Electrophysiological Interactions Between 5-Hydroxytryptamine and Thyrotropin Releasing Hormone on Rat Hippocampal CA1 Neurons

Intracellular recording from CA1 neurons of the rat hippocampal slice preparation was used to examine the possibility of functional interactions between 5-hydroxytryptamine (5-HT) and thyrotropin releasing hormone (TRH), which act as cotransmitters in other areas of the central nervous system. 5-HT (30 μM) elicited complex effects consisting of biphasic changes in membrane potential and a strong depression of the afterhyperpolarization (AHP) following a spike burst. TRH (10 μM) did not alter membrane potential or input conductance but it produced a partial block of the AHP. Under single-electrode voltage clamp, 5-HT and TRH both reduced the amplitude of voltage-activated total K⁺ currents. When the two substances were co-applied, their actions were occluded. The voltage-activated K⁺ current remaining in Ca²⁺-free solution lost its sensitivity to 5-HT and TRH, suggesting that the K⁺ current modulated by TRH and 5-HT was Ca²⁺-dependent, although TRH itself did not depress high-threshold voltage-activated Ca²⁺ currents. When a relatively small concentration (5 μM) of 5-HT was co-applied with an equimolar amount of TRH, the degree of block of the spike AHP was the sum of the two individual effects of these drugs. It is suggested that in hippocampal pyramidal cells 5-HT and TRH influenced neuronal excitability by depressing a Ca²⁺-dependent K⁺ current, a phenomenon perhaps mediated through a common intracellular second messenger pathway.     

Effects of a Cognition-enhancer, Linopirdine (DuP 996), on M-type Potassium Currents (IK(M)) Some Other Voltage- and Ligand-gated Membrane Currents in Rat Sympathetic Neurons

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K⁺ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 μM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (I_K(v)), IC50 63 μM; transient (I_A) current, IC50 69 μM] and produced no detectable inhibition of the fast and slow Ca²⁺-activated K⁺ currents I_c and I_AHP or of a hyperpolarization-activated cation current (I_Q/I_h), at 10–30 μM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl^- currents with IC50 values of 7.6 and 26 μM respectively. It is concluded that linopirdine shows some 20–fold selectivity for M-channels among different K⁺ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.     

The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.     

Skin Sympathetic Response In Subclinical Diabetic Neuropathy

Hyperglycemia and its associated Na⁺/K⁺ pump activity has been implicated in the development of diabetic neuropathy. We recently reported that high glucose in the presence of ouabain induced a progressive increase in the delayed K⁺ current which was suppressed by a blocker of Ca²⁺-activated K⁺ channels and blockers of Ca²⁺ channels in rat single myelinated nerve fibers, suggesting an increase of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the influences of high glucose with ouabain on [Ca²⁺]_i in sensory neurons remain to be elucidated. The present study was undertaken to examine the modulation of depolarization-induced Ca²⁺ transients by high glucose and ouabain in isolated adult rat dorsal root ganglion (DRG) neurons using the fluorescent Ca²⁺ indicator fura-2. Bath application of KCl (50 mM) evoked a rapid increase in [Ca²⁺]_i through voltage dependent Ca²⁺ channels ([Ca²⁺]_i: 154.2 ± 22.5 nM). This increase was enhanced under high glucose (30 mM D-glucose) in the presence of ouabain (100 M) ([Ca²⁺]_i: 764.8 ± 210.1 nM). We conclude that a combination of high glucose and decreased Na⁺/K⁺ pump activity leads to an increase in [Ca²⁺]_i in rat DRG neurons, thereby resulting in nerve dysfunction.     

Sodium, Calcium and Late Potassium Currents are Reduced in Cerebellar Granule Cells Cultured in the Presence of a Protein Complex Conferring Resistance to Excitatory Amino Acids

Whole-cell, patch-clamp recordings were used to study voltage-gated currents generated by cerebellar granule cells that were cultured in medium containing either 10% fetal calf serum (hereafter termed S + granules) or neurite outgrowth and adhesion complex (NOAC, hereafter called NOAC granules). NOAC is a protein complex found in rabbit serum that renders granules resistant to the excitotoxic action of excitatory amino acids. During depolarizing commands both S+ and NOAC granules generated Na⁺ and Ca²⁺ inward currents and an early and a late K⁺ outward currents. However, Na⁺ and Ca²⁺ Inward currents and late outward K⁺ currents recorded in NOAC granules were smaller than those seen in S+ granules. Furthermore, although of similar amplitude, early K⁺ currents displayed different kinetics in the two types of neurons. Thus, these data demonstrate that the electrophysiological properties of cerebellar granules, and probably of other neuronal populations, depend upon serum components and raise the possibility that an analogous modulation might be operative in vivo, and play a role in development, synaptic plasticity or neuropathological processes.     

HIV-1 Envelope Proteins gp120 and gp160 Potentiate NMDA-induced [Ca2+]i Increase, Alter [Ca2+]i Homeostasis and Induce Neurotoxicity in Human Embryonic Neurons

The envelope glycoprotein gp120 of the human immunodeficiency virus HIV-1 has been proposed to cause neuron death in developing murine hippocampal cultures and rat retinal ganglion cells. In the present study, cultured human embryonic cerebral and spinal neurons from 8- to 10-week-old embryos were used to study the neurotoxic effect of gp120 and gp160. Electrophysiological properties as well as N -methyl- d -aspartate (NMDA)-induced currents were recorded from neurons maintained in culture for 10–30 days. Neither voltage-activated sodium or calcium currents nor NMDA-induced currents were affected by exposure of neurons to 250 pM gp120 or gp160. In contrast, when neurons were subjected to photometric measurements using the calcium dye indo-1 to monitor the intracellular free Ca²⁺ concentration ([Ca²⁺]_i), gp120 and gp160 (20–250 pM) potentiated the large rises in [Ca²⁺]_i induced by 50 μM NMDA. The potentiation of NMDA-induced Ca²⁺ responses required the presence of Ca²⁺ in the medium, and was abolished by the NMDA antagonist d -2-amino-5-phosphonovalerate (AP5) and the voltage-gated Ca²⁺ channel inhibitor nifedipine. Moreover, exposure of a subpopulation of spinal neurons (25% of the cells tested) to 20–250 pM gp120 or gp160 resulted in an increase in [Ca²⁺]_i that followed three patterns: fluctuations not affected by AP5, a single peak, and the progressive and irreversible rise of [Ca²⁺]_i. The neurotoxicity of picomolar doses of gp120 and gp160 cultures was estimated by immuno-fluorescence and colorimetric assay. Treatment of cultures with AP5 or nifedipine reduced gp120-induced toxicity by 70 and 100% respectively.     

Cholinergic Responses in Developing Outer Hair Cells of the Rat Cochlea

Acetylcholine-evoked currents were investigated using the conventional whole-cell patch-clamp recording technique in developing outer hair cells (OHCs). The cells were isolated from the rat cochlea at different stages of postnatal development ranging from day 4 (P4) to P30. Acetylcholine-evoked currents could be recorded at P6 and P8. At this developmental stage, the majority of OHCs displayed inward nicotinic-like currents near the resting membrane potential. These cholinergic currents zeroed near 0 mV, as expected for a non-selective cation current, and could be reversibly blocked by d-tubocurarine. At P12 and adult stage, the cholinergic response of OHCs switched to an outward current reversing near E _K and displaying a bell shape peaking between -40 and -30 mV. This change in polarity of the acetylcholine response during postnatal development might be explained by progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca²⁺ and nearby Ca²⁺-activated K⁺ channels at the synaptic pole of OHCs.     

Effects of Phenytoin, Carbamazepine, and Gabapentin on Calcium Channels in Hippocampal Granule Cells from Patients with Temporal Lobe Epilepsy

Summary: Purpose: The anticonvulsants phenytoin (PHT), carbamazepine (CBZ), and gabapentin (GBP) are commonly used in the treatment of temporal lobe epilepsy. Ca²⁺ current modulation has been proposed to contribute to the antiepileptic activity of these drugs. The purpose of this study was to determine the effects of these anticonvulsants on voltage-dependent calcium channels in pathologically altered neurons from patients with chronic temporal lobe epilepsy.
Methods : Acutely isolated human hippocampal granule cells were examined by using the whole-cell configuration of the patch-clamp technique.
Results : PHT and CBZ produced a reversible, concentrationdependent inhibition of high-voltagectivated (HVA) Ca²⁺ currents without affecting voltage-dependent activation. The concentration-response curves of PHT and CBZ indicated maximal inhibition of 35 and 65%, respectively, with halfmaximal inhibition being obtained at 89 and 244 μ M , respectively. At therapeutic cerebrospinal fluid (CSF) concentrations, HVA currents were not significantly altered by PHT and CBZ. However, PHT but not CBZ showed a reduction of HVA currents of 16% at a therapeutic whole-brain concentration of 80 μ M . In ontrast to CBZ, PHT produced a small hyperpolarizing shift in the voltage dependence of steady-state inactivation. PHT, 80 μ4, shifted the potential of half-maximal inactivation by -3.1 ± 0.5 mV (p < 0.05). GBP, which was recently found to bind to the ಠsubunit of a neuronal Ca²⁺ channel, showed no modulation of Ca²⁺ conductances.
Conclusions : These results suggest that, in contrast to GBP and CBZ, modulation of postsynaptic Ca2+ channels can contribute to the anticonvulsant action of PHT in human hippocampal granule cells.     

Presynaptic Modulation of Glutamate Release Targets Different Calcium Channels in Rat Cerebrocortical Nerve Terminals

We have studied which type/s of Ca²⁺ -channel/s support glutamate exocytosis and its modulation by presynaptic receptors in cerebrocortical nerve terminals. Depolarization of nerve terminals with 30 mM KCI induced a Ca²⁺ -dependent release of 3.64 ± 0.25 nmol/mg of protein. The addition of either 2 μM ω-conotoxin-GVIA or 200 nM ω-agatoxin-IVA reduced the KCI-evoked release by 47.7 ± 3.5% and 70.4 ± 8.9% respectively, and by 85.7 ± 4.1% when both toxins were co-applied. The activation of adenosine A₁ receptors with N ⁶-cyclohexyladenosine or the activation of rnetabotropìc glutamate receptors with L(+)-2-amino-4-phosphonobutyrate inhibited the KCI-evoked release by 41.0 ± 5.9 and 54.3 ± 10% respectively. The extent of these inhibitions was not altered by the prior addition of 2 μM ω-conotoxin-GVIA but they were significantly enhanced when ω-agatoxin-IVA was added together with the adenosine A₁ receptor agonist or the metabotropic glutamate receptor agonist, suggesting that ω-conotoxin-GVIA-sensitive and not ω-agatoxin-IVA-sensitive Ca²⁺-channels are ínvolved in the action of these inhibitory receptors. By contrast, the facilitation of glutamate release that follows the activation of the protein kinase C, either with phorbol esters or with the stimulation of phospholipase C-linked metabotropic receptors, was expressed by both ω-conotoxin-GVIA-sensitive and ω-agatoxin-sensitive Ca²⁺-channels. It is concluded that different Ca²⁺-channels support the modulation of glutamate release by presynaptic receptors.     

Mechanisms for Synchronous Calcium Oscillations in Cultured Rat Cerebellar Neurons

Removal of Mg²⁺ caused oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca²⁺]_i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N -methyl-D-aspartate (NMDA). Oscillations were blocked by Ca²⁺ removal, nickel, NMDA receptor antagonists, ω-agatoxin IVA, tetrodotoxin, sodium removal and γ-aminobutyric acid, but not by dihydropyridines, ω-conotoxin M VIIA or by emptying the intracellular Ca²⁺ stores with thapsigargin or ionomycin. The upstroke of the [Ca²⁺]_i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca²⁺]_i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of ˜20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca²⁺]_i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of [Ca²⁺]_i are due to cyclic Ca²⁺ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca²⁺ entry through P-type Ca²⁺ channels activated by action potentials at the presynaptic terminal.     

Exocytosis and Selective Neurite Calcium Responses in Rat Cerebellar Granule Cells During Field Stimulation

The free calcium concentration, [Ca²⁺]_c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin-sensitive increase in Na⁺ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K⁺ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca²⁺]_c, suggesting that prolongation of the duration of depolarization is required for somatic Ca²⁺ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω-agatoxin-IVA (30 nM) but was uniformly inhibited by ω-conotoxin-GVIA (30% inhibition at 1 μM) and ω-conotoxin-MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca²⁺]_c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCl depolarization, suggesting that presynaptic Ca²⁺ channels contribute to the field-evoked neurite response.     

Modulation of Neuropeptide-lnduced Membrane Currents by Protein Kinase C in Xenopus Oocytes Injected with GH3 Pituitary Cell Poly(A)+ RNA

Protein kinase C was activated in Xenopus laevis oocytes by phorbol ester treatment and its effects on the inositol trisphosphate/Ca²⁺ transmembrane signalling pathway analysed. Induction of the pathway was achieved by ligand stimulation of TRH receptors translated from GH₃ pituitary cell mRNA. In voltage-clamped oocytes bath application of peptide, injection of guanosine 5'-(3-O-thio) triphosphate (GTPγS), inositol trisphosphate or Ca²⁺ all elicited inward membrane currents. Treatment of oocytes with tumour-promoting phorbol esters for 35 min almost completely abolished the ligand and GTPγS-induced responses. In contrast, phorbol ester treatment enhanced inositol trisphosphate-generated membrane currents. Ca²⁺-mediated responses remained unaffected by tumour promoters. The data indicate a dual role for protein kinase C in the modulation of transmembrane signalling: a feedback mechanism prevents phosphoinositide turnover whereas a feedforward reaction triggers the effect of intracellular inositol trisphosphate on the Ca²⁺ release.     

Voltage-dependent Currents in Microvillar Receptor Cells of the Frog Vomeronasal Organ

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 μm) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was −61 ± 13 mV (mean ± SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of –60 to –86 mV, with almost no spontaneous action potential. The input resistance was in the GΩ range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2 – 10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near –45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near –75 mV. An outward K⁺ current was blocked by internal Cs⁺ ions or by external tetraethylammonium or Ba²⁺ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd²⁺ ions. A voltage-dependent Ca²⁺ current was observed in an iso-osmotic BaCl₂ solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.     

Methoxyverapamil Reduction of Nicotine-induced Catecholamine Release Involves Inhibition of Nicotinic Acetylcholine Receptor Currents

The mechanism by which the putative Ca²⁺ channel blocker methoxyverapamil (D600) inhibits nicotine-induced catecholamine release was investigated in bovine adrenal chromaffin cells and in neurons from paravertebral sympathetic ganglia of chick embryos. We found D600 to prevent catecholamine release evoked by 30 s applications of nicotine with a significantly higher potency than the release induced either by 30 s K⁺ depolarizations or by electrical field stimulation of sympathetic neurons. Like the use-dependent action of D600 upon Ca²⁺ channels, the magnitude of inhibition of the K⁺-evoked secretion depended on the duration of stimulation (10 s to 5 min). Data on catecholamine release were supplemented by patch-clamp recordings. We found whole-cell currents in chromaffin cells evoked by (extrapolated) 0.5 s applications of nicotine to be significantly more sensitive to D600 than Ca²⁺ currents induced by a 0.5 s depolarization from -80 to 0 mV. In both instances, the potency of D600 depended on the duration of the (nicotinic and depolarizing) stimuli. Our data suggest that D600 inhibits nicotine-induced catecholamine release by reducing nicotinic acetylcholine receptor currents rather than voltage-gated Ca²⁺ currents. Hence, in chromaffin cells as well as in sympathetic neuronal preparations, D600 does not appear to be a suitable tool to investigate the part voltage-activated Ca²⁺ currents play in cellular events induced by nicotine.     

Switching Between Two Types of Bursting Activity of Single Ca+2-Activated K+ Channels in Dissociated Neurons

Single channel recordings of Ca²⁺-activated K⁺ currents were made from dissociated cockroach neurons by means of the gigaohm-seal patch-clamp technique. Bursts of single channel openings were composed of two distinct classes: the 'long-open burst' contained groups of long, rectangular, pulse-like openings with durations of 3.5 to 1.2 ms (depending on membrane potential), whereas the 'flickering burst' consisted of clusters of brief openings with an average duration of 0.4 ms (voltage-independent) separated by short closings with a duration of about 1.0 ms. The long-open burst and the flickering burst appeared to reflect distinct states of a single Ca²⁺-activated K⁺ channel because direct transitions between these two types of burst were often detected. We present a kinetic scheme for the gating activation pathway of a neuronal Ca²⁺-activated K⁺ channel, based on these findings.     

Analysis of the Ion Channel Complement of the Rat Oligodendrocyte Progenitor in a Commonly Studied In vitro Preparation

We have analysed the ion channel complement of the oligodendrocyte-type 2 astrocyte (O-2A) glial cell progenitor obtained from the commonly studied neonatal rat mixed brain preparation. Ionic currents, in O-2A progenitors identified on both morphological and immunological grounds, were recorded using the whole-cell variant of the patch-clamp technique. The cells had an average resting membrane potential close to -50 mV and fired single action potentials in response to suprathreshold current injections. Using voltage-clamp methods we were able to identify and characterize a voltage-activated TTX-sensitive Na⁺ current, two classes of voltage-activated outward K⁺ currents, an inactivating inwardly rectifying K⁺ current, a voltage-activated Cl^- current and at least three classes of Ca²⁺ current.     

Ca2+ Channel Expression in the Oligodendrocyte Lineage

The development of oligodendrocytes from their precursor cells through different developmental stages can be studied in vitro. These stages can be distinguished by specific monoclonal antibodies and by a characteristic K⁺ channel profile. In this study we demonstrate that the occurrence of Ca²⁺ currents also undergoes marked changes during the development of mouse oligodendrocytes. Immature precursor cells which can develop into astrocytes or oligodendrocytes expressed two different types of voltage-activated Ca²⁺ channels. The expression of Ca²⁺ channels in precursor cells was strongly correlated with the expression of Na⁺ channels. When cells started to express the O1 antigen and were committed to the oligodendrocyte lineage, Ca²⁺ and Na⁺ currents could no longer be detected. Large Ca²⁺ currents were, however, recorded later in the development of the oligodendrocytes, correlated with the expression of the O10 antigen. The Ca²⁺ channels were classified as high and low voltage-activated Ca²⁺ channels according to their range of activation, and are further described by their kinetic and pharmacological properties.     

Calcium-Dependent Regulation of Genetically Determined Spike and Waves by the Reticular Thalamic Nucleus of Rats

Summary: The pathophysiologic role of the reticular thalamic nucleus (RT) in rat generalized nonconvulsive epilepsy was investigated in the selected strain GAERS (genetic absence epilepsy rats from Strasbourg). After the RT was lesioned by the excitotoxic agent ibotenic acid stereotaxically injected in previously callosotomized rats, a disruption of ipsilateral spike and wave discharges (SWD) was observed in freely moving animals. In a second group of animals Cd²⁺ (0.5–1.5 μl, 1 m M ), which is known to block Ca²⁺ and Ca²⁺-dependent K⁺ conductances (gK⁺ (Ca²⁺), was injected into the thalamus. Cd²⁺reversibly suppressed ipsilateral SWD when injected in RT, whereas it slightly reduced SWD expression when injected in the ventrobasal (VB) complex. The difference was highly significant. We conclude that Ca²⁺-dependent oscillatory properties of the RT are critical for expression of genetically determined SWD in GAERS.     

Protein Kinase C Facilitation of Acetylcholine Release at the Rat Neuromuscular Junction

Protein kinase C (PKC) is a Ca²⁺-dependent enzyme involved in synaptic transmission, which can be experimentally activated by the phorbol ester, phorbol 12-myristate-13-acetate (TPA). We studied the effects of TPA application on acetylcholine (ACh) release at the rat neuromuscular junction by means of the focal recording technique; possible effects of TPA at the postsynaptic site had been ruled out in preliminary studies. In extracellular solutions containing 2 mM Ca²⁺ and at the stimulation frequency of 0.1 Hz, TPA increased endplate current (EPC) amplitude. In non-stimulated preparations spontaneous current frequency was increased at a similar rate. The similar time course of TPA action on evoked and spontaneous currents suggests that an increased presynaptic Ca²⁺ efficacy can be considered to be the probable mechanism of action. The interactions of PKC with ACh release were further investigated. In 0.1 mM Ca²⁺ extracellular solutions, TPA enhanced evoked currents only at stimulation frequencies (e.g. 40 Hz) that were themselves capable of inducing facilitation. This facilitation is classically associated with presynaptic Ca²⁺ accumulation, indicating that PKC interacts synergistically with Ca²⁺ to facilitate ACh release. In particular, since mean quantum size and release probability remained almost unchanged during TPA facilitation, it was concluded that PKC acted by enlarging the immediately available store. Interestingly, TPA also increased the presynaptic currents that were observed to be largely brought about by Ca²⁺-dependent K⁺ currents: evidence was obtained to suggest that increases in these currents provide negative feedback against excess release activation rather than being an expression of enhanced Ca²⁺ influx.