The contribution of increases in extracellular potassium to primary afferent depolarization in the bullfrog spinal cord

To assess the extent to which depolarization by accumulated K+ contributes to the generation of primary afferent depolarization (PAD), the isolated bullfrog spinal cord was superfused with K+-rich Ringer solutions and the resultant dorsal root depolarizations were recorded extracellularly. Action potential blockade (with tetrodotoxin) did not reduce the K+-induced depolarization of primary afferents, indicating that the depolarization was generated locally in the region around the afferents. In this respect superfusion with K+-rich solutions adequately models the localized K+ accumulation which occurs physiologically during afferent activity. K+-induced depolarizations were decreased in the presence of 20 mM Mg2+; this effect was due to a direct decrease in the membrane response to K+ and not to blockade of K+-induced transmitter release onto primary afferents. The depolarization caused by a K+ concentration comparable to a maximum estimate of the K+ accumulating around afferent terminals following a single afferent volley was found to account for no more than about one-third of the DRP height. However, higher K+ levels, comparable to those resulting from high frequency afferent stimulation, caused large depolarizations of primary afferents, sometimes greater than the DRP amplitude. Therefore, K+-induced depolarization may contribute more significantly to PAD evoked by high frequency afferent activity.     

Pathophysiology of the spinal cord studied in vitro

We describe the use of isolated hemisected mouse spinal cords for pathophysiological investigations and analyze the responses evoked and recorded with suction electrodes in spinal roots. Dorsal root (DR) recordings from preparations in control solution show a directly evoked fiber volley (FV); an early postsynaptic spike generated by neurons in spinal gray matter and picked up by volume conduction (DRR1); and a 'slow' dorsal root potential (DRP). The 'conventional' dorsal root reflex (here termed DRR2) was absent or very small in control medium but became very prominent in elevated bath [Ca2+]. DRP and DRR2 but not DRR1 are depressed by GABAA antagonists. Recordings from VR contain the electrotonically conducted VRepsp and superimposed monosynaptic reflex discharge (VRR1). Rarely in control medium but regularly in elevated bath [Ca2+] a GABA-dependent late reflex (VRR2) appears (see also Duchen, 1986). The effects of varying bath concentrations of K+, Ca2+ and Mg2+ on evoked responses are briefly summarized. Irregularly timed spontaneous discharges appear in DR and VR recordings when [Ca2+] is elevated above 1.8 or 2.4 mM, and when [Mg2+] is lowered to 0.4 mM. In hypoxic solution synaptically transmitted responses fail in 10 to 20 min, but persist longer when [Ca2+] is elevated. Unexpectedly, spreading depression (SD)-like responses were recorded in some preparations during hypoxia. Following hypoxia, after synaptically transmitted responses recovered, spontaneous activity developed in DR and VR recordings.     

Characteristics of dorsal root potentials recorded from the isolated spinal cord of the neonate rat

Summary Dorsal root potentials (DRP) and dorsal root reflexes (DRR) have been recorded from the isolated cord of the neonate rat. A single stimulus to the adjacent rostral or adjacent caudal dorsal root or dorsal columns evoked a DRP, the peak amplitude of which was reached in 110–115 msec and which decayed exponentially over most of its time course (time constant 800–850 msec). The same stimuli evoked field potentials in the dorsal horn comprising fast negative, slow negative and slow positive potentials. DRP had a lower threshold than DRR and reached a maximal amplitude at stimulus voltages sub-maximal for DRR. Increasing the intensity of stimulation shortened the latency of DRP and prolonged its time course. DRR and DRP were depressed by a prior conditioning stimulus (CS) and by the addition of Mg⁺⁺ ions to the bathing solution. A CS was more effective in producing depression of responses evoked more rostrally than more caudally.     

Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients

Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.     

The effect of peripheral nerve injury on dorsal root potentials and on transmission of afferent signals into the spinal cord

The sciatic nerve adults rats was either cut and ligated or was crushed on one side. The response of the spinal cord to stimulation of the proximal part of the injured nerve was examined at various times after the lesion and compared to the effects of stimulating the intact nerve on the other side. During the first 10 days after nerve section the following measures were not affected: (i) the size of the input volley (compound action potential, CAP, measured on a dorsal root that carried sciatic nerve afferents (L5); (ii) the volley running in the dorsal columns; (iii) the dorsal root potential (DRP) evoked on neighbouring dorsal roots which do not contain sciatic afferents (L2 and L3); (iv) the post-synaptic volleys ascending in the spinal cord. However, by the fourth day after nerve section, there was a decrease of the DRP evoked on the ipsilateral L5 dorsal root by stimulation of the cut nerve. By 10 days this DRP had decreased by 50%. There was also a decrease in the DRP on the L5 root evoked by stimulation of the contralateral intact nerve. Crush lesions of the sciatic nerve did not produce DRP charge. Beginning 10–20 days after nerve cut, there was a decrease in the amplitude of the afferent CAP and of all the measures of central response to the afferent volley. We discuss the possibility that the loss of the DRP may be associated with a disinhibition which results in novel receptive fields which we observe in cord cells deafferented by the peripheral nerve section. The decrease of DRP and the appearance of novel receptive fields do not occur if the peripheral nerve is crushed rather than cut.     

Effect of omega-conotoxin GVIA on neurotransmitter release at the mouse neuromuscular junction.

The effect of omega-conotoxin GVIA (omega-CgTx) was studied on spontaneous, K(+)-induced and electrically evoked neurotransmitter release at the neuromuscular junction of mouse diaphragm. omega-CgTx decreased the frequency and amplitude of basal and K(+)-induced miniature end plate potentials. This effect was abolished by raising the extracellular Ca2+ concentration. omega-CgTx had no effect on the quantal content of the electrically evoked release in this preparation.     

Cytosolic Ca2+ under high glucose with suppressed Na+/K+ pump activity in rat sensory neurons

Cytosolic Ca2+ concentration ([Ca2+]i) was measured in isolated rat dorsal root ganglion (DRG) neurons using the fluorescent Ca2+ indicator fura-2. Exposure to high (50 mM) extracellular K+ evoked a robust increase in [Ca2+]i, which was almost totally abolished by concomitant presence of nisoldipine (10 microM) and omega-conotoxin GVIA (10 microM). Whereas either high (30 mM) D-glucose alone or ouabain (100 microM) alone did not appreciably affect the high K+-induced [Ca2+]i elevation, neurons pretreated with high D-glucose together with ouabain exhibited a significantly larger [Ca2+]i response to high K+ stimulation, which was almost completely inhibited by nisoldipine and omega-conotoxin GVIA. These results suggest that a combination of high glucose and suppressed Na+/K+ pump activity potentiates the [Ca2+]i elevation stimulated by activation of the voltage-gated Ca2+ channels in rat DRG neurons.     

Presynaptic mechanisms of the change in segmental responses accompanying the formation of the spinal locomotor generator in the cat

It is shown on unanesthetized immobilized decorticated cats that spinalization of the animal leads to depolarization of the central afferent terminals, decrease of early polysynaptic responses in motoneurons and dorsal root potentials (DRP) evoked by stimulation of the low-threshold cutaneous and muscle afferents, increase of early polysynaptic responses and DRP evoked by stimulation of high-threshold muscle afferents, reduction in the activity of intermediate nucleus interneurons mono- and polysynaptically connected with primary afferents, rise of the activity of interneurons di- and oligosynaptically connected with primary afferents. Injection of DOPA into spinal animals leads to opposite changes. Dependence between changes in the state of segmental neuronal apparatus and the level of spinal locomotor generator activity are discussed on the basis of the data obtained.     

Time-course of ultrastructural changes in regenerated optic fiber terminals of goldfish

Extracellular potassium concentration ([K+]o) was measured, and intra- and extracellular recordings made, in the dorsal horn of rat spinal cord slices maintained in vitro during repetitive dorsal root stimulation. In about half of the dorsal horn neurons, the stimulation evoked a possibly substance P-mediated slow depolarization. [K+]o increased during stimulation, reaching its highest values approximately 150 micron from the dorsal surface. The time course of delta [K+]o was different from that of the slow depolarization. Substance P itself evoked a much smaller delta [K+]o (0.4 mM) in the dorsal horn. It is concluded that the slow depolarization is not mediated by elevated [K+]o.     

Depolarization of primary afferents evoked by cyclically modulated natural activity of proprioceptors of the hindlimb of the cat

The primary afferent depolarization (PAD) evoked during passive sinusoidal movements of a hindlimb in the ankle joint was investigated in decerebrated cats. The frequency of movements varied within 0.14-5.0 Hz, the amplitude of the joint angle with respect to the axis of the tibia changed from 90 degrees to 130 degrees. The dorsal root potential (DRP) negativity increased both during flexion and during extension of the joint. The amplitude of the evoked DRPs was about 50-100 mV. A strong negative correlation was observed between the latency and rise time of the DRP and the frequency of the joint angle changes. During flexion the latency changed from 650 ms at 0.14-0.16 Hz frequency to 100-110 ms at 2.0 Hz and higher frequencies; during extension at the same frequencies the latency changed from 300 ms to 80-85 ms. The latency and rise time became minimal at 2.0 Hz frequency and practically did not change during the further increase of the oscillation frequency. The cord dorsum potential (CDP) evoked by the cutaneous nerve stimulation was recorded in parallel with the DRP. Periodical changes of the N-component of the CDP were in the opposite phase to changes of the DRP. Mechanisms of the observed changes of the PAD and functional significance of these changes during rhythmical motor acts are discussed.     

The influence of bemegride on frog spinal cord root potentials]

The effect of the convulsant bemegride (beta-ethyl-beta-methyl-glutharimide) on the electronic potentials in the frog spinal roots was investigated in situ. Intravenous injection of the bemegride subconvulsant doses (6.8 +/- 2.7 mg/kg) depressed rapidly the electrotonic dorsal root potentials (DRP) evoked by a stimulation of the adjacent dorsal root (DR) or the ventral root (VR). They were reduced by 55--67% 3-6 min after the bemegride injection. The effect of bemegride was reversible and the DRP amplitude restored its initial value within an hour. Ventral root potentials after bemegride injection revealed only greater amplitude fluctuations. A conclusion is made that bemegride is a selective and potent depressor of the primary afferent depolarization in the frog spinal cord.     

Phase-dependent changes in the posterior root potentials during real locomotion in rats

Dorsal root potentials (DRP) were studied during two kinds of the real locomotion (swimming and stepping) of rats. Two negative DRP waves were observed in one locomotor cycle. One wave coincided with stance on extension, while the other one--with swing phase. Dependence between the DRP amplitude and intensity of afferent input during the real locomotion and passive movements of hind limbs was established. It is concluded that wave-like changes of DRP in the real locomotion are mainly due to the influences from peripheral afferents.     

Effect of chronic undernourishment on the cord dorsum potentials and the primary afferent depolarization evoked by cutaneous nerves in the rat spinal cord

The effect of chronic undernourishment on the cord dorsum potentials (CDPs) and the dorsal root potential (DRP), closely related to primary afferent depolarization (PAD) and presynaptic inhibition in the spinal cord of the rat, was analyzed in this study. Single electrical pulses applied to the sural nerve (SU) of control (n=14) and chronically undernourished (n=16) Wistar rats produced CDPs, which are composed of four components: afferent volley (AV), two negative components (N(1) and N(2)), and one positive component (P wave) and negative DRPs recorded in a small rootlet of the L6 segment of the rat. The CDPs of the control and undernourished rats with AV components of comparable amplitude (U(AV)/C(AV)=0.96), showed N(1) components of similar amplitude (U(N1)/C(N1)=0.94), but smaller P wave (U(PW)/C(PW)=0.23). A comparable reduction in the amplitude of the DRPs was obtained in the undernourished rats (U(DRP)/C(DRP)=0.36). When normalized as a function of the body mass of the animals, the CDPs and DRPs produced in undernourished rats were of significantly smaller normalized amplitude than those evoked in the control. According to these results, it is suggested that chronic undernourishment induce a depressive effect on the mechanisms generating the P wave component in the CDP and the DRPs either by decreasing the sensory input and/or the excitability of the dorsal horn neurones involved in the generation of PAD and presynaptic inhibition in the spinal cord of the rat.     

A comparison of extracellular and intracellular recording during extracellular microiontophoresis

A technique is described in which a central recording microelectrode can be moved independently of a concentrically arranged multibarrelled electrode prepared for microiontophoresis. Recordings were made from cat spinal motoneurones during microiontophoretic applications of excitatory amino acids and biogenic amines with the central electrode placed first extracellularly and then intracellularly. Recording were also made from one of the iontophoretic barrels. Both intra- and extracellular electrodes were used to record action potential firing, the ventral root field (VRF) evoked by antidromic ventral root stimulation and the membrane potential (EM). They were also used to record 'focal potentials' evoked by the extracellular application of drugs to nearby neurones. The firing pattern evoked by extracellular iontophoretic applications of DL-homocysteate and glutamate was not altered significantly following impalement of the cell by the recording microelectrode. Excitatory amino acids usually caused a reduction of the VRF negative wave and evoked an additional late positive wave. These VRF changes recovered at the same rate as the extracellularly recorded, negative 'focal potentials' (Flatman and Lambert, 1979). Iontophoretic applications of biogenic amines caused small increases, small decreases, or no change of the VRF negative wave. Variable responses were also seen during intracellular recording: hyperpolarization, no response and, occasionally, depolarizations were recorded. It is concluded that, during the drug action, VRF changes are difficult to interpret and are a poor index of drug-evoked changes in neuronal excitability or EM.     

Modulation of Gonadotropin II Release by K+ Channel Blockers in Goldfish Gonadotropes: A Novel Stimulatory Action of 4-Aminopyridine

The effects of K+ channel blockers on basal gonadotropin II (GTH-II) release were examined in cultured goldfish gonadotropes. Tetraethylammonium (TEA) inhibited basal GTH-II release, whereas 4-aminopyridine (4-AP) increased basal release, although both K+ channel blockers generated increases in [Ca2+]i. Other K+ channel blockers had no significant effect on GTH-II release. We examined whether Ca2+ entry that arises from blockade of K+ channels by 4-AP mediates the secretory response. Secretion evoked by 4-AP was slightly reduced by TEA but was unaffected by reducing Ca2+ entry using either an inhibitor of Ca2+ channels, verapamil, or nominally Ca2+-free medium. In contrast, the Ca2+ signal evoked by 4-AP was largely blocked by Ca2+-free medium, as predicted by its inhibitory action on K+ channels. Together, these data suggest that the hormone release response to 4-AP is independent of entry of extracellular Ca2+. Finally, the mechanism of hormone release evoked by 4-AP appeared to be independent of mechanism(s) evoked by caffeine since 4-AP did not affect caffeine-evoked release and caffeine did not affect 4-AP evoked release. That both 4-AP and TEA generated Ca2+ signals but affected hormone release in either an extracellular Ca2+ independent (4-AP) or inhibitory (TEA) manner suggests that Ca2+ entry is linked to GTH-II secretion in a highly nonlinear fashion.     

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.     

Evaluation of degree of nerve root injury by dermatomal somatosensory evoked potential following lumbar spinal stenosis*☆

BACKGROUND： Computed Tomography （CT） and Magnetic Resonance Imaging （MRI） can display the site of lumbar spinal stenosis and predict nervous compression at the morphological level; however, pure morphological changes cannot reflect functional alterations in a compressed nerve root. Dermatomal somatosensory evoked potential （DSEP） provides a means to assess the functional state of a nerve root. OBJECTIVE： To evaluate the clinical significance of DSEP, assessing the degree of nerve root injury following lumbar spinal stenosis. DESIGN, TIME AND SETTING： A case-control study was performed in the Department of Orthopaedic Surgery, Hainan People＇s Hospital, China, between September 2004 and December 2007. PARTICIPANTS： Forty-seven patients diagnosed with lumbar spinal stenosis by CT or MRI were selected as the case group; fifty healthy subjects were collected as the control group. METHODS： A KEYPOINT myoelectric evoked potential apparatus （DANTEC Company, Denmark） was used to measure DSEP, and stimulative spots were determined in accordance with the skin key sensory spot standards established by The American Spinal Injury Association： L4 in the medial malleolus, L5 in the third metatarsophalangeal joint of the dorsum of foot and S1 in the lateral heel. The needle electrode used as the recording electrode was located at the Cz point of the cranium, and the reference electrode at the Fz point. MAIN OUTCOME MEASURES： Latency of the P40 peak of DSEP, P1-N1 amplitude, P40 waveform and differentiation and disappearance of various waves. RESULTS： The sensitivity and diagnostic concurrence with surgery of nerve root injury following lumbar spinal stenosis evaluated by DSEP was 95.7 %. P40 latencies at L4, L5 and S1 in the case group were significantly longer than in the control group （P 〈 0.05）, and the P1-N1 amplitude in the case group was significantly lower than the control group （P 〈 0.05-0.01）. Nerve root injury was categorized according to DSEP latency as follows： severe da     

Epileptiform activity induced by lowering extracellular [Mg2+] in combined hippocampal-entorhinal cortex slices: modulation by receptors for norepinephrine and N-methyl-D-aspartate

Reduction of extracellular Mg2+ concentration induced spontaneous and evoked epileptiform activity in the entorhinal cortex (EC) and dentate gyrus (DG) of combined hippocampus (HC)-EC slices. Extracellular field potentials, as well as changes in extracellular Ca2+ and K+ concentrations, were measured in EC and DG with ion-selective/reference electrodes during both repetitive and single stimuli. In the EC, lowering extracellular [Mg2+] induces both spontaneous and single stimulus evoked ictal events consisting of extracellular negative potential shifts (up to 5 mV, 30 sec), decreases in [Ca2+]0 and increases in [K+]0. In the DG, spontaneous events were much shorter, but similar changes in [Ca2+]0, [K+]0 and field potentials (FPs) could be evoked by brief high-frequency stimulation. In both areas, the N-methyl-D-aspartate (NMDA) receptor antagonist 2-aminophosphonovalerate (2-APV) completely blocked spontaneous as well as stimulus evoked epileptiform events. The neurotransmitter norepinephrine (NE), which has previously been shown to modulate long-term potentiation in the DG, was found to exhibit differential modulation of epileptiform activity in the EC and DG. In the EC, NE, acting via alpha 1-receptors, completely blocked low Mg2+-induced epileptiform activity. In contrast, in the DG, NE exhibited a beta-receptor mediated prolongation of the low Mg2+-induced ictal events, and enhanced the stimulus-induced ionic and field potential changes. From these results, we conclude that lowering extracellular [Mg2+], acting in large part through the removal of the Mg2+ voltage-dependent blockade of NMDA receptors, leads to induction of epileptiform activity in both the EC and DG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial buffering of K+ by the retinal pigment epithelium in frog

Using K+-specific microelectrodes in the isolated retinal pigment epithelium preparation from frog eye, we have examined changes in extracellular K+ in the unstirred layer between the choroid and basal membrane. We found, using agents to depolarize apical and basal membranes, that membrane potential modulates K+ efflux from the basal membrane into the choroid. [K+] in the choroid was found to be higher than in the bathing medium. From the basal (scleral) surface of the choroid to the basal membrane of the retinal pigment epithelium, [K+] increased from 2 mM (bath concentration) to an average of 2.84 mM near the basal membrane. When [K+] was increased on the apical side, epithelial membranes depolarized and produced [K+] efflux from the basal membrane that led to an increase in choroidal [K+]. Ba2+, 2 mM, ouabain, 0.1 mM, also evoked increases in choroidal [K+] concomitant with membrane depolarization. Thus, apical Na+/K+ pump transport and K+ leakage due to tissue damage were eliminated as possible sources of observed increases in choroidal [K+] since apical Ba2+ and ouabain depolarize epithelial membranes without increasing apical [K+]. The effects of depolarizing agents were blocked by basal Ba2+, demonstrating the requirement for a large K+ conductance at the basal membrane. Theoretical analysis shows that shunt resistance and isolation of apical and basal compartments are limiting factors in the transfer of K+. In the retinal pigment epithelium, shunt resistance favors the transfer of electrical potentials between membranes while the paracellular pathway selectively impedes movement of K+ between compartments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ role on the effect of phorbol esters on the spontaneous quantal release of neurotransmitter at the mouse neuromuscular junction

The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the release process was studied in presence of different extracellular Ca2+ concentrations, in the mouse phrenic-diaphragm preparation. Hemidiaphragms were incubated for 2 h at room temperature in the presence or absence of TPA. TPA increased the basal frequency of miniature end plate potentials (mepp's) in a dose-dependent manner, resulting in a maximal increase of 280% at a concentration of 0.5 microM. An inverse relationship between extracellular Ca2+ concentration and TPA effect was observed: at high extracellular concentrations of Ca2+ the action of TPA decreased significatively, while at low Ca2+ concentrations the effect of TPA was remarkably augmented. The highest effect of TPA was obtained when tested in a calcium-free medium. TPA also increased mepp frequency stimulated by 10 mM K+. As at basal conditions, the effect of TPA was higher at lower concentrations of extracellular calcium. The results suggest that the effect of stimulation of PKC on neurotransmitter release at the mice neuromuscular junction is not exerted at the level of calcium influx to the nerve terminal. Moreover the action of calcium and TPA seems to be superimposed. The effect of K+ on neurotransmitter release could be explained not only by depolarization of the nerve terminal but by increasing the pool of activable PKC.