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.

Postsynaptic mechanism of depression of GABAergic synapses by oxytocin in the supraoptic nucleus of immature rat.

1. Oxytocin is known to act on autoreceptors of oxytocin neurones in the supraoptic nucleus (SON). We investigated whether oxytocin modulates putative oxytocin neurones by suppressing the GABAA receptor-mediated synaptic inputs on these cells. 2. GABAergic inhibitory postsynaptic currents (IPSCs) were recorded from SON neurones in hypothalamic slices from young rats. Oxytocin specifically reduced the amplitude of both spontaneous and evoked IPSCs, without altering their current kinetics. 3. The effect of oxytocin was observed in 70% of the magnocellular neurones recorded from the dorsomedial part of the SON. d(CH2)5OVT, a specific antagonist of oxytocin receptors, blocked the effect of oxytocin on the IPSCs. Vasopressin had no effect on oxytocin-sensitive SON neurones. 4. The intervals between spontaneous IPSCs were not affected by oxytocin. This suggested that oxytocin had a postsynaptic effect on SON neurones. 5. This postsynaptic origin was further substantiated by application of TTX, which blocked all evoked release but did not prevent the suppressive effect of oxytocin on the amplitude of the spontaneous IPSCs still present in the recording. The selective effect of oxytocin on IPSC amplitude was also maintained in nominally zero extracellular calcium. 6. Intracellular perfusion of SON neurones with GTP gamma S mimicked the effect of oxytocin on IPSCs, while GDP beta S, similarly applied, abolished the effect of oxytocin. 7. Application of calcium mobilizers such as thapsigargin and caffeine also reduced the amplitude of spontaneous IPSCs without significantly altering the frequency at which IPSCs occurred. 8. Thus, oxytocin depresses GABAergic synapses in the SON via modulation of the postsynaptic GABAA receptors. This would lead to disinhibition of SON neurones sensitive to oxytocin and could, therefore, be a powerful means of controlling the firing of oxytocin neurones.

The role of membrane properties, synaptic input and electrical junction in determination of spike output of a pair of peptidergic neurons in the mollusc Lymnaea stagnalis.

The two electrotonically coupled peptidergic neurons, VD1 and RPD2 show in the isolated central nervous system (CNS) a patterned activity. The cells fire in almost perfect synchrony in CNS's derived from animals of moderate age, while in old animals disturbances in synchrony are observed. The firing pattern varies from beating to bursting. Isolated VD1's show a beating firing pattern, indicating that the cell possesses pacemaker properties, while the isolated RPD2 is almost always silent. Hybrid current/voltage clamp experiments show that in the intact CNS spike generation in the two cells is due to VD1, indicating that the pacemaker properties of VD1 constitute the main driving force for the firing rhythm of the two cell system. In low calcium/high magnesium solutions the rhythm is a beating one, while in solutions which do not suppress synaptic input, sometimes a bursting pattern is observed. The idea that the bursting pattern is due to synaptic modulation of these cells is corroborated by the observation that application of certain transmitters induces a very prominent burst of activity in these neurons. These bursts are very reminiscent of the bursts, observed in semi-intact preparations, which are accompanied by penumostome movements. Increase in coupling resistance, which occurs at older age, is accompanied by irregularities in firing rhythm. Apparently the electrotonic junction is essential for the patterned output of the two cell system.

Inhibitory modulation by FMRFamide of the voltage-gated sodium current in identified neurones in Lymnaea stagnalis.

1. The putative neurotransmitter FMRFa (Phe-Met-Arg-Phe-amide) caused an inhibitory modulation of the voltage-gated sodium current (INa) in central neurones, the peptidergic caudo dorsal cells (CDCs) of the mollusc Lymnaea stagnalis. FMRFa reduced INa at all command potentials tested (ranging from -35 to +20 mV), but the amplitude of the effect of FMRFa was voltage dependent, inhibition being stronger at more negative potentials (50 +/- 5% reduction at half-maximal INa activation versus 25 +/- 8% at the peak of the I-V curve). 2. INa current traces were well fitted by a Hodgkin & Huxley based model, using m3 activation kinetics and two time constants for inactivation. 3. The steady-state inactivation curve of INa was characterized by half-maximal inactivation at -42.5 +/- 1.81 mV and a slope factor of 4.6 +/- 0.28 mV. The fastest time constant of inactivation ran from 100 +/- 5 to 0.8 +/- 0.32 ms and the slower time constant from 505 +/- 45 to 4.8 +/- 1.40 ms in the range -40 to -5 mV. 4. FMRFa had no significant effect on either component of inactivation, nor on the voltage dependence of steady-state inactivation, nor on the maximal conductance. 5. FMRFa affected the activation of INa. The activation time constant was increased, ranging from 0.75 +/- 0.050 to 0.22 +/- 0.017 ms under control and from 0.91 +/- 0.043 to 0.31 +/- 0.038 ms with FMRFa in the voltage range -25 to +5 mV. The steady-state activation curve was shifted to less negative potentials: half-maximal activation occurred at -26.5 +/- 1.2 mV under control and at 23.6 +/- 1.4 mV with FMRFa; the slope factor (4.6 +/- 1.4 mV in control experiments) was not affected. The combination of slower activation kinetics and a shift in the voltage dependence of activation in the Hodgkin & Huxley based model, adequately explained the reduction of INa by FMRFa. 6. The physiological consequence is that the spiking threshold is increased, causing an arrest of on-going firing activity and a decrease in excitability.

The role of pacemaker properties and synaptic input in generation and modulation of spiking activity in a pair of electrically coupled peptidergic neurons.

The origin of patterned electrical activity in two electronically coupled peptidergic neurons, VD1 and RPD2, in the CNS of Lymnaea stagnalis was investigated. VD1 proved to have intrinsic beating pacemaker properties. Hybrid current/voltage clamp experiments demonstrated that in the intact CNS generation of spike activity in the coupled cell system is dominated by VD1. Modulation of spiking activity of VD1/RPD2 appears to originate mainly from chemical synaptic input. The electrical coupling of VD1 and RPD2 proved essential for spike synchronization between the cells.

Age-related changes in junctional and non-junctional conductances in two electrically coupled peptidergic neurons of the mollusc Lymnaea stagnalis.

Age-related changes in electrotonic coupling ratio of two identified neurons in Lymnaea stagnalis were studied together with the underlying changes in the steady-state conductance properties of the network. Two phases were distinguished in the development of coupling ratio across lifespan. During the first phase (age of 3-13 months), coupling ratio decreased from decreased from 60% to 30%. The second phase (age 13-20 months) was characterized by an increase in coupling ratio. Values of up to 60% were reached again in the oldest animals. Voltage clamp measurements showed that the biphasic trend of the age-related changes in coupling ratio is paralleled by changes in conductance properties of the junction between VD1 and RPD2. During the first phase junctional conductance decreased, whereas during the second phase junctional conductance increased. In addition to the decrease in junctional conductance, a growth-related increase in non-junctional conductance of VD1 and RPD2 contributed to the decrease in coupling ratio observed during the first phase. Thus our results indicate that in Lymnaea junctional connections between neurons may undergo considerable and discontinuous changes after sexual maturation. In addition to these changes in steady-state electrical properties, indications were obtained that age-related changes of kinetically slower conductance(s) may occur in the non-junctional membrane of VD1 and RPD2.

Localization of dopamine and its relation to the growth hormone producing cells in the central nervous system of the snail Lymnaea stagnalis.

The distribution of dopamine in the central nervous system of the pond snail Lymnaea stagnalis was investigated by using immunocytochemistry and HPLC measurements. With both methods it was demonstrated that dopamine is predominantly present in the cerebral and pedal ganglia. The dopamine-immunoreactivity was mainly observed in nerve-fibers in the neuropile of the ganglia. Relatively few dopamine-immunopositive cell bodies (diameters 10-30 microns) were found. A large cell in the right pedal ganglion (the so-called RPeD1) stained positively with the dopamine antibody. It has previously been demonstrated that the growth hormone producing cells (GHCs) possess dopamine receptors on their cell bodies. However, dopamine-immunopositive fibers were observed only in the vicinity of the GHC nerve-endings and not close to the GHC cell bodies.

Comparative toxicokinetics of 2,2'- and 4,4'-dichlorobiphenyls in the pond snail Lymnaea stagnalis (L.).

Pond snails (Lymnaea stagnalis (L.)) were treated with 2,2'-dichlorobiphenyl (DCB) or 4,4'-DCB, to examine the toxicokinetic profile of these compounds. Snails were treated orally with 210 micrograms 4,4'-DCB (impregnated on food) for 14 hr, or snails were injected with 50 micrograms of 2,2'-DCB or 4,4'-DCB in the foot. At different times after starting feeding or injection, tissues (albumen gland, digestive gland and digestive tube, central nervous system, remainder parts), hemolymph and faeces were analyzed for unchanged 2,2'- or 4,4'-DCB. The results showed that in case of oral administration of 4,4'-DCB after 144 hr, 97.5% of the dose was excreted unchanged in the faeces. During the first 48 hr 4,4'-DCB was found in all tissues. Thereafter, an exponential elimination was found (the rate constant of elimination (kel) varied from 0.010-0.021 per hr, t1/2 from 33-60 hr and the apparent clearance from 0.02-0.3 g/hr for the different tissues). After injection, the compounds were found in all the above mentioned tissues, especially in the digestive gland. There was a clear difference between snails injected with 2,2'- and 4,4'-DCB. Firstly, kel for 2,2'-DCB was higher (0.028 per hr vs 4,4'-DCB: 0.001 per hr). Secondly, 2,2'-DCB was lethal; 63% of the animals died after 72 hr.

Inhibitory modulation of neuronal voltage-dependent sodium current by Phe-Met-Arg-Phe-amide.

The tetrodotoxin (TTX)-sensitive, voltage-gated Na(+)-current (INa) in a cluster of peptidergic neurons, involved in egg laying, in the CNS of the mollusc Lymnaea stagnalis, is modulated by the neuropeptide FMRFa (Phe-Met-Arg-Phe-NH2). Application of FMRFa reversibly reduced the isolated INa in a dose-dependent fashion. The physiological consequence is that the threshold for action potential generation is increased, causing an arrest of ongoing firing activity. The inhibitory action of FMRFa reported here is the first known example of modulation of the voltage-gated INa by a putative neurotransmitter in intact nerve cells. This finding underlines the importance of modulation of ionic currents as a mechanism of regulation of neuronal excitability and includes the voltage dependent Na current in the range of currents subject to transmitter modulation.

Indications for a hormonal function of dopamine in the central nervous system of the snail Lymnaea stagnalis.

In the present paper we collected evidence for the occurrence of D2-like dopamine receptors on the cell bodies of the neuroendocrine growth hormone-producing cells (GHCs) in the central nervous system (CNS) of the snail Lymnaea stagnalis. Measurements of the membrane potential of GHCs in situ as well as isolated GHCs revealed that stimulation of these dopamine receptors results in a hyperpolarization. Although immunohistochemical analysis of the CNS of L. stagnalis clearly revealed the occurrence of dopamine containing cells and nerve fibers, no projections of dopamine immunopositive fibers to the GHC cell bodies could be observed. By using HPLC with electrochemical detection we found that the blood concentration of dopamine in L. stagnalis is in the range of concentrations hyperpolarizing GHCs in vitro (0.1-10 microM). On the basis of these findings it is proposed that dopamine is involved in hormonal communication in the CNS of L. stagnalis.

Further pharmacological characterization of a D-2-like dopamine receptor on growth hormone producing cells in Lymnaea stagnalis.

A preliminary study has revealed that a mammalian D-2-like dopamine (DA) receptor mediates hyperpolarization of the neuroendocrine growth hormone-producing cells (GHCs) in the snail Lymnaea stagnalis. An extensive pharmacological characterization of this receptor was performed in the present study. Several mammalian D-2 receptor agonists (e.g. aminotetralins) and antagonists (e.g.(-)-sulpiride) showed agonistic and antagonistic effects, respectively. However, some selective D-2 receptor agonists (e.g. N 0437) and antagonists (e.g. domperidone) failed to show agonistic or antagonistic effects, respectively. It is concluded that the dopamine receptor mediating hyperpolarization of the GHCs displays, besides some similarities, several differences from the mammalian D-2 receptor.

Dopamine receptor stimulation induces a potassium dependent hyperpolarizing response in growth hormone producing neuroendocrine cells of the gastropod mollusc Lymnaea stagnalis.

Of several putative transmitters used, dopamine was the only one which caused (at low concentrations) a hyperpolarizing response (H-response) in growth hormone producing cells (GHCs) of the freshwater snail Lymnaea stagnalis. Membrane resistance changes, and shifts in the reversal potential of this H-response in different K+-concentrations, indicate that the response is due to an increase in potassium conductance. The dopamine induced H-response is blocked by (-)-sulpiride, 4-aminopyridine, dibutyryl cAMP, 8CPT-cAMP, forskolin and IBMX. These data suggest that dopamine induces the H-response by stimulating a receptor resembling the mammalian D-2 receptor and that this effect of dopamine is mediated by a decrease in the formation of intracellular cAMP.

Insect adipokinetic hormones.

Peptides with adipokinetic (and usually carbohydrate-mobilizing) potency have been demonstrated in various insects, including Locusta migratoria, Schistocerca gregaria, Manduca sexta, Danaus plexippus and Periplaneta americana. As far as characterized by now the adipokinetic factors are blocked peptides, consisting of eight to ten amino acid residues. In locusts the adipokinetic hormones are synthesized in the glandular lobe of the corpus cardiacum and released into the haemolymph in response to flight stimuli. This release is under direct control of neurons, the cell bodies of which are located in the lateral areas of the protocerebrum, while their axons run via the nervi corporis cardiaci II into the glandular lobe. Hormone release is modulated by axons present in the nervi corporis cardiaci I as well as by the haemolymph trehalose concentration. Trehalose apparently exerts its influence via a neuronal network present in the corpus cardiacum. The fat body is the main target organ of the adipokinetic hormones, which are involved in both mobilization and release of flight substrates from fat body stores, i.e., trehalose from glycogen and diacylglycerol from triacylglycerol. Lipid release is accompanied by haemolymph lipoprotein conversions.

Opposing roles for D-1 and D-2 dopamine receptors in regulating the excitability of growth hormone-producing cells in the snail Lymnaea stagnalis.

Dopamine hyperpolarizes growth hormone-producing cells (GHC) in the CNS of Lymnaea stagnalis. This effect of dopamine was mimicked by the D-2 receptor agonist LY 141865 and antagonized by the D-2 receptor antagonists (-)-sulpiride and YM 09151-2. SKF 38393, a selective D-1 receptor agonist, increased the excitability of the GHC. This effect was mimicked by intracellular injection of cyclic AMP and antagonized by the D-1 receptor antagonist SCH 23390. Dopamine (in the presence of (-)-sulpiride) also increased the excitability of the GHC. It is concluded that both a D-1 and a D-2 receptor regulate the electrical activity of the GHC in the CNS of Lymnaea stagnalis.

Morphology and electrophysiology of the ovulation hormone producing neuro-endocrine cells of the freshwater snail Lymnaea stagnalis (L.).

The ovulation hormone producing neuro-endocrine cells of Lymnaea stagnalis, the caudo-dorsal cells (CDC), are comparable to the bag cells of Aplysia. Both cell types are capable of the production of a long-lasting activity (afterdischarge) during which an ovulation hormone is released. The CDC (30 cells in the left cerebral ganglion and 70 cells in the right) are usually electrically silent but an afterdischarge can be brought about in all cells of both groups by direct, repetitive electrical stimulation of single CDC. This is not possible in every preparation, indicating that the CDC can be in different states of excitability. All cells participate in the afterdischarge and fire approximately synchronously. All CDC are electrotonically connected. Results of experiments in which neurones were injected with horseradish peroxidase suggest that the demonstrated electrotonic connexions between the two opposite groups of CDC are brought about by 10-12 special axons.