Action potentials recorded with patch-clamp amplifiers: are they genuine?

A growing number of experimental studies have used patch-clamp amplifiers (PCAs) in the current-clamp (CC) mode to investigate classical excitability. In this paper we show that the measurements obtained in this way are affected by errors due to the electronic design of the PCA input section. We present experimental evidence of such errors, and demonstrate that they derive from PCA current absorption. Moreover, we propose a new PCA input-circuit configuration for the CC mode, which is suitable for accurately recording physiological voltage signals and is perfectly compatible with the standard voltage-clamp mode.

High conductance sustained single-channel activity responsible for the low-threshold persistent Na(+) current in entorhinal cortex neurons.

Stellate cells from entorhinal cortex (EC) layer II express both a transient Na(+) current (I(Na)) and a low-threshold persistent Na(+) current (I(NaP)) that helps to generate intrinsic theta-like oscillatory activity. We have used single-channel patch-clamp recording to investigate the Na(+) channels responsible for I(NaP) in EC stellate cells. Macropatch (more than six channels) recordings showed high levels of transient Na(+) channel activity, consisting of brief openings near the beginning of depolarizing pulses, and lower levels of persistent Na(+) channel activity, characterized by prolonged openings throughout 500 msec long depolarizations. The persistent activity contributed a noninactivating component to averaged macropatch recordings that was comparable with whole-cell I(NaP) in both voltage dependence of activation (10 mV negative to the transient current) and amplitude (1% of the transient current at -20 mV). In 14 oligochannel (less than six channels) patches, the ratio of transient to persistent channel activity varied from patch to patch, with 10 patches exhibiting exclusively transient openings and one patch showing exclusively persistent openings. In two patches containing only a single persistent channel, prolonged openings were observed in >50% of test depolarizations. Moreover, persistent openings had a significantly higher single-channel conductance (19.7 pS) than transient openings (15.6 pS). We conclude that this stable high-conductance persistent channel activity is responsible for I(NaP) in EC stellate cells. This persistent channel behavior is more enduring and has a higher conductance than the infrequent and short-lived transitions to persistent gating modes that have been described previously in brain neurons.

Biophysical properties and slow voltage-dependent inactivation of a sustained sodium current in entorhinal cortex layer-II principal neurons: a whole-cell and single-channel study.

The functional and biophysical properties of a sustained, or "persistent," Na(+) current (I(NaP)) responsible for the generation of subthreshold oscillatory activity in entorhinal cortex layer-II principal neurons (the "stellate cells") were investigated with whole-cell, patch-clamp experiments. Both acutely dissociated cells and slices derived from adult rat entorhinal cortex were used. I(NaP), activated by either slow voltage ramps or long-lasting depolarizing pulses, was prominent in both isolated and, especially, in situ neurons. The analysis of the gating properties of the transient Na(+) current (I(NaT)) in the same neurons revealed that the resulting time-independent "window" current (I(NaTW)) had both amplitude and voltage dependence not compatible with those of the observed I(NaP), thus implying the existence of an alternative mechanism of persistent Na(+)-current generation. The tetrodotoxin-sensitive Na(+) currents evoked by slow voltage ramps decreased in amplitude with decreasing ramp slopes, thus suggesting that a time-dependent inactivation was taking place during ramp depolarizations. When ramps were preceded by increasingly positive, long-lasting voltage prepulses, I(NaP) was progressively, and eventually completely, inactivated. The V(1/2) of I(NaP) steady state inactivation was approximately -49 mV. The time dependence of the development of the inactivation was also studied by varying the duration of the inactivating prepulse: time constants ranging from approximately 6.8 to approximately 2.6 s, depending on the voltage level, were revealed. Moreover, the activation and inactivation properties of I(NaP) were such as to generate, within a relatively broad membrane-voltage range, a really persistent window current (I(NaPW)). Significantly, I(NaPW) was maximal at about the same voltage level at which subthreshold oscillations are expressed by the stellate cells. Indeed, at -50 mV, the I(NaPW) was shown to contribute to >80% of the persistent Na(+) current that sustains the subthreshold oscillations, whereas only the remaining part can be attributed to a classical Hodgkin-Huxley I(NaTW). Finally, the single-channel bases of I(NaP) slow inactivation and I(NaPW) generation were investigated in cell-attached experiments. Both phenomena were found to be underlain by repetitive, relatively prolonged late channel openings that appeared to undergo inactivation in a nearly irreversible manner at high depolarization levels (-10 mV), but not at more negative potentials (-40 mV).

Dual effect of Zn2+ on multiple types of voltage-dependent Ca2+ currents in rat palaeocortical neurons.

The effects of Zn(2+) were evaluated on high-voltage-activated Ca(2+) currents expressed by pyramidal neurons acutely dissociated from rat piriform cortex. Whole-cell, patch-clamp experiments were carried out using Ba(2+) (5 mM) as the charge carrier. Zn(2+) blocked total high-voltage-activated Ba(2+) currents with an IC(50) of approximately 21 microM. In addition, after application of non-saturating Zn(2+) concentrations, residual currents activated with substantially slower kinetics than control Ba(2+) currents. Both of the above-mentioned effects of Zn(2+) were also observed in high-voltage-activated currents recorded in the presence of nearly-physiological concentrations of extracellular Ca(2+) (1 and 2 mM) rather than Ba(2+). Under the latter conditions, 30 microM Zn(2+) inhibited high-voltage-activated currents somewhat less than observed in extracellular Ba(2+) (approximately 47% and approximately 41%, respectively, vs. approximately 59%), but slowed Ca(2+)-current activation to very similar degrees. All of the pharmacological components in which Ba(2+) currents could be dissected (L-, N-, P/Q-, and R-type) were inhibited by Zn(2+), the percentage of current blocked by 30 microM Zn(2+) ranging from 34 to 57%. Moreover, the activation kinetics of all pharmacological Ba(2+) current components were slowed by Zn(2+). Hence, the lower activation speed observed in residual Ba(2+) currents after Zn(2+) block is due to a true slowing of macroscopic Ca(2+)-current activation kinetics and not to the preferential inhibition of a fast-activating current component. The inhibitory effect of Zn(2+) on Ba(2+) current amplitude was voltage-independent over the whole voltage range explored (-60 to +30 mV), hence the Zn(2+)-dependent decrease of Ba(2+) current activation speed is not the consequence of a voltage- and time-dependent relief from block. Zn(2+) also caused a slight, but significant, reduction of Ba(2+) current deactivation speed upon repolarization, which is further evidence against a depolarization-dependent unblocking mechanism. Finally, the slowing effect of Zn(2+) on Ca(2+)-channel activation kinetics was found to result in a significant, extra reduction of Ba(2+) current amplitude when action-potential-like waveforms, rather than step pulses, were used as depolarizing stimuli. We conclude that Zn(2+) exerts a dual action on multiple types of voltage-gated Ca(2+) channels, causing a blocking effect and altering the speed at which channels are delivered to conducting states, with mechanism(s) that could be distinct.

Persistent sodium channel activity mediates subthreshold membrane potential oscillations and low-threshold spikes in rat entorhinal cortex layer V neurons.

Entorhinal cortex layer V occupies a critical position in temporal lobe circuitry since, on the one hand, it serves as the main conduit for the flow of information out of the hippocampal formation back to the neocortex and, on the other, it closes a hippocampal-entorhinal loop by projecting upon the superficial cell layers that give rise to the perforant path. Recent in vitro electrophysiological studies have shown that rat entorhinal cortex layer V cells are endowed with the ability to generate subthreshold oscillations and all-or-none, low-threshold depolarizing potentials. In the present study, by applying current-clamp, voltage-clamp and single-channel recording techniques in rat slices and dissociated neurons, we investigated whether entorhinal cortex layer V cells express a persistent sodium current and sustained sodium channel activity to evaluate the contribution of this activity to the subthreshold behavior of the cells. Sharp-electrode recording in slices demonstrated that layer V cells display tetrodotoxin-sensitive inward rectification in the depolarizing direction, suggesting that a persistent sodium current is present in the cells. Subthreshold oscillations and low-threshold regenerative events were also abolished by tetrodotoxin, suggesting that their generation also requires the activation of such a low-threshold sodium current. The presence of a persistent sodium current was confirmed in whole-cell voltage-clamp experiments, which revealed that its activation "threshold" was negative by about 10mV to that of the transient sodium current. Furthermore, stationary noise analysis and cell-attached, patch-clamp recordings indicated that whole-cell persistent sodium currents were mediated by persistent sodium channel activity, consisting of relatively high-conductance ( approximately 18pS) sustained openings. The presence of a persistent sodium current in entorhinal cortex layer V cells can cause the generation of oscillatory behavior, bursting activity and sustained discharge; this might be implicated in the encoding of memories in which the entorhinal cortex participates but, under pathological situations, may also contribute to epileptogenesis and neurodegeneration.

Oscillatory activity in entorhinal neurons and circuits. Mechanisms and function.

Layers II and V of the entorhinal cortex (EC) occupy a privileged anatomical position in the temporal lobe memory system that allows them to gate the main flow of information in and out of the hippocampus, respectively. In vivo studies have shown that layer II of the EC is a robust generator of theta as well as gamma activity. Theta may also be present in layer V, but the layer V network is particularly prone to genesis of short-lasting high-frequency oscillations ("ripples"). Interestingly, in vitro studies have shown that EC layers II and V, but not layer III, have the potential to act as independent pacemakers of population oscillatory activity. Moreover, it has also been shown that subgroups of principal neurons both within layers II and V, but not layer III, are endowed with autorhythmic properties. These are characterized by subthreshold oscillations where the depolarizing phase is driven by the activation of "persistent" Na+ channels. We propose that the oscillatory properties of layer II and V neurons and local circuits are responsible for setting up the proper temporal dynamics for the coordination of the multiple sensory inputs that converge onto EC and thus help to generate sensory representations and memory encoding.

Long-term survival of cortical neurones from adult guinea-pig maintained in low-density cultures.

In vitro survival of neurones isolated from adult mammalian brain is normally scarce and the postnatal age limit for obtaining viable cultures of cortical, hippocampal and diencephalic neurones is commonly two weeks. Here we describe a novel procedure for the establishment and long-term maintenance of cortical neurones of the adult mammalian brain in low-density cultures. Neurones isolated from the piriform cortex of 30- to 90-day-old guinea-pigs were initially grown in a chemically defined medium enriched with basic fibroblast growth factor (bFGF); later, a small quantity of foetal bovine serum (FBS) was added to facilitate cell differentiation. Under these conditions cells could be maintained in culture for at least 3 weeks, when indirect immunocytochemistry and whole-cell patch-clamp recordings were performed. Cells exhibiting neuronal morphology expressed the neuronal marker microtubule associated protein-2 (MAP2) and generated action potentials. Moreover, about 70% of the MAP2-immunoreactive cells were simultaneously labelled with anti-gamma-aminobutyric acid (GABA) antibody. Cells expressing neuronal antigens were never labelled by antibody raised against the glial marker glial fibrillary acidic protein (GFAP). These results indicate that long-term survival of adult neurones can be achieved under definite culture conditions.

The relationship between synaptogenesis and expression of voltage-dependent currents in cerebellar granule cells in situ.

In this work we consider the ontogenetic changes of membrane currents and their relationship with synaptogenesis in cerebellar granule cells. Recordings were performed in whole-cell patch-clamp configuration from cerebellar slices obtained from 4 to 31-day-old rats. Granule cells in the external granular layer, and non-connected granule cells in the internal granular layer expressed outward currents, and inconstantly also small Ca2+ currents, but no fast Na+ currents. Most connected granule cells expressed Ca2+ and Na+ currents. These data indicate that Ca2+ and Na+ current development occurs after synapse formation, while outward (K+) currents begin their development before. Mixed NMDA/non-NMDA synaptic currents were observed at all stages, while synaptic currents with a prominent NMDA component were observed exclusively at immature stages. At P4, ie 1-2 days after the arrival of the first granule cells in the internal granular layer, some granule cells already expressed mature synaptic and voltage-dependent currents, suggesting that establishment of mossy fibre synapses and development of membrane properties takes just 1-2 days to complete. Starting at P4, the probability of activating mossy fibre currents, and sizeable Ca2+ and Na+ currents increased at a similar rate, attaining a plateau level around P20. Average amplitude of Na+ and outward currents decreased until P10 and then increased attaining plateau soon beyond P20. Average amplitude of Ca2+ currents increased monotonically. The time courses of probability and average current amplitude curves are likely explained by changes in the rate of accumulation of migrating granule cells in the internal granular layer, and by changes in granule cell membrane surface extension. These data suggest a relevant role for the process of synapse formation in inducing the expression of new channels in the developing granule cells, which may involve Ca2+ influx through the NMDA channel.

Intercellular K⁺ accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx.

Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K(+) current, I(K,L), which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells. The functional meaning of I(K,L) and associated calyx is still enigmatic. By combining the patch-clamp whole-cell technique with the mouse whole crista preparation, we have recorded the current- and voltage responses of in situ hair cells. Outward K(+) current activation resulted in K(+) accumulation around Type I hair cells, since it induced a rightward shift of the K(+) reversal potential the magnitude of which depended on the amplitude and duration of K(+) current flow. Since this phenomenon was never observed for Type II hair cells, we ascribed it to the presence of a residual calyx limiting K(+) efflux from the synaptic cleft. Intercellular K(+) accumulation added a slow (τ>100ms) depolarizing component to the cell voltage response. In a few cases we were able to record from the calyx and found evidence for intercellular K(+) accumulation as well. The resulting depolarization could trigger a discharge of action potentials in the afferent nerve fiber. Present results support a model where pre- and postsynaptic depolarization produced by intercellular K(+) accumulation cooperates with neurotransmitter exocytosis in sustaining afferent transmission arising from Type I hair cells. While vesicular transmission together with the low Rm of Type I hair cells appears best suited for signaling fast head movements, depolarization produced by intercellular K(+) accumulation could enhance signal transmission during slow head movements.

A fast transient outward current in layer II/III neurons of rat perirhinal cortex.

The perirhinal cortex (PRC) is a supra-modal cortical area that collects and integrates information originating from uni- and multi-modal neocortical regions, transmits it to the hippocampus, and receives a feedback from the hippocampus itself. The elucidation of the mechanisms that underlie the specific excitable properties of the different PRC neuronal types appears as an important step toward the understanding of the integrative functions of PRC. In this study, we investigated the biophysical properties of the transient, I (A)-type K(+) current recorded in pyramidal neurons acutely dissociated from layers II/III of PRC of the rat (P8-P16). The current activated at about -50 mV and showed a fast monoexponential decay (tau(h) >> 14 ms at -30 to +10 mV). I (A) recovery from inactivation also had a monoexponential time course. No significant differences in the biophysical properties or current density of I (A) were found in pyramidal neurons from rats of different ages. Application of 4-AP (1-5 mM) reversibly and selectively blocked I (A), and in current clamp conditions it increased spike duration and shortened the delay of the first spike during repetitive firing evoked by sustained depolarizing current injection. These properties are similar to those of the I (A) found in thalamic neurons and other cortical pyramidal neurons. Our results suggest that I (A) contributes to spike repolarization and to regulate both spike onset timing and firing frequency in PRC neurons.

Sodium-activated potassium current in sensory neurons: a comparison of cell-attached and cell-free single-channel activities.

Single-channel currents from Na(+)-dependent K+ channels (KNa) were recorded from cell-attached and inside-out membrane patches of cultured avian trigeminal ganglion neurons by means of the patch-clamp technique. Single-channel properties, such as the high elementary conductance and the occurrence of sub-conductance levels, were unchanged after the patches had been excised from the cells, indicating that they are not under the control of soluble cytoplasmic factors. In cell-attached recordings at the cell resting potential the degree of KNa activity, measured as the probability of the channel being open, Po, was low in most cases (around 0.01) and similar to that observed in the inside-out configuration when the bath solution contained concentrations of Na+ around 30 mM and of K+ close to the physiological intracellular levels. However, in some cell-attached patches Po was high (around 0.2) and comparable to the values measured in cell-free recordings with high Na+ concentrations in the bath (100 mM). The excision of a high-activity patch in the presence of 30 mM Na+ resulted in a fall of Po in about 20 s, which is consistent with the wash-out of a soluble cytoplasmic molecule. After the excision, all KNa displayed a similar Na+ sensitivity, irrespective of the degree of activation observed in the cell-attached mode. In inside-out patches the Po values observed in the presence of either low or high concentrations of Na+ in bath solutions were not modified by internal Ca2+ (0.8-8.5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex.

A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 +/- 6.2 (SD) pF] than in neurons from layers II and III (8.6 +/- 2.8 and 7.9 +/- 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to -40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11-16 pA/pF at -30 mV, 2) inactivated rapidly with a time constant of approximately 22 ms at -30 mV, and 3) was completely steady-state inactivated at -60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.

Glucose transport stimulation by bradykinin in Swiss 3T3 fibroblasts: a pertussis toxin-sensitive mechanism operates without involvement of arachidonic acid and cyclic AMP.

The possible role of cAMP and/or arachidonic acid (and metabolites) in the stimulation of glucose transport elicited by bradykinin in Swiss 3T3 fibroblasts was investigated with particular attention to the part of this effect inhibitable by pertussis toxin. Application of the membrane permeant cAMP analog 8-BrcAMP modified neither basal nor stimulated transport observed after bradykinin, insulin, or the combination of the two, indicating that [cAMP]i fluctuations are probably not involved. In contrast, arachidonic acid, which is released by the cells exposed to bradykinin, was able to markedly stimulate glucose transport, however, only at relatively high concentrations (EC50 approximately 30 microM). The stimulation by arachidonic acid was insensitive to pertussis toxin and was largely inhibited by both the cyclooxygenase blocking drug, indomethacin, and the [Ca2+]i clamping at the resting level (by ionomycin administered in a Ca2(+)-free incubation medium). Neither of the last treatments affected the glucose transport activated by bradykinin to a great extent. Moreover, the bradykinin-induced arachidonic acid release was unaffected by pertussis toxin and markedly inhibited by two treatments ineffective on glucose transport, the blockade of [Ca2+]i increases elicited by the peptide and the administration of the phospholipase A2 blocker, quinacrine. These results exclude that glucose transport stimulation by bradykinin is mediated intracellularly via arachidonic acid release. Since the involvement of Ca2+ and diacylglycerol can also be ruled out by present and previous results, this effect of the peptide appears to be independent of the generation of known second messengers and might be triggered by the direct interaction of a pertussis toxin-sensitive G protein with the glucose transporter in the plane of the plasma membrane.

Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons.

The effects of three divalent metal cations (Mn2+, Co2+, and Cu2+) on high-voltage-activated (HVA) Ca2+ currents were studied in acutely dissociated pyramidal neurons of rat piriform cortex using the patch-clamp technique. Cu2+, Mn2+, and Co2+ blocked HVA currents conducted by Ba2+ ( IBa) with IC50 of approximately 920 nM, approximately 58 micro M, and approximately 65 micro M, respectively. Additionally, after application of non-saturating concentrations of the three cations, residual currents activated with substantially slower kinetics than control IBa. As a consequence, the current fraction abolished by the blocking cations typically displayed, in its early phase, an unusually fast-decaying transient. The latter phenomenon turned out to be a subtraction artifact, since none of the pharmacological components (L-, N-, P/Q-, and R-type) that constitute the total HVA currents under study showed a similarly fast early decay: hence, the slow activation kinetics of residual currents was not due to the preferential inhibition of a fast-activating/inactivating component, but rather to a true slowing effect of the blocker cations. The percent IBa-amplitude inhibition caused by Mn2+, Co2+, and Cu2+ was voltage-independent over the whole potential range explored (up to +30 mV), hence the slowing of IBa activation kinetics was not due to a mechanism of voltage- and time-dependent relief from block. Moreover, Mn2+, Co2+, and Cu2+ significantly reduced I(Ba) deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The above results show that 1) Cu2+ is a particularly potent HVA Ca2+-channel blocker in rat palaeocortical neurons; and 2) Mn2+, Co2+, and Cu2+, besides exerting a blocking action on HVA Ca2+-channels, also modify Ca2+-current activation and deactivation kinetics, most probably by directly interfering with channel-state transitions.

Ni2+ slows the activation kinetics of high-voltage-activated Ca2+ currents in cortical neurons: evidence for a mechanism of action independent of channel-pore block.

The effects of Ni2+ were evaluated on slowly-decaying, high-voltage-activated (HVA) Ca2+ currents expressed by pyramidal neurons acutely dissociated from guinea-pig piriform cortex. Whole-cell, patch-clamp recordings were performed with Ba2+ as the charge carrier. Ni2+ blocked HVA Ba2+ currents (IBas) with an EC50 of approximately 60 microM. Additionally, after application of nonsaturating Ni2+ concentrations, residual currents activated with substantially slower kinetics than both total and Ni2+-sensitive I(Ba)s. None of the pharmacological components of slowly decaying, HVA currents activated with kinetics significantly different from that of total currents, indicating that the effect of Ni2+ on I(Ba)s kinetics cannot be attributed to the preferential inhibition of a fast-activating component. The effect of Ni2+ on I(Ba) amplitude was voltage-independent over the potential range normally explored in our experiments (-60 to +20 mV), hence the Ni2+-dependent decrease of I(Ba) activation rate is not due to a voltage- and time-dependent relief from block. Moreover, Ni2+ significantly reduced I(Ba) deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The dependence on Ni2+ concentration of the I(Ba) activation-rate reduction was remarkably different from that found for I(Ba) block, with an EC50 of approximately 20 microM and a Hill coefficient of approximately 1.73 vs. approximately 1.10. These results demonstrate that Ni2+, besides inhibiting the I(Ba)s under study probably by exerting a blocking action on the pore of the underlying Ca2+ channels, also interferes with Ca2+-channel gating kinetics, and strongly suggest that the two effects depend on Ni2+ occupancy of binding sites at least partly distinct.

A blocker-resistant, fast-decaying, intermediate-threshold calcium current in palaeocortical pyramidal neurons.

The whole-cell patch-clamp technique was used to record Ca2+ currents in acutely dissociated neurons from layer II of guinea-pig piriform cortex (PC). Ba2+ (5 mM) was used as charge carrier. In a subpopulation of layer II cells ( approximately 22%) total Ba2+ currents (IBas) displayed a high degree (> 70%) of inactivation after 300 ms of steady depolarization. The application of L-, N- and P/Q-type Ca2+-channel blockers to these high-decay IBas left their fast inactivating component largely unaffected. The inactivation phase of the blocker-resistant, fast-decaying IBa thus isolated had a bi-exponential time course, with a fast time constant of approximately 20 ms and a slower time constant of approximately 100 ms at voltage levels positive to -10 mV. The voltage dependence of activation of the blocker-resistant, fast-decaying IBa was shifted by approximately 7-9 mV in the negative direction in comparison with those of other pharmacologically and/or kinetically different high-voltage-activated Ca2+ currents. We named this blocker-resistant, fast-decaying, intermediate-threshold current IRfi. The amplitude of IRfi decreased only slightly (by approximately 9%) when extracellular Ca2+ was substituted for Ba2+, in contrast with that of slowly decaying, high-voltage-activated currents, which was reduced by approximately 41% on average. Moreover, IRfi was substantially inhibited by low concentrations of Ni2+ (50 microM). We conclude that IRfi, because of its fast inactivation kinetics, intermediate threshold of activation and resistance to organic blockers, represents a definite, identifiable Ca2+ current different from classical high-voltage-activated currents and clearly distinguishable from classical IT. The striking similarity found between IRfi and Ca2+ currents resulting from heterologous expression of alpha1E-type channel subunits is discussed.

Biophysical and pharmacological diversity of high-voltage-activated calcium currents in layer II neurones of guinea-pig piriform cortex.

1. High-voltage-activated calcium currents were studied with the whole-cell, patch-clamp technique in acutely dissociated pyramidal neurones from guinea-pig piriform cortex layer II. Barium ions were used as charge carriers. 2. Barium currents (IBa) displayed a remarkable kinetic diversity in different neurones. The ratio between the current amplitude at the end of the test pulses and the peak amplitude (Re/p) showed two frequency-distribution peaks at approximately 0.4 and 0.8. The index of current activation speed (rise time 10-90 %) directly correlated with the index of current persistence, Re/p. 3. The half-activation potential (V ) of total IBas positively correlated with the Re/p of the corresponding currents. This implied that the high-decay IBas also had a more negative voltage range of activation than the more persistent ones. 4. The L- and N-type channel blockers nifedipine (10 microM) and omega-conotoxin GVIA (omega-CTx GVIA, 0.5-1 microM) additively blocked 20 and 25 % of the total IBa, respectively. The P/Q-type calcium channel blockers omega-agatoxin IVA (100 nM), omega-conotoxin MVIIC (1 microM) and 3.3 funnel toxin (1 microM), had little effect on IBa. 5. The nifedipine- and omega-CTx GVIA-sensitive current had a Re/p > 0.55 and their voltage dependence of activation was of the high-voltage-activated type (V approximately 0 mV). 6. High-, intermediate- and low-decay blocker-resistant currents were observed in different neurones. Their Re/p values highly correlated with those of the corresponding total IBas and with the voltage dependence of activation of the underlying conductances. Exponential fittings of the inactivation phase of blocker-resistant currents returned very fast time constants (lower than 30 ms) for high-decay currents (Re/p < 0.25). The intermediate-decay currents (Re/p approximately 0.55) could not derive from variable combinations of high- and low-decay current components. 7. Our data demonstrate a remarkable variety in voltage-activated calcium currents expressed by piriform cortex neurones, that include currents resistant to high-voltage-activated calcium-channel blockers.

Direct demonstration of persistent Na+ channel activity in dendritic processes of mammalian cortical neurones.

1. Single Na+ channel activity was recorded in patch-clamp, cell-attached experiments performed on dendritic processes of acutely isolated principal neurones from rat entorhinal-cortex layer II. The distances of the recording sites from the soma ranged from approximately 20 to approximately 100 microm. 2. Step depolarisations from holding potentials of -120 to -100 mV to test potentials of -60 to +10 mV elicited Na+ channel openings in all of the recorded patches (n = 16). 3. In 10 patches, besides transient Na+ channel openings clustered within the first few milliseconds of the depolarising pulses, prolonged and/or late Na+ channel openings were also regularly observed. This 'persistent' Na+ channel activity produced net inward, persistent currents in ensemble-average traces, and remained stable over the entire duration of the experiments ( approximately 9 to 30 min). 4. Two of these patches contained < or = 3 channels. In these cases, persistent Na+ channel openings could be attributed to the activity of one single channel. 5. The voltage dependence of persistent-current amplitude in ensemble-average traces closely resembled that of whole-cell, persistent Na+ current expressed by the same neurones, and displayed the same characteristic low threshold of activation. 6. Dendritic, persistent Na+ channel openings had relatively high single-channel conductance ( approximately 20 pS), similar to what is observed for somatic, persistent Na+ channels. 7. We conclude that a stable, persistent Na+ channel activity is expressed by proximal dendrites of entorhinal-cortex layer II principal neurones, and can contribute a significant low-threshold, persistent Na+ current to the dendritic processing of excitatory synaptic inputs.

Modalities of distortion of physiological voltage signals by patch-clamp amplifiers: a modeling study.

An extensive evaluation of the possible alterations affecting physiological voltage signals recorded with patch-clamp amplifiers (PCAs) working in the current-clamp (CC) mode was carried out by following a modeling approach. The PCA output voltage and current signals obtained during CC recordings performed under simplified experimental conditions were exploited to determine the equations describing the generation of error currents and voltage distortions by PCAs. The functions thus obtained were used to construct models of PCAs working in the CC mode, which were coupled to numerical simulations of neuronal bioelectrical behavior; this allowed us to evaluate the effects of the same PCAs on different physiological membrane-voltage events. The models revealed that rapid signals such as fast action potentials are preferentially affected, whereas slower events, such as low-threshold spikes, are less altered. Prominent effects of model PCAs on fast action potentials were alterations of their amplitude, duration, depolarization and repolarization speeds, and, most notably, the generation of spurious afterhyperpolarizations. Processes like regular firing and burst firing could also be altered, under particular conditions, by the model PCAs. When a cell consisting of more than one single intracellular compartment was considered, the model PCAs distorted fast equalization transients. Furthermore, the effects of different experimental and cellular parameters (series resistance, cell capacitance, temperature) on PCA-generated artifacts were analyzed. Finally, the simulations indicated that no off-line correction based on manipulations of the error-current signals returned by the PCAs can be successfully performed in the attempt to recover unperturbed voltage signals, because of alterations of the overall current flowing through the cell-PCA system.