Ca-dependent adaptive properties in the solitary olfactory receptor cell of the newt

The time-dependent decay of the olfactory receptor potential was analyzed with a solitary cell preparation by using the whole-cell patch clamp technique. During prolonged stimulation by 10 mMN-amylacetate under standard conditions, 17 out of 63 isolated olfactory cells responded with slow depolarization. Of these 17 cells, response amplitudes in 14 cells (‘phasic/tonic’ response) gradually decayed within 9 s, with a half-decay time of1.71 ± 1.10s(mean±S.D.). The relative amplitude (ratio of tonic component to peak amplitude,V_tonic/V_max) was0.29 ± 0.10. The response decay was attributed to the inactivation of the odorant-activated conductance. The recovery after inactivation, which was determined with double pulse experiments, was dependent on the resting interval. The inactivation of the odorant-activated conductance was found to be observed only when the external medium contained Ca²⁺. In addition, it was found that the odorant-activated conductance was capable of permeating Ca²⁺ (P_Ca/P_Na= 6.5), and a rise in the internal EGTA concentration (to 50 mM) inhibited the inactivation. These observations suggest that the decay of the olfactory response to prolonged stimulation is mediated by Ca²⁺ influx.     

A prolonged post-tetanic hyperpolarization in rat hippocampal pyramidal cells in vitro

The post-tetanic sequelae of trains of synaptic stimuli (50 pulses at 5 or 10 Hz) were studied with intracellular recordings from rat hippocampal neurons in vitro. In a large proportion of CA1 neurons, stimulation of afferent fibers was followed by a prolonged membrane hyperpolarization (peak amplitude approximately 6 mV) that was associated with a decrease in neuronal input resistance (approximately 33%) that lasted from tens of seconds to over 1 min. Antidromic stimulation or activation of cells with intracellular current injection did not elicit this post-tetanic hyperpolarization (PTH). The PTH could be elicited in chloride (Cl-)-loaded cells, its null potential shifted in response to changes in extracellular potassium ([K+]o), and it was significantly reduced by 5-10 mM extracellular cesium (Cs+). The K(+)-dependent PTH may also be calcium (Ca2+) dependent as its amplitude and associated conductance increase were sensitive to changes in [Ca2+]o. The PTH was enhanced by treatments that increase Ca2+ entry into cells including perfusion with elevated [Ca2+]o, with picrotoxin or with tetraethylammonium ion (TEA). The K+ conductance blocker 4-AP had no consistent effect on the PTH. The PTH was potently blocked by the membrane-permeant forms of cAMP, dibutyryl- and 8-bromo-cAMP. However, phorbol esters that activate protein kinase C and carbachol, which usually block the same potential that is blocked by cAMP, did not depress the PTH. The cardiac glycosides dihydro-ouabain and strophanthidin had only small and variable effects on the PTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Ruthenium ions on the sensory terminal discharges of the frog muscle spindle

The presence of a mixed Na+-Ca2+ spike along the sensory terminal of the frog muscle spindle was verified. When the terminal was perfused with Ringer's solution containing 0.1-0.5 mM ruthenium red (RuR), the amplitude and duration of the spike were increased, occurring as a prolonged or a long-lasting depolarization of up to 20-30 s duration following individual afferent spikes evoked spontaneously or antidromically by electrical stimulation. In an isotonic TEA solution, the amplitude and duration of the afferent spikes were increased; however, no prolonged depolarization occurred. Adding 0.2 mM RuR to the TEA solution produced the prolonged and long-lasting depolarization. All responses disappeared in the presence of 3 microM TTX or Na+-free Ringer's solution. An impedance decrease along the terminal was observed during the prolonged or long-lasting depolarization. The prolonged depolarization was blocked by the addition of Ca2+-blockers; the afferent spikes remained. In preparations preincubated with 0.1 mM RuR, increasing CaCl2 in Ringer's solution from 0.2 mM, resulted in shortening of the duration of individual spikes with prolonged depolarization and in increase in the maximum rate of rise (MRR) of the spikes. Preincubation with higher concentrations of RuR produced higher sensitivities in the modifications of the duration and MRR to the change in [Ca2+]O. The responses were retained by adding RuR or RuCl3 to Ca2+-free Ringer's solution containing 0.1-5 mM EGTA, although all responses disappeared in Ca2+-free EGTA Ringer's solution. It is concluded that the RuR-induced prolonged response is produced by an influx of Na+.     

A hyperpolarization-activated inward current in heart interneurons of the medicinal leech

Heart interneurons (HN cells) in isolated ganglia of the medicinal leech were voltage-clamped with single microelectrodes. Hyperpolarizing voltage steps elicited a slow inward current (Ih), which underlies the characteristic depolarizing response of HN cells to injection of prolonged hyperpolarizing current pulses (Arbas and Calabrese, 1987a). The conductance underlying Ih begins to activate near -mV and is fully activated between -70 and -80 mV. The activation kinetics of Ih are slow and voltage dependent. The activation time constant (tau h) ranges from approximately 2 sec at -60 mV to near 700 msec at -100 mV. Ih persists in low Ca2+ (0.1 mM), 5 mM Mn2+ saline and exhibits a reversal potential of -21 +/- 5 mV. The reversal potential is shifted by altering [Na+]o or [K+]o but is unaffected by changes in [Cl-]o. Ih is blocked by extracellular Cs+ (1-5 mM) but not Ba2+ (5 mM) or TEA (25 mM). Low concentrations of Cs+ (100-200 microM) cause a partial block that exhibits strong voltage dependence. Temperature changes were also shown to affect Ih. Both the rate of activation and the steady-state amplitude of Ih are enhanced by temperature increases. HN cells are interconnected by inhibitory chemical synapses, and their normal electrical activity consists of bursts of action potentials separated by periods of inhibition. During the inhibitory phase of rhythmic bursting activity, HN cells hyperpolarize to a voltage range where Ih is activated. Block of Ih with extracellular Cs+ (4 mM) disrupted the normal bursting activity of HN cells. These results are consistent with the hypothesis that Ih contributes to escape from inhibitory inputs during normal bursting activity.     

Hypoxic and hypercapnic responses of [Ca]0 and [K]0 in the cat carotid body in vitro

Measurements of extracellular Ca2+ and K+ activities [( Ca2+]o, [K+]o) in the superfused cat carotid body in vitro with triple-barrelled ion-selective electrodes have shown that hypoxia induced a decrease in [Ca2+]o of 0.035 +/- 0.17 mM (mean +/- S.D.; n = 17) and a biphasic change in [K+]o which consisted of an increase of 2.3 +/- 1.8 mM followed by an undershoot of -0.52 +/- 0.34 mM (mean +/- S.D.; n = 17). Hypercapnia induced a monophasic upward deflection increase of both [Ca2+]o and [K+]o of about 0.037 +/- 0.013 mM and 0.33 +/- 0.15 mM, respectively (n = 17). During hypoxia, lowering [Ca2+] in the medium to 0.1 mM resulted in a reversed [Ca2+]o response, attenuated [K+]o increase and absence of chemosensory nerve discharges. TTX generally did not affect the hypoxic and hypercapnic induced ionic changes, although the [K+]o undershoot was reduced by 30%. Co2+ competitively blocked the changes in [Ca2+]o and the increase in the sensory nerve discharge elicited by hypoxia and, not competitively, the changes of [K+]o. The ionic changes to hypercapnia were less affected by Co2+. Ouabain inhibited the [K+]o undershoot induced by hypoxia, as did the removal of Na+ from medium. It is concluded that changes in extracellular free Ca2+ and K+ ions concentration induced by hypoxia and hypercapnia represent ionic fluxes related to the transduction process of carotid body cells (glomus and/or sustentacular).     

Voltage-gated currents in identified rat olfactory receptor neurons

Whole-cell recording techniques were used to characterize voltage-gated membrane currents in neonatal rat olfactory receptor neurons (ORNs) in cell culture. Mature ORNs were identified in culture by their characteristic bipolar morphology, by retrograde labeling techniques, and by olfactory marker protein (OMP) immunoreactivity. ORNs did not have spontaneous activity, but fired action potentials to depolarizing current pulses. Action potentials were blocked by tetrodotoxin (TTX), which contrasts with the TTX-resistant action potentials in salamander olfactory receptor cells (e.g., Firestein and Werblin, 1987). Prolonged, suprathreshold current pulses evoked only a single action potential; however, repetitive firing up to 35 Hz could be elicited by a series of brief depolarizing pulses. Under voltage clamp, the TTX-sensitive sodium current had activation and inactivation properties similar to other excitable cells. In TTX and 20 mM barium, sustained inward current were evoked by voltage steps positive to -30 mV. This current was blocked by Cd (100 microM) and by nifedipine (IC50 = 368 nM) consistent with L-type calcium channels in other neurons. No T-type calcium current was observed. Voltage steps positive to -20 mV also evoked an outward current that did not inactivate during 100-msec depolarizations. Tail current analysis of this current was consistent with a selective potassium conductance. The outward current was blocked by external tetraethylammonium but was unaffected by Cd or 4-aminopyridine (4-AP) or by removal of external calcium. A transient outward current was not observed. The 3 voltage-dependent conductances in cultured rat ORNs appear to be sufficient for 2 essential functions: action potential generation and transmitter release. As a single odorant-activated channel can trigger an action potential (e.g., Lynch and Barry, 1989), the repetitive firing seen with brief depolarizing pulses suggests that ORNs do not integrate sensory input, but rather act as high-fidelity relays such that each opening of an odorant-activated channel reaches the olfactory bulb glomeruli as an action potential.     

Active and Passive Membrane Properties of Spinal Cord Neurons that Are Rhythmically Active during Swimming in Xenopus Embryos

Cellular properties have been examined in ventrally located Xenopus spinal cord neurons that are rhythmically active during fictive swimming and presumed to be motoneurons. Resting potentials and input resistances of such neurons are - 75 +/- 2 mV (mean +/- standard error) and 118 +/- 17 M ohm respectively. Most cells fire a single impulse, 0.5 to 2.0 ms in duration and 48.5 +/- 1.8 mV in amplitude, in response to a depolarizing current step. A minority fire several spikes of diminishing amplitude to more strongly depolarizing current. Cells held above spike, threshold fire on rebound from brief hyperpolarizing pulses. Spikes are blocked by 0.1 to 1.0 microM tetrodotoxin (TTX) and are therefore Na+-dependent. Current/voltage (I/V) plots to injected current are approximately linear near the resting potential but become non-linear at more depolarized levels. Cells recorded in TTX with CsCI-filled microelectrodes show a linearized I/V plot at depolarized membrane potentials suggesting the normal presence of a voltage-dependent K+ conductance activated at relatively depolarized levels. Most cells recorded in this way but without TTX fire long trains of spikes of near constant amplitude, pointing to a role of the K+ conductance in limiting firing in normal cells. Spike blockage with TTX reveals, in some cells, a transient depolarizing Cd2+-sensitive and therefore presumably Ca2+-dependent potential that increases in amplitude with depolarization. Cells in TTX, Cd2+, and strychnine, and recorded with CsCI-filled microelectrodes to block active conductances respond to hyperpolarizing current steps with a two component exponential response. The cell time constant (tau0) obtained from the longer of these by exponential peeling is relatively long (mean 15.7 ms). These findings contribute to an increased understanding of the cellular properties involved in spinal rhythm generation in this simple vertebrate.     

The neuropeptide egg-laying hormone modulates multiple ionic currents in single target neurons of the abdominal ganglion of Aplysia

The bag cell neurons of the abdominal ganglion of Aplysia are a useful system for the study of peptidergic neurotransmission. A 20 min burst of impulse activity in the bag cells induces or augments repetitive firing in LB and LC neurons in the abdominal ganglion for up to several hours. Previous experiments have indicated that this effect is mediated by the putative bag cell transmitter egg-laying hormone (ELH). Using voltage-clamp analysis we found that bag cell bursts (BCBs) evoke long-lasting changes in membrane current in these neurons that are mimicked by the application of ELH. The combined ELH-evoked current is inward at all membrane potentials between -110 and -10 mV and consists of 3 separable currents persisting for 30-120 min. They include (1) a depolarizing current that is activated at membrane potentials above -40 mV. This current, termed ISI, is blocked by prolonged exposure to 10 mM Ni2+/0 mM Ca2+ and is not abolished by 0 mM Na+ or 100 mM TEA+/0 mM Na+ in the bathing medium. It is therefore a Ca2+-sensitive current and does not involve Na+ as a charge carrier. (2) There is a hyperpolarizing current that is activated at membrane potentials below approximately -70 mV. This current, termed IR, is blocked by external Rb+ (5 mM) and Cs+ (10 mM) and has a chord-conductance that shifts with the external [K+] according to the Nernst potential for potassium. It is therefore an inwardly rectifying K+ current. (3) There is a small, steady depolarizing current, termed Ix. This current is the only one that remains after prolonged exposure to 10 mM Ni2+/0 mM Ca2+-containing bathing medium. It is Na+ dependent and is associated with a small increase in membrane conductance that is largely independent of membrane voltage. All 3 currents are slow to inactivate; they appear to sum algebraically to produce the net BCB- or ELH-evoked current.     

Extracellular trypsin increases ASIC1a selectivity for monovalent versus divalent cations

Sustained proton activation of native ASIC channels in primary sensory neurons or HEK293 cells leads to a reduction in the peak amplitude of transient inward currents and the progressive development of a persistent component, which hinders titration experiments in pharmacological studies. Here we report that extracellular trypsin applied for 5 min at 10-45 microg/ml and/or a short exposure to high Ca2+ (75 mM for less than 1 min) alleviate the persistent component, improving reproducibility of acid-elicited transients. Selectivity measurements performed in current clamp mode, in essentially bi-ionic conditions, prove that these two treatments decrease hASIC1a permeability for divalent but not for monovalent cations, producing a significant change in P(Na)/P(Ca) from 8.2+/-2.1 (mean+/-S.D.) to 26.0+/-7.8 (trypsin) or 24.5+/-11.1 (high Ca2+). The slope conductance of the unit inward Ca2+ transient was also lowered from 5.7 to 2.7 pS after trypsin.     

Glial hyperpolarization upon nerve root stimulation in the leech Hirudo medicinalis.

Hyperpolarizing responses in neuropil glial cells evoked by nerve root stimulation were studied in the central nervous system of the leech Hirudo medicinalis using intracellular recording and extracellular stimulation techniques. From a mean resting potential of -60.5 +/- 1.0, the glial membrane was hyperpolarized by -8.6 +/- 0.8 mV, via stimulation of the dorsal posterior nerve root in an isolated ganglion. Nerve root stimulation evoked biphasic or depolarizing responses in glial cells with resting potentials around -70 mV (Rose CR, Deitmer JW. J. Neurophysiol. 73:125-131, 1995). The hyperpolarizing response was reduced by the ionotropic glutamate receptor antagonist CNQX (50 microM) to 58% of its initial amplitude. In 15 mM Ca2+/15 mM Mg(2+)-saline the hyperpolarization was reduced by 44%. The hyperpolarization that persisted in high-divalent cation saline was not affected by CNQX. Bath-applied glutamate (500 microM) and kainate (2 microM) elicited glial hyperpolarizations that were sensitive to CNQX and 10 mM Mg2+/1 mM Ca(2+)-saline. The 5-HT-antagonist methysergide did not affect the hyperpolarizations evoked by nerve root stimulation. The results show that in the leech glial membrane responses to neuronal activity include not only depolarizations, as shown previously, but also hyperpolarizations, which are mediated by direct and indirect neuron-glial communication pathways. In the indirect pathway, glutamate is a transmitter between neurons.     

Saccharin activates cation conductance via inositol 1,4,5-trisphosphate production in a subset of isolated rod taste cells in the frog

The transduction mechanism of the conductance activated by saccharin was analysed in isolated bullfrog taste cells under whole-cell voltage-clamp. Bath application of 30 mM saccharin induced an inward current of -34 +/- 12 pA (mean +/- SEM, n = 10) at a membrane potential of -50 mV in 10 (23%) of 44 rod cells. The concentration-response relationship for the saccharin-gated current was consistent with that of the gustatory neural response. The saccharin-induced current was accompanied with a conductance increase under internal low Cl- condition (E(Cl) = -56 mV), suggesting that saccharin activated a cation conductance. The reversal potential of the saccharin-induced current was -17 +/- 2 mV (n = 10). Intracellular dialysis of 0.5 mM guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) completely blocked the saccharin-induced response, suggesting the involvement of a G protein in the transduction. The dialysis of heparin (1 mg/mL) also inhibited the response almost completely, but the dialysis of 1 mM 8-Br-cAMP did not affect the response significantly. Intracellular 50 microM inositol 1,4,5-trisphosphate (1,4,5 InsP(3)) also induced the inward current in five (38%) of 13 rod cells, but intracellular Pasteurella multocida toxin (5 microg/mL, G alpha q-coupled PLC activator) did not elicit any response in the cells. The results suggest that saccharin mainly activates a cation conductance in frog taste cells through the mediation of IP3 production.     

Elevation of intracellular Ca2+ modulates A-currents in rat cerebellar granule neurons

In the brain, the transient-inactivating voltage-gated potassium channel currents (called I(K(A)) or A-currents) are activated at subthreshold membrane potentials to control the excitability of neurons. In the current study, the effect of intracellular calcium on the A-current and the action mechanism of intracellular calcium was investigated by using the whole-cell voltage-clamp technique. Elevation of intracellular calcium by addition of 2 mM CaCl2 in the pipette solution significantly modulated both the peak amplitude and the kinetics of the A-current in rat granule neurons. The peak amplitudes of the A-current were 1,060 +/- 87 pA and 1,972 +/- 16 pA under conditions of no Ca2+ and elevated intracellular Ca2+, respectively. The time to peak, the time course of fast inactivation, and the steady-state inactivation property of the A-current were all significantly altered by elevating the intracellular Ca2+. Replacement of the Ca2+ in the pipette solution with the same concentration of Co2+ did not mimic the effects of intracellular Ca2+ on the A-current amplitude and kinetics. These effects are similar to the behavior of the reconstituted Kv4/KChIP (K(V) channel-interacting proteins) current induced by expression of KChIP and Kv4 together in a cell expression system. Application of 10 microM arachidonic acid, which can bind to the Kv4/KChIP complex, inhibited the A-current and eliminated the effects of intracellular Ca2+ on the A-current, suggesting that KChIP may be involved in the effects of Ca2+ on the A-current. Collectively, our results indicate that elevated intracellular Ca2+ modulates the amplitude, fast activation, and steady-state inactivation characteristics of the A-current in rat cerebellar granule neurons, and this may occur via KChIP.     

Characterisation of the L- and N-type calcium channels in differentiated SH-SY5Y neuroblastoma cells: calcium imaging and single channel recording.

We have used single cell imaging of [Ca2+]i and single channel cell-attached patch clamp recording to characterise the Ca2+ channels present on the plasma membrane of retinoic acid-differentiated human neuroblastoma (SH-SY5Y) cells. Exposure to raised K+ (45 or 60 mM) for 1 min resulted in a transient rise in [Ca2+]i which was abolished by cadmium (100 microM). The amplitude of the evoked rise varied from cell to cell. Both omega-Conus toxin (500 nM) and nifedipine (10 microM) reduced, but did not abolish, the rise in [Ca2+]i whereas Bay K 8644 (3 microM) potentiated it. In single channel records both L- and N-type Ca2+ channel openings were observed during membrane depolarisations from a holding potential of -90 mV. L-type channel openings (unitary conductance 22.5 pS) were prolonged by S(+)-PN 202-791 (500 nM) and could still be evoked from a depolarised holding potential (-40 mV). N-type channel openings (unitary conductance 12.5 pS) were unaffected by the dihydropyridine agonist but were inactivated at a holding potential of -40 mV. These results indicate that, in contrast to previous observations using whole cell recording, retinoic acid-differentiated SH-SY5Y cells express both L- and N-type Ca2+ channels.     

Bicuculline- and Phaclofen-Resistant Hyperpolarizations Evoked by Glutamate Applications to Stratum Lacunosum-Moleculare in CA1 Pyramidal Cells of the Rat Hippocampus In Vitro

In the hippocampus, different types of interneurons may mediate distinct gamma-aminobutyric acid (GABA) responses, i.e. the early and late inhibitory postsynaptic potentials (IPSPs). To verify this hypothesis, intracellular recordings were obtained from CA1 pyramidal cells (n=63) in rat hippocampal slices. Glutamate (1 mM) was locally ejected in stratum lacunosum-moleculare to activate interneurons in this region. Glutamate-evoked hyperpolarizing responses were characterized in pyramidal cells and compared to the early IPSP and the late IPSP elicited by stratum radiatum electrical stimulation. Several characteristics were similar for the glutamate-evoked IPSPs and late IPSPs: their amplitude was small (-3.4 versus -4.9 mV, respectively), each was associated with a small conductance increase (5.0 versus 9.3 nS, respectively), their peak latency was slow (124.4 versus 129.8 ms, respectively) and in the majority of cells, each displayed little response reversal. However, the equilibrium potential of the glutamate IPSP (-76.5 mV) was similar to that of the early IPSP (-73.8 mV). Perfusion with a low Ca2+ (0.5 mM)/high Mg2+ (8 mM) medium or with tetrodotoxin (1 microM), which blocked synaptic transmission, also reduced the glutamate IPSP. Therefore the glutamate IPSP may be mediated indirectly by inhibitory interneurons. The GABAA antagonist bicuculline (10 microM), or picrotoxin (10-20 microM), blocked the early IPSP, but not the glutamate IPSP. The GABAB antagonist phaclofen (1 mM) attenuated the late IPSP, but did not affect the glutamate IPSP. The results of these experiments suggest that glutamate stimulation of interneurons in stratum lacunosum-moleculare evokes a slow IPSP different from the GABA-mediated early and late IPSPs in CA1 pyramidal cells of the hippocampus.     

Electrophysiological characterization of laminar synaptic inputs to the olfactory tubercle of the rat studied in vitro: modulation of glutamatergic transmission by cholinergic agents is pathway-specific

We have exploited the complementary arrangement of afferents in a coronal slice (300-400 microm) of the rat olfactory tubercle (OT) maintained in vitro to investigate transmission in two separate synaptic pathways. We recorded extracellular responses within the OT dense cell layer in slices and stimulated either the outermost layer to activate primary olfactory fibres or deeper to activate secondary input. Superficial stimulation produced a synaptic potential with superimposed population spike. This interpretation was based on blockade by calcium removal from the bathing medium and the use of the glutamate antagonist DNQX (10 microM); the spike was found to be selectively suppressed by tetrodotoxin applied near the cells. The spike, but not the synaptic wave, was depressed by 12 mM Ca2+ and enhanced by 1 mM Ba2+ in the bathing medium. Deep stimulation to activate association and intrinsic fibres elicited a nerve volley followed by a later response, also blocked by Ca2+ removal or 10 microM DNQX. It was unaffected by high Ca2+ or Ba2+, hence resulting from synaptic and not action current flow. Removal of Mg2+ from the bathing medium revealed an NMDA component of synaptic transmission at both loci that was selectively blocked by D-AP-5. The deep synaptic response, only, was depressed by carbachol IC50 7 microM or muscarine IC50 13 microM. This depression was also induced by AChE inhibitors eserine or tacrine and was antagonized by 1 microM atropine or 5-10 microM clozapine. These results characterize transmission in the OT and demonstrate a role for muscarinic modulation of deeper synapses in the OT that is influenced by psychotherapeutic drugs.     

Cellular mechanisms underlying the rhythmic bursts induced by NMDA microiontophoresis at the apical dendrites of CA1 pyramidal neurons

This article reports the cellular mechanisms underlying a form of intracellular "theta-like" (theta-like) rhythm evoked in vitro by microiontophoresis of N-methyl-D-aspartate (NMDA) at the apical dendrites of CA1 pyramidal neurons. Rhythmic membrane potential (Vm) oscillations and action potential (AP) bursts (approximately 6 Hz; approximately 20 mV; approximately 2-5 APs) were evoked in all cells. The response lasted approximately 2 s, and the initial oscillations were usually small (< 20 mV) and below AP threshold. Rhythmic bursts were never evoked by imposed depolarization in the absence of NMDA. Block of Na+ conductance with tetrodotoxin (TTX) (1.5 microM), of non-NMDA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and of synaptic inhibition by bicuculline (50 microM) and picrotoxin (50 microM) did not prevent NMDA oscillation. Inhibition of the voltage dependence of the NMDA conductance in Mg2+-free Ringer's solution blocked oscillations. Preventing Ca2+ influx with Ca2+-free and Co2+ (2-mM) solutions and block of the slow Ca2+-dependent afterhyperpolarization (sAHP) by carbamilcholine (5 microM), isoproterenol (10 microM), and intracellular BAPTA blocked NMDA oscillations. Inhibition of L-type Ca2+ conductance with nifedipine (30 microM) reduced oscillation amplitude. Block of tetraethylammonium (TEA) (10 mM) and 4AP (10 mM)-sensitive K+ conductance increased the duration and amplitude, but not the frequency, of oscillations. In conclusion, theta-like bursts relied on the voltage dependence of the NMDA conductance and on high-threshold Ca2+ spikes to initiate and boost the depolarizing phase of oscillations. The repolarization is initiated by TEA-sensitive K+ conductance and is controlled by the sAHP. These results suggest a role of interactions between NMDA conductance and intrinsic membrane properties in generating the CA1 theta-rhythm.     

The generation of nonadrenergic inhibiting synaptic potentials in the smooth muscles of the gastrointestinal tract by substituting cesium ions for potassium ions

Nonadrenergic inhibitory junction potentials (IJPs) evoked by intramural stimulation were investigated in smooth muscle of guinea pig stomach, caecum and colon by means of sucrose-gap method. IJPs disappeared in the smooth muscle preexposed to K-free Krebs solution for 4-9 h and restored by addition 6mM Cs+. The amplitude of IJPs was half as much as one in normal conditions but the latency and duration were significantly prolonged. In most cases apamin blocks IJPs in these muscles. The results presented suggest that IJPs generation is due to Cs+ ions permeated through Ca(2+)-activated apamin-sensitive potassium channels of small conductance in these conditions. As the responses to ATP were affected in parallel with IJPs, these results are consistent with the purinergic hypothesis.     

Voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671

A characterization of the properties of voltage-gated sodium channels expressed in the human cerebellar medulloblastoma cell line TE671 is presented. Membrane currents were recorded under voltage clamp conditions using the patch clamp technique in both the whole-cell and the excised-patch configurations. Macroscopic sodium currents display a typical transient time course with a sigmoidal rise to a peak followed by an exponential decay. The rates of early activation and subsequent inactivation accelerate and approach a maximum in response to test potentials, V, of greater depolarization. The magnitude of peak sodium current increased from negligible values below V = -50 mV and reached a maximum at V = -3.6 mV +/- 2.7 mV (mean +/- S.E.M., n = 12). Sodium currents reversed at V = + 70 mV, near the predicted Nernst equilibrium potential for a Na+ selective channel. The peak sodium conductance, gpeak increased with depolarizing voltages to a maximum at V = approximately 0 mV, exhibiting half-activation voltage at V approximately equal to -36.8 mV and an e-fold change in gpeak/9.5 mV. The Hodgkin-Huxley inactivation parameter h infinity indicates that at V = -73.6 mV half of the sodium currents were inactivated. Single channel current recordings demonstrated the occurrence of discrete events: the latency for first opening was shorter as the depolarizing pulse became more positive. The single-channel current amplitude was ohmic with a slope conductance, gamma = 17.13 pS +/- 0.66 pS. Sodium channel currents were reversibly blocked by tetrodotoxin (TTX).(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic properties of an inositol 1,4,5-trisphosphate-gated channel in carp olfactory cilia

In addition to the activation of cAMP-dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5-trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip-dip method). In the presence of InsP3 (10 microM) and Ca2+ (100 nM), a current of 1.6 +/- 0.1 pA (mean +/- SEM, n = 4) was measured, using Ba2+ as charge carrier. The I/V curve displayed a slope conductance of 45 +/- 5 pS and a reversal potential of -29 mV indicating a higher selectivity for divalent cations. This current was characterized by two mean open times (3.0 +/- 0.4 ms and 42.0 +/- 2.6 ms, n = 4) and was strongly inhibited by ruthenium red (30 microM) or heparin (10 microg/mL). Importantly, the channel activity was closely dependent on the Ca2+ concentration, with the highest open probability (Po) at 100 nM Ca2+ (Po = 0.50 +/- 0.02, n = 4). Po is lower at both higher and lower Ca2+ concentrations. A structural identification of the channel was attempted by using a large panel of antibodies, raised against several InsP3 receptor (InsP3R)/Ca2+ release channel isoforms. The type 1 InsP3R was detected in carp cerebellum and whole brain, while a lower molecular mass InsP3R, which may correspond to type 2 or 3, was detected in heart, whole brain and the soma of the olfactory neurons. None of the antibodies, however, cross-reacted with olfactory cilia. Taken together, these results indicate that in carp olfactory cilia an InsP3-dependent channel is present, distinct from the classical InsP3Rs localized on intracellular membranes.