A glia-derived acetylcholine-binding protein that modulates synaptic transmission.

There is accumulating evidence that glial cells actively modulate neuronal synaptic transmission. We identified a glia-derived soluble acetylcholine-binding protein (AChBP), which is a naturally occurring analogue of the ligand-binding domains of the nicotinic acetylcholine receptors (nAChRs). Like the nAChRs, it assembles into a homopentamer with ligand-binding characteristics that are typical for a nicotinic receptor; unlike the nAChRs, however, it lacks the domains to form a transmembrane ion channel. Presynaptic release of acetylcholine induces the secretion of AChBP through the glial secretory pathway. We describe a molecular and cellular mechanism by which glial cells release AChBP in the synaptic cleft, and propose a model for how they actively regulate cholinergic transmission between neurons in the central nervous system.

Activation of protein kinase C by oxytocin-related conopressin underlies pacemaker current in Lymnaea central neurons.

The vasopressin/oxytocin-related neuropeptide Lys-conopressin activates two pacemaker currents in central neurons of the mollusk Lymnaea stagnalis. A high-voltage-activated current (I-HVA) is activated at potentials greater than -40 mV and resembles pacemaker currents found in many molluscan neurons. A low-voltage-activated current (I-LVA) activates throughout the range of -90 to 0 mV. Based on sequence homologies, Lymnaea conopressin receptors are thought to couple to Q-type G proteins and protein kinase C (PKC). Alternatively, agonist-induced pacemaker currents in molluscan neurons have traditionally been attributed to cAMP-dependent protein kinase (PKA) activation. Accordingly, this study aimed at resolving possible involvement of cAMP/PKA and diacylglycerol/PKC in the conopressin response. Injection of cAMP into anterior lobe neurons induced a slow inward current with a voltage dependence resembling that of I(LVA) (and not I(HVA)). However, lack of effect of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the absence of cross-desensitization between cAMP and conopressin suggest that neither current is dependent on intracellular cAMP. The PKC-activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate (but not inactive phorbol 12-myristate 13-acetate) mimicked activation of I(HVA), but not I(LVA), and occluded subsequent responses to conopressin. Activation of I(HVA) was blocked by general protein kinase inhibitors and the PKC-inhibitor GF-109203X. Modulation of the calcium buffering capacity of the pipette medium did not affect the conopressin response, suggesting that calcium dynamics are not of major importance. We conclude that conopressin activates the ion channels carrying I(LVA) and I(HVA) through different second-messenger cascades and that PKC-dependent phosphorylation underlies activation of I(HVA).

Regulation of exocytosis in neuroendocrine cells: spatial organization of channels and vesicles, stimulus-secretion coupling, calcium buffers and modulation.

Neuroendocrine cells display a similar calcium dependence of release as synapses but a strongly different organization of channels and vesicles. Biophysical and biochemical properties of large dense core vesicle release in neuroendocrine cells suggest that vesicles and channels are dissociated by a distance of 100-300 nm. This distinctive organization relates to the sensitivity of the release process to mobile calcium buffers, the resulting relationship between calcium influx and release and the modulatory mechanisms regulating the efficiency of excitation-release coupling. At distances of 100-300 nm, calcium buffers determine the calcium concentration close to the vesicle. Notably, the concentration and diffusion rate of mobile buffers affect the efficacy of release, but local saturation of buffers, possibly enhanced by diffusion barriers, may limit their effects. Buffer conditions may result in a linear relationship between calcium influx and exocytosis, in spite of the third or fourth power relation between intracellular calcium concentration and release. Modulation of excitation-secretion coupling not only concerns the calcium channels, but also the secretory process. Transmitter regulation mediated by cAMP and PKA, as well as use-dependent regulation involving calcium, primarily stimulates filling of the releasable pool. In addition, direct effects of cAMP on the probability of release have been reported. One mechanism to achieve increased release probability is to decrease the distance between channels and vesicles. GTP may stimulate release independently from calcium. Thus, while in most cases primary inputs triggering these pathways await identification, it is evident that large dense core vesicle release is a highly controlled and flexible process.

All classes of calcium channel couple with equal efficiency to exocytosis in rat melanotropes, inducing linear stimulus-secretion coupling.

1. The contribution of low voltage-activated (LVA) T-type Ca2+ channels and four different types of high voltage-activated (HVA) Ca2+ channel to exocytosis, and the relationship between calcium influx and exocytosis during action potentials (APs) were studied in pituitary melanotropes. 2. Selective HVA Ca2+ channel blockers reduced exocytosis, monitored by membrane capacitance measurements, proportional to the reduction in Ca2+ influx. The efficacy of Ca2+ in stimulating exocytosis did not change in the presence of the Ca2+ channel blockers, indicating that all HVA Ca2+ channels act together in stimulating exocytosis. 3. The relationship between Ca2+ influx and exocytosis during the AP was examined using APs recorded from spontaneously active melanotropes as command templates under voltage clamp. Under voltage clamp, multiphasic Ca2+ currents were activated over the entire duration of the APs, i.e. during the rising phase as well as the plateau phase. The maximum amplitude of the Ca2+ current coincided with the peak of the AP. 4. The relationship between Ca2+ entry and exocytosis was linear for the different phases of the AP. Also, the influx of Ca2+ through LVA T-type channels stimulated exocytosis with the same efficacy as through the HVA channels. 5. APs of increasing duration ( approximately 50 to approximately 300 ms) evoked increasing amounts of exocytosis. The number of entering Ca2+ ions and the capacitance change were linearly related to AP duration, resulting in a fixed relationship between Ca2+ entry and exocytosis. 6. The results show that Ca2+ ions, entering a melanotrope, couple with equal strength to exocytosis regardless of the channel type involved. We suggest that the linear relationship between Ca2+ entry and secretion observed under physiological conditions (during APs), results from the equal strength with which LVA and HVA channels in melanotropes couple to exocytosis. This guarantees that secretion takes place over the entire duration of the AP.

Calcium channels and the release of large dense core vesicles from neuroendocrine cells: spatial organization and functional coupling.

The release of large dense core vesicles (LDCV) by neuroendocrine cells displays a very similar calcium dependence as found in synapses, yet, the organization of channels and vesicles is quite different. Various biophysical properties of the release process, notably a large delay (>10 ms) between excitation and release and a high impact of mobile calcium buffers, suggest that, generally, vesicles and channels do not co-localize as in synapses, but are separated by a distance of 100-300 nm. This review focuses on the consequences of this organization for the functional coupling of calcium channels to LDCV-release in neuroendocrine cells. The large distance between LDCV and calcium channels in neuroendocrine cells obviates molecular interactions between channels and fusion peptides and implies that each type of calcium channel may be involved in release. Thus, preferential functional coupling of specific calcium channel types to the exocytotic process may be completely lacking, as in melanotropes. Alternatively, it may be present to some extent to induce differences in coupling efficacy between channel types, as in calf chromaffin cells and mouse pancreatic beta-cells. Physiological mechanisms, like recruitment of channels through facilitation processes or suppression of channels through inactivation, may change coupling characteristics during activity. Due to the large distance between channels and vesicles, single action potentials (APs) are usually insufficient to elicit release, and the coupling between individual APs and release is loose. Most neuroendocrine cells are therefore seen to fire in bursts, like pancreatic beta-cells. Furthermore, a large variation in shape and duration of the APs, with APs of up to 300 ms as in melanotropes, acts as another mechanism to enhance stimulus secretion coupling.

Progesterone-metabolite prevents protein kinase C-dependent modulation of gamma-aminobutyric acid type A receptors in oxytocin neurons.

Gonadal steroid feedback to oxytocin neurons during pregnancy is in part mediated via the neurosteroid allopregnanolone (3alpha-OH-DHP), acting as allosteric modulator of postsynaptic gamma-aminobutyric acid type A (GABA(A)) receptors. We describe here a form of nongenomic progesterone signaling by showing that 3alpha-OH-DHP not only potentiates GABA(A) receptor-channel activity but also prevents its modulation by protein kinase C (PKC). Application of oxytocin or stimulation of PKC suppressed the postsynaptic GABA responses of oxytocin neurons in the absence, but not in the presence of 3alpha-OH-DHP. This finding was true at the juvenile stage and during late pregnancy, when the GABA(A) receptor is sensitive to 3alpha-OH-DHP. In contrast, after parturition, when the GABA(A) receptors expressed by oxytocin neurons are less sensitive to 3alpha-OH-DHP, this neurosteroid no longer counteracts PKC. The change in GABA(A)-receptor responsiveness to 3alpha-OH-DHP helps to explain the onset of firing activity and thus the induction of oxytocin release at parturition.

Roles of G-protein beta gamma, arachidonic acid, and phosphorylation inconvergent activation of an S-like potassium conductance by dopamine, Ala-Pro-Gly-Trp-NH2, and Phe-Met-Arg-Phe-NH2.

Dopamine and the neuropeptides Ala-Pro-Gly-Trp-NH2 (APGWamide or APGWa) and Phe-Met-Arg-Phe-NH2 (FMRFamide or FMRFa) all activate an S-like potassium channel in the light green cells of the mollusc Lymnaea stagnalis, neuroendocrine cells that release insulin-related peptides. We studied the signaling pathways underlying the responses, the role of the G-protein betagamma subunit, and the interference by phosphorylation pathways. All responses are blocked by an inhibitor of arachidonic acid (AA) release, 4-bromophenacylbromide, and by inhibitors of lipoxygenases (nordihydroguaiaretic acid and AA-861) but not by indomethacin, a cyclooxygenase inhibitor. AA and phospholipase A2 (PLA2) induced currents with similar I-V characteristics and potassium selectivity as dopamine, APGWa, and FMRFa. PLA2 occluded the response to FMRFa. We conclude that convergence of the actions of dopamine, APGWa, and FMRFa onto the S-like channel occurs at or upstream of the level of AA and that formation of lipoxygenase metabolites of AA is necessary to activate the channel. Injection of a synthetic peptide, which interferes with G-protein betagamma subunits, inhibited the agonist-induced potassium current. This suggests that betagamma subunits mediate the response, possibly by directly coupling to a phospholipase. Finally, the responses to dopamine, APGWa, and FMRFa were inhibited by activation of PKA and PKC, suggesting that the responses are counteracted by PKA- and PKC-dependent phosphorylation. The PLA2-activated potassium current was inhibited by 8-chlorophenylthio-cAMP but not by 12-O-tetradecanoylphorbol 13-acetate (TPA). However, TPA did inhibit the potassium current induced by irreversible activation of the G-protein using GTP-gamma-S. Thus, it appears that PKA targets a site downstream of AA formation, e.g., the potassium channel, whereas PKC acts at the active G-protein or the phospholipase.

Changes in properties and neurosteroid regulation of GABAergic synapses in the supraoptic nucleus during the mammalian female reproductive cycle.

1. GABAA receptor-mediated synaptic innervation of oxytocin neurones in the supraoptic nucleus (SON) was analysed in adult female rats going through their first reproductive cycle by recording the spontaneous inhibitory postsynaptic currents (sIPSCs) at six stages of female reproduction. 2. During pregnancy we observed a reduction in the interval between monoquantal sIPSCs. The synaptic current amplitude, current decay and neurosteroid sensitivity of postsynaptic GABAA receptors observed at this stage were not distinguishable from those measured in virgin stage SON. 3. Upon parturition an increase in monoquantal synaptic current decay occurred, whereas potentiation by the progesterone metabolite allopregnanolone (3alpha-OH-DHP) was suppressed. 4. Throughout a substantial part of the lactation period the decay of synaptic currents remained attenuated, whilst the potentiation by 3alpha-OH-DHP remained suppressed. 5. Several weeks after the end of lactation sIPSC intervals, their current decay velocity as well as the potentiation by 3alpha-OH-DHP were restored to pre-pregnancy levels, which is indicative of the cyclical nature of synaptic plasticity in the adult SON. 6. Competitive polymerase chain reaction (PCR) analysis showed that virgin animals expressed alpha1 and alpha2 GABAA receptor subunit mRNA at a relative ratio of 2 : 1 compared with beta-actin. After pregnancy both alpha1 and alpha2 subunit mRNA levels were transiently increased, although at a relative ratio of 1 : 4, in line with the hypothesis that alpha2 plays a large role in postsynaptic receptor functioning. During post-lactation both alpha subunits were downregulated. 7. We propose that synaptic remodelling in the SON during pregnancy includes changes in the putative number of GABA release sites per neurone. At parturition, and during the two consecutive weeks of lactation, a subtype of postsynaptic GABAA receptors was observed, distinct from the one being expressed before and during pregnancy. Synaptic current densities, calculated in order to compare the impact of synaptic inhibition, showed that, in particular, the differences in 3alpha-OH-DHP potentiation of these two distinct GABAA receptor subtypes produce robust shifts in the impact of synaptic inhibition of oxytocin neurones at the different stages of female reproduction.

Diffusion barriers limit the effect of mobile calcium buffers on exocytosis of large dense cored vesicles.

Fast exocytosis in melanotropic cells, activated by calcium entry through voltage-gated calcium channels, is very sensitive to mobile calcium buffers (complete block at 800 microM ethylene glycol bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA)). This indicates that calcium diffuses a substantial distance from the channel to the vesicle. Surprisingly, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), having a similar KD for calcium as EGTA but a approximately 100 times faster binding rate, blocked exocytosis only twice as effectively as EGTA. Using computer simulations, we demonstrate that this result cannot be explained by free diffusion and buffer binding rates. We hypothesized that local saturation of calcium buffers is involved. A diffusion barrier for both calcium and buffer molecules, located 50-300 nm from the membrane and reducing diffusion 1000 to 10,000 times, generated similar calcium concentrations for specific concentrations of EGTA and BAPTA. With such barriers, calcium rise phase kinetics upon short step depolarizations (2-20 ms) were faster for EGTA than for BAPTA, implying that short depolarizations should allow exocytosis with 50 microM EGTA but not with 25 microM BAPTA. This prediction was confirmed experimentally with capacitance measurements. Coupling exocytosis to calcium dynamics in the model, we found that a barrier with a approximately 3000 times reduced diffusion at approximately 130 nm beneath the membrane best explains the experimentally observed effects of EGTA and BAPTA on block and kinetics of release.

Conopressin affects excitability, firing, and action potential shape through stimulation of transient and persistent inward currents in mulluscan neurons.

The molluscan vasopressin/oxytocin-related neuropeptide conopressin activates two persistent inward currents in neurons from the anterior lobe of the right cerebral ganglion of Lymnaea stagnalis that are involved in the control of male copulatory behavior. The low-voltage-activated (LVA) current is activated at a wide range of membrane potentials, its amplitude being only weakly voltage dependent. The high-voltage-activated (HVA) current is activated at potentials positive to -40 mV only and shows a steep voltage dependence. Occurrence of both currents varies from cell to cell, some expressing both and others only the HVA current. In most neurons that have the LVA current, a conopressin-independent persistent inward current (INSR) is found that resembles the HVA current in its voltage dependence. The functional importance of the LVA and HVA currents was studied under current-clamp conditions in isolated anterior lobe neurons. In cells exhibiting both current types, the effect of activation of the LVA current alone was investigated as follows: previously recorded LVA current profiles were injected into the neurons, and the effects were compared with responses induced by conopressin. Both treatments resulted in a strong depolarization and firing activity. No differences in firing frequency and burst duration were observed, indicating that activation of the LVA current is sufficient to evoke bursts. In cells exhibiting only the HVA current, the effect of conopressin on the response to a depolarizing stimulus was tested. Conopressin reversibly increased the number of action potentials generated by the stimulus, suggesting that the HVA current enhances excitability and counteracts accommodation. Conopressin enhanced action potential broadening during depolarizing stimuli in many neurons. Voltage-clamp experiments performed under ion-selective conditions revealed the presence of transient sodium and calcium currents. Using the action potential clamp technique, it was shown that both currents contribute to the action potential. The calcium current, which is activated mainly during the repolarizing phase of the action potential, is augmented by conopressin. Thus conopressin may directly modulate the shape of the action potential. In summary, conopressin may act simultaneously on multiple inward currents in anterior lobe neurons of Lymnaea to affect firing activity, excitability, and action potential shape.

gamma-Conotoxin-PnVIIA, a gamma-carboxyglutamate-containing peptide agonist of neuronal pacemaker cation currents.

A novel gamma-carboxyglutamate-containing peptide, designated gamma-conotoxin-PnVIIA, is described from the venom of the molluscivorous snail Conus pennaceus. gamma PnVIIA, triggers depolarization and firing of action potential bursts in the caudodorsal neurons of Lymnaea. This effect is due to activation or enhancement of a slow inward cation current that may underly endogenous bursting activity of these neurons. The amino acid sequence of gamma PnVIIA was determined as DCTSWFGRCTVNS gamma CCSNSCDQTYC gamma-LYAFOS (where gamma is gamma-carboxyglutamate, O is trans-4-hydroxyproline), thus gamma PnVIIA belongs to the six cysteine four loop structural family of conotoxins, and is most homologous to the previously described excitatory conotoxin-TxVIIA. Interestingly, TxVIIA did not induce action potentials in Lymnaea caudodorsal neurons. gamma PnVIIA is the prototype of a new class of gamma-conotoxins that will provide tools for the study of voltage-gated pacemaker channels, which underly bursting processes in excitable systems.

The relation of exocytosis and rapid endocytosis to calcium entry evoked by short repetitive depolarizing pulses in rat melanotropic cells.

Melanotropic cells release predocked, large, dense-cored vesicles containing alpha-melanocyte stimulating hormone in response to calcium entry through voltage-gated calcium channels. Our first objective was to study the relationship between exocytosis, rapid endocytosis, and calcium entry evoked by short step depolarizations in the order of duration of single action potentials (APs). Exocytosis and rapid endocytosis were monitored by capacitance measurements. We show that short step depolarizations (40 msec) evoke the fast release of only approximately 3% of the predocked release-ready vesicle pool. Second, we asked what the distance is between voltage-gated calcium channels and predocked vesicles in these cells by modulating the intracellular buffer capacity. Exocytosis and rapid endocytosis were differentially affected by low concentrations of the calcium chelator EGTA. EGTA slightly attenuated exocytosis at 100 microM relative to 50 microM, but exocytosis was strongly depressed at 400 microM, showing that calcium ions have to travel a large distance to stimulate exocytosis. Nevertheless, the efficacy of calcium ions to stimulate exocytosis was constant for pulse durations between 2 and 40 msec, indicating that in melanotropes, exocytosis is related linearly to the amount and duration of calcium entry during a single AP. Rapid endocytosis was already strongly depressed at 100 microM EGTA, which shows that the process of endocytosis itself is calcium dependent in melanotropic cells. Furthermore, rapid endocytosis proceeded with a time constant of approximately 116 msec at 33 degrees C, which is three times faster than at room temperature. There was a strong correlation between the amplitude of endocytosis and the amplitude of exocytosis immediately preceding endocytosis. Both this correlation and the fast time constant of endocytosis suggest that the exocytotic vesicle is retrieved rapidly.

Plasticity in fast synaptic inhibition of adult oxytocin neurons caused by switch in GABA(A) receptor subunit expression.

We found that magnocellular oxytocin neurons in adult female rats exhibit an endogenous GABA(A) receptor subunit switch around parturition: a decrease in alpha1:alpha2 subunit mRNA ratio correlated with a decrease in allopregnanolone potentiation and increase in decay time constant of the GABA(A) receptor-mediated IPSCs in these cells. The causal relationship between changes in alpha1:alpha2 mRNA ratio and the ion channel kinetics was confirmed using in vitro antisense deletion. Further, GABA(A) receptors exhibited a tonic inhibitory influence upon oxytocin release in vivo, and allopregnanolone helped to restrain oxytocin neuron in vitro firing only before parturition, when the alpha1:alpha2 subunit mRNA ratio was still high. Such observations provide evidence for the physiological significance of GABA(A) receptor subunit heterogeneity and plasticity in the adult brain.

Phe-Met-Arg-Phe-amide activates a novel voltage-dependent K+ current through a lipoxygenase pathway in molluscan neurones.

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED50 = 23 nM) activated a K+ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K+]o = 1.7 mM), only outward current responses occurred. In high K+ salines ([K+]o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K+. In both salines, no responses were measured below -120 mV. Between -120 mV and the K+ reversal potential, currents were inward with maximal amplitudes at approximately -60 mV. Thus, U-shaped current-voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K+ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per approximately 14 mV at all [K+]o. Since this result was also obtained in nearly symmetrical K+ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K+ concentrations. Onset kinetics of the response were slow (rise time approximately 650 ms at -40 mV). However, when FMRFa was applied while holding the cell at -120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to -40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at -120 mV, the time constant of activation during the subsequent test pulse decreased from approximately 36 ms at -60 mV to approximately 13 ms at -30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from -120 to -50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba2+, and, partially, Cd2+ and Cs+. The response to FMRFa was affected by intracellular GTPgammaS. The response was inhibited by blockers of phospholipase A2 and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K+ current distinguish it from other receptor-driven K+ currents such as the S-current- and G-protein-dependent inward rectifiers.

Vasopressin/oxytocin-related conopressin induces two separate pacemaker currents in an identified central neuron of Lymnaea stagnalis.

The molluscan vasopressin/oxytocin analogue Lys-conopressin excites neurons in the anterior lobe of the right cerebral ganglion of the snail Lymnaea stagnalis. Persistent inward currents that underlie the excitatory response were studied with the use of voltage-ramp protocols in the identified neuron RCB1 and other anterior lobe neurons. Under whole cell voltage-clamp conditions, two types of conopressin-activated current could be distinguished on the basis of their voltage dependence: 1) a pacemaker-like current that was activated at potentials above -40 mV (high-voltage-activated current, I(HVA)) and 2) an inward current that was activated at all potentials between -90 and +10 mV (low-voltage-activated current, I(LVA)). Ion substitution experiments indicate that sodium is the main charge carrier for I(HVA) and I(LVA). Both currents are differentially affected by cadmium. I(HVA) and I(LVA) differ in dose dependence, with median effective concentration values of 7.7 x 10(-8) M and 2.2 x 10(-7) M, respectively. Vasopressin and oxytocin act as weak agonists for the conopressin responses. The kinetics of desensitization and washout of I(HVA) and I(LVA) are different. The HVA response shows little desensitization, whereas the LVA response desensitizes within minutes (time constant 80 +/- 28 s, mean +/- SD). The time constant of washout on removal of conopressin is 159 +/- 63 s for I(HVA) and 36 +/- 13 s for I(LVA). These results suggest that two distinct conopressin receptors are involved in the activation of both currents. The conopressin-activated currents induce or enhance a region of negative slope resistance in the steady-state current-voltage relation. They differ from a third persistent inward current that is carried by calcium and completely blocked by cadmium. The presumed functional roles of these currents, possibly including autoregulation, are discussed.

High intracellular calcium levels during and after electrical discharges in molluscan peptidergic neurons.

Intracellular calcium levels ([Ca2+]i) during and following electrical activity of the neuroendocrine caudodorsal cells of the pond snail (Lymnaea stagnalis) were measured in situ and in dissociated cells by combining electrical recordings and Fura-2 measurements. Caudodorsal cells are typical neuroendocrine cells that control egg laying via the release of a set of peptides during a stereotyped discharge of action potentials. Single action potentials or short trains of spikes in dissociated caudodorsal cells induced only small but consistent increases in [Ca2+]i. With longer or repeated spike trains, larger [Ca2+]i transients were measured, indicating accumulation of calcium. The calcium channel blocker Ni2+ suppressed the calcium elevation, suggesting that calcium influx occurred through voltage-activated calcium channels. Calcium levels in caudodorsal cells in situ were measured before, during and after the stereotyped firing pattern, a approximately 35-min discharge of regular spiking. Basal calcium levels in caudodorsal cells in situ were about 125 nM. During the initial phase of the discharge, the intracellular calcium level increased to approximately 250 nM. Maximal calcium levels (300-600 nM) were only reached at the final phase of the discharge or several minutes after the cessation of firing. Calcium levels remained elevated for up to 1 h after the end of the discharge. During this time, caudodorsal cells were characterized by very low excitability. We suggest that the prolonged, elevated level of calcium following the discharge need not be directly dependent on action potentials. The long-lasting [Ca2+]i elevation may cause the release of neuropeptides to outlast the duration of electrical activity, thus uncoupling release from spiking. In addition, it may cause reduced excitability of neuroendocrine cells following a period of spiking, thereby inducing a refractory period.

High intracellular calcium levels during and after electrical discharges in molluscan peptidergic neurons.

Intracellular calcium levels ([Ca2+]i) during and following electrical activity of the neuroendocrine caudodorsal cells of the pond snail (Lymnaea stagnalis) were measured in situ and is dissociated cells by combining electrical recordings and Fura-2 measurements. Caudodorsal cells are typical neuroendocrine cells that control egg laying via the release of a set of peptides during a stereotyped discharge of action potentials. Single action potentials or short trains of spikes in dissociated caudodorsal cells induced only small but consistent increases in [Ca2+]i. With longer or repeated spike trains, larger [Ca2+]i transients were measured, indicating accumulation of calcium. The calcium channel blocker Ni2+ suppressed the calcium elevation, suggesting that calcium influx occurred through voltage-activated calcium channels. Calcium levels in caudodorsal cells in situ were measured before, during and after the stereotyped firing pattern, a approximately 35-min discharge of regular spiking. Basal calcium levels in caudodorsal cells in situ were about 125 nM. During the initial phase of the discharge, the intracellular calcium level increased to approximately 250 nM. Maximal calcium levels (300-600 nM) were only reached at the final phase of the discharge or several minutes after the cessation of firing. Calcium levels remained elevated for up to 1 h after the end of the discharge. During this time, caudodorsal cells were characterized by very low excitability. We suggest that the prolonged, elevated level of calcium following the discharge need not be directly dependent on action potentials. The long-lasting [Ca2+]i elevation may cause the release of neuropeptides to outlast the duration of electrical activity, thus uncoupling release from spiking. In addition, it may cause reduced excitability of neuroendocrine cells following a period of spiking, thereby inducing a refractory period.

Cloning and expression of a complementary DNA encoding a molluscan octopamine receptor that couples to chloride channels in HEK293 cells.

A cDNA encoding a G-protein-coupled receptor was cloned from the central nervous system of the pond snail Lymnaea stagnalis. The predicted amino acid sequence of this cDNA most closely resembles the Drosophila tyramine/octopamine receptor, the Locusta tyramine receptor, and an octopamine receptor (Lym oa1) that we recently cloned from Lymnaea. After stable expression of the cDNA in HEK293 cells, we found that [3H]rauwolscine binds with high affinity to the receptor (KD = 6.2.10(-9) M). Octopamine appears to be the most potent naturally occurring agonist to displace the [3H]rauwolscine binding (Ki = 3.0.10(-7) M). Therefore, the receptor is considered to be an octopamine receptor and is consequently designated Lym oa2. The novel receptor shares little pharmacological resemblance with Lym oa1, indicating that the two receptors represent different octopamine receptor subfamilies. Octopaminergic stimulation of Lym oa2 does not induce changes in intracellular concentrations of cAMP or inositol phosphates. However, electrophysiological experiments indicate that octopamine is able to activate a voltage-independent Cl- current in HEK293 cells stably expressing Lym oa2. Although opening of this chloride channel most probably does not require the activation of either protein kinase A or C, it can be blocked by inhibition of protein phosphorylation.

Convergence of multiple G-protein-coupled receptors onto a single type of potassium channel.

The light green cells (LGCs) in the central nervous system of the pond snail Lymnaea stagnalis form a homogeneous group of neuroendocrine cells that are involved in the control of growth and metabolism. These cells are inhibited by dopamine and the neuropeptides APGWamide, FMRFamide and GGSLFRFamide. Thus, the LGCs form an endogenous system in which processing and integration of different inputs into a physiological response can be studied. In this study we characterize the current(s) that are responsible for the inhibition of the LGCs by dopamine, APGWamide, FMRFamide and GGSLFRFamide. The responses are G-protein dependent, as follows from experiments with GTP-gamma-S. Several experiments indicate that the four agonists activate a single type of potassium channel. First, the currents evoked by the agonists have the same ion selectivity and voltage dependence. Potassium is the predominant charge carrier and the responses are weakly voltage sensitive, with conductance decreasing at potentials below approximately - 100 mV. Second, the currents activated by the four agonists display similar sensitivity towards several blockers. Internal and external TEA (10 mM), and extracellular Ba2+ (1 mM) cause a block of approximately 60-90%. External 4AP (1 mM) causes approximately 30% block and external Cs+ (1 mM) causes a voltage sensitive block. There is no sensitivity towards apamine and glibenclamide. Third, there is no summation of the responses to dopamine, APGWamide and GGSLFRFamide with maximal FMRFamide responses. Together, these data indicate that the responses induced by dopamine, APGWamide, FMRFamide and GGSLFRFamide are G-protein mediated and converge onto a single type of potassium channel in the LGCs of Lymnaea stagnalis.