Retzius Neurons Research Articles

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca2+ transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca2+ transient in axon, dendrites and cell body of the Retzius neuron. This Ca2+ transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5–12mM. The Ca2+ transient was suppressed in the absence of extracellular Ca2+ and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca2+ channel blocker Co2+ (2mM). Injection of the non-hydrolysable GTP analogue GTPγS increased and prolonged the dendritic 5-HT-induced Ca2+ transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500mM) did not affect the Ca2+ transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca2+ influx by removal of external Ca2+ improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca2+ influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca2+ increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.

Modulation of Ca2+ influx in leech Retzius neurons. I. Effect of extracellular pH.

We investigated the cytosolic free Ca2+ concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca2+]i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K+ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca2+]i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca2+]i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca2+]i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca2+]i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane.

Modulation of Ca2+ influx in leech Retzius neurons II. Effect of extracellular Ca2+.

We investigated the cytosolic free calcium concentration ([Ca2+]i) of leech Retzius neurons in situ while varying the extracellular Ca2+ concentration via the bathing solution ([Ca2+]B). Changing [Ca2+]B had only an effect on [Ca2+]i if the cells were depolarized by raising the extracellular K+ concentration. Surprisingly, raising [Ca2+]B from 2 to 10 mm caused a decrease in [Ca2+]i, and an increase was evoked by reducing [Ca2+]B to 0.1 mm. These changes were not due to shifts in membrane potential. At low [Ca2+]B moderate membrane depolarizations were sufficient to evoke a [Ca2+]i increase, while progressively larger depolarizations were necessary at higher [Ca2+]B. The changes in the relationship between [Ca2+]i and membrane potential upon varying [Ca2+]B could be reversed by changing extracellular pH. We conclude that [Ca2+]B affects [Ca2+]i by modulating Ca2+ influx through voltage-dependent Ca2+ channels via the electrochemical Ca2+ gradient and the surface potential at the extracellular side of the plasma membrane. These two parameters are affected in a counteracting way: Raising the extracellular Ca2+ concentration enhances the electrochemical Ca2+ gradient and hence Ca2+ influx, but it attenuates Ca2+ channel activity by shifting the extracellular surface potential to the positive direction, and vice versa.

Spread of synaptic potentials through electrical synapses in Retzius neurones of the leech.

We studied the spread of excitatory postsynaptic potentials (EPSPs) through electrical synapses in Retzius neurones of the leech Haementeria officinalis. The pair of Retzius neurones in each ganglion is coupled by a non-rectifying electrical synapse. Both neurones displayed synchronous EPSPs of varying amplitudes and rise times. The kinetics of synchronous EPSPs was similar in 79 % of the EPSP pairs. In the remaining 21 %, one EPSP was smaller and slower than the other, suggesting its passive spread from the other neurone. The proportion of these events increased to 75 % in the presence of Mg(2+) in the bathing fluid. This spread of EPSPs from one neurone to another was tested by producing artificial EPSPs by current injection into the soma of one Retzius neurone. The artificial EPSPs were smaller and arrived more slowly at the soma of the coupled neurone. The coupling ratios for the EPSPs were proportional to the coupling ratio for long steady-state pulses in different neuronal pairs. Our results showed that EPSPs spread from one Retzius neurone to the other and support the idea that EPSP spread between electrically coupled neurones may contribute to the integration processes of neurones.

Feedback control of intracellular pH by means of iontophoretic H+/OH- injection.

A new method for controlling intracellular pH (pH(i)) within a single cell has been developed that is based on the iontophoretic injection of the pH-affecting ions H+ and OH-. The H+/OH- injection currents are governed by an electronic feedback system, which compares the actual pH(i), monitored by pH-sensitive microelectrodes, with a command value. To avoid membrane potential (Em) alterations caused by the H+/OH- currents, the pH(i) control is performed under voltage-clamp conditions, using a two-electrode voltage-clamp design. This feedback-controlled iontophoretic injection system was capable of adjusting pH(i) between pH 6.8 and 8.0 with a precision of 0.031+/-0.026 pH units (n=51) and without affecting Em. We illustrate the application of this system by investigating the pH(i) dependence of glutamate receptors in identified leech Retzius neurons. Stimulation of these receptors with the glutamatergic agonist kainate (50 microM) under control conditions induced an intracellular acidification of 0.14+/-0.07 pH units (n=13). The feedback control system for pH(i) reduced this intracellular acidification to 0.02+/-0.01 pH units (n=15). The kainate-induced inward current was not affected by pH(i) alterations. This result demonstrates that the feedback system for pH(i) control was feasible for investigating the pH(i) dependence of physiological processes and suggests that the glutamate receptors are not modulated by pH(i).

Potentiometric measurement of cell volume changes and intracellular ion concentrations in leech Retzius neurones

A potentiometric method to monitor cell volume changes in intact biological tissues is based on ion-selective microelectrodes filled with the classical K + exchanger Corning 477317, which is very sensitive to large quaternary ammonium ions. After introduction into a single cell, these cations can be used as volume marker by relating their changes in concentration to changes in cell volume, provided that the ions are neither metabolized nor transported out of the cell. In this article we tested the applicability of this method in leech Retzius neurones. In addition to the cell volume, by using triple-barreled microelectrodes we also measured simultaneously the intracellular concentrations of physiologically relevant ion species as well as the membrane potential. We found that Retzius neurones possess an uptake mechanism which allowed for the intracellular accumulation of tetramethylammonium, tetraethylammonium or choline, simply by adding these ions to the bathing solution. In contrast, tetrapropylammonium was not taken up and therefore, we iontophoretically injected this ion species via conventional microelectrodes. The isoosmotic elevation of the extracellular K + concentration from 4 to 30 mM (Na + replaced by K +) induced a swelling of the cells. The changes in the intracellular concentrations of K + and Na + suggest that cell swelling is primarily due to the uptake of K +, probably with Cl − as counterion.

Calcium transients in subcompartments of the leech Retzius neuron as induced by single action potentials

Regional Ca(2+) influx into neurons plays an essential role for fast signal processing, yet it is little understood. We have investigated intracellular Ca(2+) transients induced by a single action potential (AP) in Retzius neurons in situ of isolated ganglia of the leech Hirudo medicinalis using confocal laser scanning microscopy in the cell body, in different axonal branches, and in dendrites. In the cell body, a single AP induced a Ca(2+) transient in submembrane regions, while in central regions no fluorescence change was detected. Burst activity evoked a much larger Ca(2+) influx, which elicited Ca(2+) signals in central somatic regions, including the cell nucleus. A single AP induced a Ca(2+) transient in distal branches of the axon and in dendrites that was significantly larger than in the proximal axon and in the cell body (p <.05), and the recovery of the Ca(2+) transient was significantly faster in axonal branches than in dendrites (p <.01). The AP-induced Ca(2+) transient was inhibited by Co(2+) (2 mM). The P/Q-type Ca(2+) channel blocker omega-agatoxin TK (500 nM) and the L-type Ca(2+) channel blocker nifedipine (20 microM) had no effect on the Ca(2+) transient, whereas the L-type Ca(2+) channel blocker methoxyverapamil (D600, 0.5-1 mM) irreversibly reduced the Ca(2+) transient by 37% in axons and by 42% in dendrites. Depletion of intracellular Ca(2+) stores following inhibition of endoplasmic Ca(2+)-ATPases by cyclopiazonic acid (10 microM) decreased the AP-induced Ca(2+) transient in the dendrites by 21% (p <.01), but not in axons, and increased the Ca(2+) recovery time constant (tau) in the axonal branches by 129% (p <.01), but not in dendrites. The results indicate that an AP evokes a voltage-gated Ca(2+) influx into all subcompartments of the Retzius neuron, where it produces a Ca(2+) signal of different size and/or kinetics. This may contribute to the modulation of electrical excitation and propagation of APs, and to different modes of synaptic and nonsynaptic processes.

Bursting activity in leech Retzius neurons induced by low external chloride.

We have investigated the bursting activity of Retzius neurons in the central nervous system of the leech Hirudo medicinalis as induced in Cl(-)-free saline by measuring membrane potential, membrane current and the intracellular calcium concentration ([Ca2+]i), using fura-2 or Oregon-Green488-Bapta-1. The Retzius neurons changed their low tonic firing to rhythmical bursting activity when the extracellular Cl- concentration ([Cl-]o) was lowered to 1 mM or less. In Cl(-)-free saline (Cl- exchanged by gluconate), bursting was accompanied by a rise in intracellular Ca2+ in both cell body and axon, which oscillated in synchrony with the bursts. The Ca2+ transients depended on the amplitude and duration of the depolarization underlying the burst, and were presumably due to Ca2+ influx through voltage-dependent Ca2+ channels. In Ca(2+)-free, EGTA-buffered saline or in the presence of Ca2+ channel blockers verapamil (1 mM) or diltiazem (500 microM) the depolarizations underlying the bursts in Cl(-)-free saline were enhanced in amplitude and duration. Bursting was not affected by depleting the intracellular Ca2+ stores with cyclopiazonic acid. The depolarization in Cl(-)- and Ca(2+)-free saline did not evoke intracellular Ca2+ changes. The burst-underlying membrane depolarization induced by Cl- removal was found to be due to a Na(+)-dependent persistent inward current and could be inhibited by saxitoxin (25-50 microM). The results suggest that a persistent Na+ current is generated in Cl(-)-free saline and induces the depolarization underlying rhythmic activity, and that presumably Ca(2+)-induced K+ currents modulate the bursting behaviour.

Apparent Intracellular Mg 2+ Buffering in Neurons of the Leech Hirudo medicinalis

The apparent intracellular Mg 2+ buffering, or muffling (sum of processes that damp changes in the free intracellular Mg 2+ concentration, [Mg 2+] i, e.g., buffering, extrusion, and sequestration), was investigated in Retzius neurons of the leech Hirudo medicinalis by iontophoretic injection of H +, OH −, or Mg 2+. Simultaneously, changes in intracellular pH and the intracellular Mg 2+, Na +, or K + concentration were recorded with triple-barreled ion-selective microelectrodes. Cell volume changes were monitored measuring the tetramethylammonium (TMA) concentration in TMA-loaded neurons. Control measurements were carried out in electrolyte droplets (diameter 100–200 μm) placed on a silver wire under paraffin oil. Droplets with or without ATP, the presumed major intracellular Mg 2+ buffer, were used to quantify the pH dependence of Mg 2+ buffering and to determine the transport index of Mg 2+ during iontophoretic injecton. The observed pH dependence of [Mg 2+] i corresponded to what would be expected from Mg 2+ buffering through ATP. The quantity of Mg 2+ muffling, however, was considerably larger than what would be expected if ATP were the sole Mg 2+ buffer. From the decrease in Mg 2+ muffling in the nominal absence of extracellular Na + it was estimated that almost 50% of the ATP-independent muffling is due to the action of Na +/Mg 2+ antiport.

The ouabain-induced [Ca 2+] i increase in leech Retzius neurones is mediated by voltage-dependent Ca 2+ channels

In leech Retzius neurones the inhibition of the Na +–K + pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca 2+] i). To elucidate the mechanism of this increase we investigated the changes in [Ca 2+] i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na +–K + pump in bathing solutions of different ionic composition. The results show that Na +–K + pump inhibition induced a [Ca 2+] i increase only if the cells depolarized sufficiently in the presence of extracellular Ca 2+. Specifically, the relationship between [Ca 2+] i and the membrane potential upon Na +–K + pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca 2+ channels by raising the extracellular K + concentration. It is concluded that the [Ca 2+] i increase caused by inhibiting the Na +–K + pump in leech Retzius neurones is exclusively due to Ca 2+ influx through voltage-dependent Ca 2+ channels.

Excitotoxicity of Lathyrus Sativus Neurotoxin in Leech Retzius Neurons

The effects of Lathyrus sativus neurotoxin were studied on the cell membrane potential and cellular cation composition in Retzius nerve cells of the leech Haemopis sanguisuga, with ion-selective microelectrodes using liquid ion-exchangers. Bath application of 10-4 mol/l Lathyrus sativus neurotoxin for 3 min depolarized the cell membrane potential and decreased the input resistance of directly polarized membrane in Retzius neurons. At the same time the cellular Na+ activity increased and cellular K+ activity decreased with slow but complete recovery, while the intracellular Ca2+ concentration was not changed. Na+-free Ringer solutions inhibited the depolarizing effect of the neurotoxin on the cell membrane potential. Zero-Ca2+ Ringer solution or Ni2+-Ringer solution had no influence on the depolarizing effect of the neurotoxin on the cell membrane potential. It is obvious that the increase in membrane conductance and depolarization of the cell membrane potential are due to an influx of Na+ into the cell accompanied by an efflux of K+ from the cell.

Quantal Release of Serotonin

We have studied the origin of quantal variability for small synaptic vesicles (SSVs) and large dense-cored vesicles (LDCVs). As a model, we used serotonergic Retzius neurons of leech that allow for combined amperometrical and morphological analyses of quantal transmitter release. We find that the transmitter amount released by a SSV varies proportionally to the volume of the vesicle, suggesting that serotonin is stored at a constant intravesicular concentration and is completely discharged during exocytosis. Transmitter discharge from LDCVs shows a higher degree of variability than is expected from their size distribution, and bulk release from LDCVs is slower than release from SSVs. On average, differences in the transmitter amount released from SSVs and LDCVs are proportional to the size differences of the organelles, suggesting that transmitter is stored at similar concentrations in SSVs and LDCVs.

Presenilin-1 deficiency leads to loss of Cajal–Retzius neurons and cortical dysplasia similar to human type 2 lissencephaly

Background: Presenilin-1 (PS1) is a transmembrane protein that is located in the endoplasmic reticulum and the cis Golgi apparatus. Missense mutations of PS1 that modify γ-secretase function, leading to a pathologic processing of amyloid precursor protein, are an important cause of familial Alzheimer's disease. Physiologically, the presenilins are involved in the Notch and Wnt–β-catenin signaling pathways.Results: PS1-deficient mice develop a cortical dysplasia resembling human type 2 lissencephaly, with leptomeningeal fibrosis and migration of cortical-plate neurons beyond their normal position into the marginal zone and subarachnoid space. This disorder of neuronal migration is associated with the disappearance of the majority of the cells of the marginal zone, notably most of the Cajal–Retzius pioneer neurons, between embryonic days E14 and E18, and is preceded and accompanied by disorganization of Notch-1 immunoreactivity on the neuronal cell membranes. The marginal zone also becomes depleted of the extracellular matrix protein reelin and chondroitin sulfate proteoglycans. At that stage PS1 is transiently expressed in leptomeningeal fibroblasts, which are mandatory for the trophic support of Cajal–Retzius neurons.Conclusions: In agreement with models in which neuronal migration disorders have been linked to a defect in Cajal–Retzius cells, the loss of most of these cells in PS1-deficient mice leads to cortical dysplasia. Because PS1 is normally expressed in the leptomeninges, and these become fibrotic in the PS1-knockout mice, we favor the hypothesis that the loss of Cajal–Retzius cells is caused by a defective trophic interaction with leptomeningeal cells, possibly involving disruption of Notch signaling.

Multi-barrelled ion-sensitive microelectrodes and their application in micro-droplets and biological systems

Multi-barrelled ion-sensitive microelectrodes were developed to enable simultaneous measurement of the free concentrations of up to three ion species and the reference potential in living cells. These electrodes were used for studies in Retzius neurones of the leech Hirudo medicinalis to investigate the effect of intracellular alkalinization on the release of Mg 2+ from intracellular stores. The investigations were complemented by control measurements in micro-droplets containing ATP, which is presumed to be a major intracellular Mg 2+ buffer. The droplets were placed on a silver wire under paraffin oil and had approximately the same size (about 100 μm in diameter) as the Retzius neurones. H + or OH − were injected iontophoretically into living cells and micro-droplets from separate glass microelectrodes while the resulting changes in pH and Mg 2+ concentration were recorded with a pH/Mg 2+-sensitive microelectrode. Our results demonstrate that an alkalinization of the cytosol caused by an injection of OH − had no effect on the intracellular Mg 2+ concentration ([Mg 2+] i) and thus did not change cytosolic Mg 2+ buffering. When, however, alkalinization was elicited by the withdrawal of propionate from the bath solution, a method that also leads to the alkalinization of intracellular organelles, [Mg 2+] i increased, possibly by release of stored Mg 2+ from mitochondria.

Mechanism of the kainate-induced intracellular acidification in leech Retzius neurons

We examined the effect of the glutamatergic agonist kainate on the membrane potential, the intracellular Na + concentration ([Na +] i), the intracellular-free Ca 2+ concentration, and on the intracellular pH of Retzius neurons of the medicinal leech, Hirudo medicinalis, in order to investigate the mechanism responsible for the intracellular acidification caused by glutamatergic stimulation. The recordings were made with Na +- and pH-sensitive microelectrodes and iontophoretically injected Fura-2. Bath application of kainate evoked a marked membrane depolarization, a [Na +] i increase, and an intracellular acidification. The intracellular acidification was unaffected by reversal of the electromotive force for H +, suggesting that an influx of H + from the interstitial space does not contribute to the acidification. While the Ca 2+ channel blockers La 3+ and Co 2+ had no effect on the kainate-induced intracellular acidification, suggesting that a Ca 2+ influx via voltage-dependent Ca 2+ channels was not relevant, the acidification was reduced in Ca 2+-free saline solution. In Na +-free saline solution the kainate-induced intracellular acidification was absent, suggesting the involvement of Na + influx in generating the acidification. When injected iontophoretically Na + induced an intracellular acidification but Li +, K +, Rb + or Cs + did not. Furthermore, a [Na +] i increase induced by blocking the Na +/K + pump also led to an intracellular acidification. We conclude that the [Na +] i increase is the crucial event underlying the kainate-induced intracellular acidification. Possible mechanisms linking the [Na +] i increase to the intracellular acidification are discussed.

Laser-induced native fluorescence (LINF) imaging of serotonin depletion in depolarized neurons

Since certain neurotransmitters exhibit native fluorescence we can monitor this property to disclose intracellular changes that result from neurotransmitter release. Isolated Retzius neurons of the leech are known to release serotonin (5-HT) during depolarization. Using intensified CCD technology coupled with UV laser (305 nm) excitation we observed depolarization and calcium-dependent reductions in native fluorescence in the axon, as well as in the cortex of the cell body. When taken together with data obtained from single-cell capillary electrophoresis, we demonstrate that this laser-induced native fluorescence can be reliably used to study spatial and temporal changes in intracellular transmitter content that accompany calcium-dependent secretion.

ATP-inhibited K + channels and membrane potential of identified leech neurons

The effect of the ATP-inhibited K + channel on the membrane potential of leech Retzius neurons was analyzed using electrolyte-filled single-barrelled microelectrodes. The membrane potential was independent of the external nutrient supply during a period of 11 h, probably because the internal energy reserves were sufficient. The K + channel activator HOE 234 ((3 S,4 R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl)-6-phenylsulfonylchromane hemihydrate, 500 μM) induced a membrane hyperpolarization. In the presence of HOE 234, action potentials occurred with a reduced after-hyperpolarization and were discharged in bursts, possibly because of an inhibition of Ca 2+ channels. The blocker of ATP-inhibited K + channels tolbutamide did not significantly alter the membrane potential. In the absence of tolbutamide, the metabolic inhibitors iodoacetate, azide and cyanide (10 mM) evoked membrane hyperpolarizations, but in the presence of 1 mM tolbutamide their hyperpolarizing actions were reduced or abolished while membrane depolarizations were intensified. We conclude that ATP-inhibited K + channels in the soma membrane of leech Retzius neurons provide coupling of cellular metabolism to electrical activity and ionic fluxes.

Long-Lasting Depolarization Of Leech Neurons Mediated By Receptors With A Nicotinic Binding Site

The serotonergic Retzius neurons of the leech midbody ganglia respond in a complex manner to pressure pulses of acetylcholine (ACh) applied onto their soma with a fast depolarization followed by a slower hyperpolarization and an additional delayed long-lasting depolarization. The delayed depolarization is the subject of the present study. The delayed depolarization could be elicited by long (> 1 s) ACh pressure pulses or by short pulses (10 ms) of carbachol, nicotine and DMPP, but not by muscarinic agonists. It was inhibited by bath application of nicotine (10-100 mumol l-1), strychnine (100 mumol l-1) and atropine (10-100 mumol l-1). Nicotinic antagonists that blocked the fast depolarization and the slow hyperpolarization (100 mumol l-1 mecamylamine and d-tubocurarine) did not affect the delayed depolarization induced by carbachol. Partial replacement of the extracellular Na+ by glucamine caused a decrease in the amplitude of the response and a shift of its reversal potential to more negative values. Carbachol pulses applied to Retzius neurons of the ganglia innervating the reproductive segments elicited delayed depolarizations of much smaller amplitude than the ones recorded in Retzius neurons from standard segments. The delayed depolarization could be elicited by the application of short agonist pulses onto different loci over the surface of the ganglion, at a distance from the soma. Isolated cultured Retzius neurons did not exhibit the delayed depolarization although they readily expressed the earlier phases of the complex cholinergic response. Carbachol pulses applied to the soma of other neurons in the leech ganglion produced a variety of specific responses. The results suggest that the delayed depolarization was produced by the activation of a cationic conductance mediated by receptors with a pharmacological profile similar to that of the alpha 9 nicotinic receptors and was not a byproduct of the early phases of the cholinergic response. The response seemed to be initiated in the extensive neuropilar processes of the Retzius cell, enabling a persistent excitatory signal.

Basal Acetylcholine Release In Leech Ganglia Depolarizes Neurons Through Receptors With A Nicotinic Binding Site

The response of Retzius neurons, the main neuronal source of serotonin in the leech nervous system, to cholinergic agonists has been extensively investigated. In this study, we analyzed the effects of inhibiting the acetylcholinesterase (AChE) activity in the leech midbody ganglion on the electrophysiological activity of the Retzius neurons. Bath application of neostigmine and physostigmine (0.1-100 mumol l-1) produced, after a delay, a strong depolarization of the Retzius neurons with a dose-dependent amplitude and latency. The amplitude of this depolarization increased as the extracellular level of Ca2+ increased and decreased as the extracellular level of Ca2+ decreased. The response to neostigmine and physostigmine was inhibited by curare (100 mumol l-1), nicotine (10 mumol l-1), atropine (100 mumol l-1) and strychnine (100 mumol l-1), but was not affected by mecamylamine (100 mumol l-1) or hexamethonium (100 mumol l-1). Superfusion with solutions containing 100 mumol l-1 strychnine or atropine produced a progressive hyperpolarization of the Retzius neurons, while superfusion with 100 mumol l-1 curare did not. The hyperpolarization induced by atropine was inhibited in the presence of curare. Other neurons in the ganglion showed distinctive responses to the AChE inhibitors that were coincident with their responses to cholinergic agonists. The results suggest the existence of a basal level of acetylcholine (ACh) release in the leech ganglion that is powerfully counteracted by endogenous AChE activity. Under control conditions, this basal release appears to be sufficient to generate an ACh tonus that regulates the membrane potential of Retzius neurons. Since these neurons can support a sustained firing rate, which is dependent on the membrane potential, the results presented in this report suggest that the basal ACh tonus regulates the output of these neuromodulatory serotonergic neurons.

Neuron-silicon junction with voltage-gated ionic currents.

We recorded the signals of firing Retzius neurons from Hirudo medicinalis by field-effect transistors. The axon stump of dissociated cells was attached to an open gate coated with concanavalin A. We observed a new type of neuron-transistor coupling: the extracellular voltage transients beneath the neuron were dominated by a negative peak during the rising phase of the action potential with a weaker positive transient in the falling phase. The biphasic response was opposite to the signal of capacitive coupling. We simulated the junction on the basis of the Hodgkin-Huxley equations. We found that the negative transient corresponded to an inward flow of sodium and the positive response to an outward flow of potassium. The field-effect transistors are able to probe the local flow of ionic currents in a membrane which is hidden in the region of cell adhesion. They may become a novel tool in neuroscience.