influx in a model cell.

Fixed and diffusible calcium (Ca) buffers shape the spatial and temporal distribution of free Ca following Ca entry through voltage-gated ion channels. This modeling study explores intracellular Ca levels achieved near the membrane and in deeper locations following typical Ca currents obtained with patch clamp experiments. Ca ion diffusion sets an upper limit on the maximal average Ca concentration achieved near the membrane. Fixed buffers restrict Ca elevation spatially to the outermost areas of the cell and slow Ca equilibration. Fixed buffer bound with Ca near the membrane can act as Ca source after termination of Ca influx. The relative contribution of fixed versus diffusible buffers to shaping the Ca transient is determined to a large extent by the binding rate of each buffer, with diffusible buffer dominating at equal binding rates. In the presence of fixed buffers, diffusible buffers speed Ca equilibration throughout the cell. The concentration profile of Ca-bound diffusible buffer differs from the concentration profile of free Ca, reflecting theoretical limits on the temporal resolution which can be achieved with commonly used diffusible Ca indicators. A Ca indicator which is fixed to an intracellular component might more accurately report local Ca concentrations.

COMPENSATORY AND EXCESS RETRIEVAL : TWO TYPES OF ENDOCYTOSIS FOLLOWING SINGLE STEP DEPOLARIZATIONS IN BOVINE ADRENAL CHROMAFFIN CELLS

1 Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated‐patch voltage clamp recordings. 2 Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre‐stimulus level (‘compensatory retrieval’) or far below the pre‐stimulus level (‘excess retrieval’). During excess retrieval, > 10 % of the cell surface could be internalized in under 5 s. 3 Compensatory retrieval was equal in magnitude to stimulus‐evoked exocytosis for membrane additions > 100 fF (about fifty large dense‐cored vesicles). In contrast, excess retrieval surpassed both the stimulus‐evoked exocytosis, and the initial capacitance level recorded at the onset of phase‐tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre‐stimulus levels, even in the absence of stimulation. 4 A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval. 5 Compensatory retrieval could occur with any amount of Ca2+ entry, but excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. 6 The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5.7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval. 7 The speed of compensatory retrieval was Ca2+ dependent: the largest mono‐exponential time constants occurred for the smallest amounts of Ca2+ entry and decreased with increasing Ca2+ entry. The Ca2+ dependence of mono‐exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca2+‐ and calmodulin‐dependent phosphatase calcineurin. 8 CsA also reduced the proportion of responses with excess retrieval, but this action was caused by a shift in Ca2+ entry values below the threshold for activation. The lower total Ca2+ entry in the presence of CsA was due to an increase in the rate of Ca2+ current inactivation rather than a reduction in peak amplitude. 9 Our data suggest that compensatory and excess retrieval represent two independent, Ca2+‐regulated mechanisms of rapid membrane internalization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between two distinct modes of behaviour.

Dopaminergic neurons: Role of presynaptic receptors in the regulation of transmitter biosynthesis

Abstract 1. 1. Presynaptic dopamine receptors appear to modulate the increase of dopamine synthesis which is observed after both cessation and an increase in impulse flow. Stimulation of presynaptic receptors limits synthesis in both cases by suppressing the activation of tyrosine hydroxylase. 2. 2. Two in vivo models for studying presynaptic receptors are described. In one, impulse flow is abolished by mechanical lesion of the dopaminergic fibers or by administration of γ-butyrolactone, a drug which reversibly inhibits all impulse flow. In the second model, dopaminergic fibers are stimulated at supra-physiological rates. Once the rate of impulse flow is experimentally stablized by either of these methods, various dopaminergic agonists and antagonists are administered. The observed drug-induced changes of dopamine synthesis activity must then be the result of their interaction with receptors on the terminals since the drugs can no longer affect the rate of impulse flow. 3. 3. Using these in vivo models, it was found that dopamine agonists depress synthesis; dopamine antagonists reverse the effects of the agonists. In the stimulation model dopamine antagonists further enhance synthesis above the stimulation-induced increase, presumably by blocking the effects of released dopamine. These pharmacological studies suggest that by responding to the amount of dopamine present in the synaptic cleft, presynaptic receptors may function to modulate changes in transmitter synthesis which normally occur in response to alterations in impulse flow. Some evidence is presented which is consistent with the hypothesis that these changes may be mediated by the ability of presynaptic receptors to gate calcium entry into the dopamine terminals. 4. 4. The two in vivo models have been utilized to demonstrate that presynaptic receptors in different brain regions vary in sensitivity to agonists. Like postsynaptic receptors, presynaptic receptors become supersensitive after chronic treatment and withdrawal of antipsychotic drugs. Some antipsychotic drugs such as clozapine and pimozide were found to be less effective than other antipsychotic drugs in blocking presynaptic receptors. These results suggest that pre- and postsynaptic dopamine receptors may not be identical. The pharmacological implications of the above observations as well as the possible physiological role played by presynaptic receptors are discussed.

Dopaminergic neurons: effect of acute and chronic morphine administration on single cell activity and transmitter metabolism.

At various time points following acute and chronic administration of morphine to rats, dopamine transmitter metabolism and neuronal activity were determined. Following acute injection of morphine (20 mg/kg intraperitoneally), dopamine cell firing rates increased slowly and steadily. This slow increase was accompanied by a similar slow increase in the accumulation of the dopamine metabolite, dihydroxyphenylacetic acid (DOPAC). Apparentin vivo tyrosine hydroxylase activity, measured by dopa accumulation following inhibition of dopa decarboxylase, also increased. In chronically treated animals the average firing rate of dopamine cells was measured two hours after the last injection of morphine. The distribution of dopamine cell firing rates was significantly higher than in controls. DOPAC levels andin vivo tyrosine hydroxylase activity were also increased at this time. When morphine (100 mg/kg intraperitoneally) was administered to chronically treated animals 12 hours after the last injection a slow increase of firing rates was observed similar to that seen in naive animals after an acute morphine injection. In chronically morphine treated animals naloxone caused a rapid dose-dependent decrease in firing rates and DOPAC levels.In vivo tyrosine hydroxylase activity was not changed.

Electrophysiological analysis of mitral cells in the isolated turtle olfactory bulb.

1. An in vitro preparation of the turtle olfactory bulb has been developed. Electrophysiological properties of mitral cells in the isolated bulb have been analysed with intracellular recordings. 2. Mitral cells have been driven antidromically from the lateral olfactory tract, or activated directly by current injection. Intracellular injections of horseradish peroxidase (HRP) show that turtle mitral cells have long secondary dendrites that extend up to 1800 micrometer from the cell body and reach around half of the bulbar circumference. There are characteristically two primary dendrites, each supplying separate olfactory glomeruli. 3. Using intracellular current pulses, the whole‐neurone resistance was found to range from 33 to 107 M omega. The whole‐neurone charging transient had a slow time course. The membrane time constant was estimated to be 24‐93 msec by the methods of Rall. The electrotonic length of the mitral cell equivalent cylinder was estimated by Ralls methods to be 0.9‐1.9. 4. The spikes generated by turtle mitral cells were only partially blocked by tetrodotoxin (TTX) in the bathing medium. The TTX‐resistant spikes were enhanced in the presence of tetraethylammonium (TEA), and blocked completely by cobalt. 5. The implications of the electrical properties for impulse generation in turtle mitral cells are discussed. The mitral cells have dendrodendritic synapses onto granule cells, and the TTX‐resistant spikes may therefore play an important role in presynaptic transmitter release at these synapses.

Induction of Calcium Influx through TRPC5 Channels by Cross-Linking of GM1 Ganglioside Associated with α5β1 Integrin Initiates Neurite Outgrowth

Previous studies demonstrated that cross-linking of GM1 ganglioside with multivalent ligands, such as B subunit of cholera toxin (CtxB), induced Ca2+ influx through an unidentified, voltage-independent channel in several cell types. Application of CtxB to undifferentiated NG108-15 cells resulted in outgrowth of axon-like neurites in a Ca2+ influx-dependent manner. In this study, we demonstrate that CtxB-induced Ca2+ influx is mediated by TRPC5 channels, naturally expressed in these cells and primary neurons. Both Ca2+ influx and neurite induction were blocked by TRPC5 small interfering RNA (siRNA). Pretreatment of NG108-15 cells with neuraminidase increased cell-surface GM1 and greatly enhanced the signal. GM1 was not directly associated with TRPC5 but rather with α5β1 integrin, which opened the channel through a signaling sequence after cross-linking of the GM1/integrin complex. This cascade included autophosphorylation of focal adhesion kinase and subsequent activation of phospholipase Cγ (PLCγ) and phosphoinositide-3 kinase [PI(3)K]. Pharmacological blockers that inhibited tyrosine kinase, PLC, and PI(3)K suppressed both CtxB-induced Ca2+ influx and neurite outgrowth. These were also suppressed by SK&F96365, a nonspecific transient receptor potential channel blocker. Confocal immunocytochemistry revealed that GM1 cross-linking induced colocalization of GM1 with these signaling elements in sprouting regions of plasma membrane. In primary cerebellar granular neurons (CGNs), TRPC5 was detected at 2 d in vitro (2 DIV), a stage corresponding to CtxB-stimulated Ca2+ influx. Neurite outgrowth in CGNs, determined at 3 DIV, was accelerated by CtxB and suppressed by TRPC5 siRNA and the above blockers. The crucial role of GM1 was indicated with CGNs from ganglio-series null mice, in which growth of axons was significantly retarded.

Brain‐derived neurotrophic factor facilitates maturation of mesenchymal stem cell‐derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain‐derived neurotrophic factor (BDNF) in the maturation of the MSC‐derived DA progenitors. 9‐day induced MSCs show significant tropomyosin‐receptor‐kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF‐treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post‐synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC‐derived DA progenitors.

Reciprocal amplification of ROS and Ca2+ signals in stressed mdx dystrophic skeletal muscle fibers

Muscular dystrophies are among the most severe inherited muscle diseases. The genetic defect is a mutation in the gene for dystrophin, a cytoskeletal protein which protects muscle cells from mechanical damage. Mechanical stress, applied as osmotic shock, elicits an abnormal surge of Ca2+ spark-like events in skeletal muscle fibers from dystrophin deficient (mdx) mice. Previous studies suggested a link between changes in the intracellular redox environment and appearance of Ca2+ sparks in normal mammalian skeletal muscle. Here, we tested whether the exaggerated Ca2+ responses in mdx fibers are related to oxidative stress. Localized intracellular and mitochondrial Ca2+ transients, as well as ROS production, were assessed with confocal microscopy. The rate of basal cellular but not mitochondrial ROS generation was significantly higher in mdx cells. This difference was abolished by pre-incubation of mdx fibers with an inhibitor of NAD(P)H oxidase. In addition, immunoblotting showed a significantly stronger expression of NAD(P)H oxidase in mdx muscle, suggesting a major contribution of this enzyme to oxidative stress in mdx fibers. Osmotic shock produced an abnormal and persistent Ca2+ spark activity, which was suppressed by ROS-reducing agents and by inhibitors of NAD(P)H oxidase. These Ca2+ signals resulted in mitochondrial Ca2+ accumulation in mdx fibers and an additional boost in cellular and mitochondrial ROS production. Taken together, our results indicate that the excessive ROS production and the simultaneous activation of abnormal Ca2+ signals amplify each other, finally culminating in a vicious cycle of damaging events, which may contribute to the abnormal stress sensitivity in dystrophic skeletal muscle.