Isolated Crayfish Stretch Receptor Research Articles

Exogenous recombinant Hsp70 mediates neuroprotection after photothrombotic stroke.

Ischaemic stroke is an acute interruption of the blood supply to the brain, which leads to rapid irreversible damage to nerve tissue. Ischaemic stroke is accompanied by the development of neuroinflammation and neurodegeneration observed around the affected brain area. Heat shock protein 70 (Hsp70) facilitates cell survival under a variety of different stress conditions. Hsp70 may be secreted from cells and exhibits cytoprotective activity. This activity most likely occurs by decreasing the levels of several proinflammatory cytokines through interaction with a few receptors specific to the innate immune system. Herein, we demonstrated that intranasal administration of recombinant human Hsp70 shows a significant twofold decrease in the volume of local ischaemia induced by photothrombosis in the mouse prefrontal brain cortex. Our results revealed that intranasal injections of recombinant Hsp70 decreased the apoptosis level in the ischaemic penumbra, stimulated axonogenesis and increased the number of neurons producing synaptophysin. Similarly, in the isolated crayfish stretch receptor, consisting of a single sensory neuron surrounded by the glial envelope, exogenous Hsp70 significantly decreased photoinduced apoptosis and necrosis of glial cells. The obtained data enable one to consider human recombinant Hsp70 as a promising compound that could be translated from the bench into clinical therapies.

The effect of axotomy on firing and ultrastructure of the crayfish mechanoreceptor neurons and satellite glial cells

Neurotrauma is among main causes of human disability and death. We studied effects of axotomy on ultrastructure and neuronal activity of a simple model object – an isolated crayfish stretch receptor that consists of single mechanoreceptor neurons (MRN) enwrapped by multilayer glial envelope. After isolation, MRN regularly fired until spontaneous activity cessation. Axotomy did not change significantly MRN spike amplitude and firing rate. However, the duration of neuron activity from MRN isolation to its spontaneous cessation decreased in axotomized MRN relative to intact neuron. [Ca2+] in MRN axon and soma increased 3–10 min after axotomy. Ca2+ entry through ion channels in the axolemma accelerated axotomy-stimulated firing cessation. MRN incubation with Ca2+ionophore ionomycin accelerated MRN inactivation, whereas Ca2+-channel blocker Cd2+ prolonged firing. Activity duration of either intact, or axotomized MRN did not change in the presence of ryanodine or dantrolene, inhibitors of ryanodin-sensitive Ca2+ channels in endoplasmic reticulum. Thapsigargin, inhibitor of endoplasmic reticulum Ca2+-ATPase, or its activator ochratoxin were ineffective. Ultrastructural study showed that the defect in the axon transected by thin scissors is sealed by fused axolemma, glial and collagen layers. Only the 30–50 μm long segment completely lost microtubules and contained swelled mitochondria. The microtubular bundle remained undamaged at 300 μm away from the axotomy site. However, mitochondria within the 200–300 μm segment were strongly condensed and lost matrix and cristae. Glial and collagen layers exhibited greater damage. Swelling and edema of glial layers, collagen disorganization and rupture occurred within this segment. Thus, axotomy stronger damages glia/collagen envelope, axonal microtubules and mitochondria.

The Localization of p53 in the Crayfish Mechanoreceptor Neurons and Its Role in Axotomy-Induced Death of Satellite Glial Cells Remote from the Axon Transection Site.

Neuron and glia death after axon transection is regulated by various signaling proteins. Protein p53 is a key regulator of diverse cell functions including stress response, DNA repair, proliferation, and apoptosis. We showed that p53 was overexpressed in crayfish ganglia after bilateral axotomy. In the isolated crayfish stretch receptor, a simple natural neuroglial preparation, which consists of a single mechanoreceptor neuron (MRN) enveloped by glial cells, p53 regulated axotomy-induced death of glial cells remote from the axon transection site. In MRN, p53 immunofluorescence was highest in the nucleolus and in the narrow cytoplasmic ring around the nucleus; its levels in the nucleus and cytoplasm were lower. After axotomy, p53 accumulated in the neuronal perikaryon. Its immunofluorescence also increased in the neuronal and glial nuclei. However, p53 immunofluorescence in the most of neuronal nucleoli disappeared. Axotomy-induced apoptosis of remote glial cells increased in the presence of p53 activators WR-1065 and nutlin-3 but reduced by pifithrin-α that inhibits transcriptional activity of p53. Pifithrin-μ that inhibits p53 effect on mitochondria increased axotomy-induced apoptosis of remote glial cells but reduced their necrosis. Therefore, axotomy-induced apoptosis of remote glial cells was associated with p53 effect on transcription processes, whereas glial necrosis was rather associated with transcription-independent p53 effect on mitochondria. Apparently, the fate of remote glial cells in the axotomized crayfish stretch receptor is determined by the balance between different modalities of p53 activity.

Involvement of MAPK, Akt/GSK-3β and AMPK/mTOR signaling pathways in protection of remote glial cells from axotomy-induced necrosis and apoptosis in the isolated crayfish stretch receptor.

Severe mechanical nerve injury such as axotomy can lead to neuron degeneration and death of surrounding glial cells. We showed that axotomy not only mechanically injures glial cells at the cutting location, but also induces necrosis or apoptosis of satellite glial cells remote from the transection site. Therefore, axon integrity is necessary for survival of surrounding glial cells. We used the crayfish stretch receptor that consists of a single mechanoreceptor neuron enveloped by satellite glial cells as a simple, but informative model object in the study of the role of various signaling proteins in axotomy-induced death of remote glial cells. After axon transection, stretch receptors were isolated and incubated in saline in the presence or without specific inhibitors of various signaling proteins. Inhibition of MEK1/2, p38, Akt, GSK-3β and mTOR increased axotomy-induced apoptosis of remote glial cells, whereas inhibition of ERK1/2 and GSK-3β enhanced necrosis. This suggests the involvement of these signaling proteins in protective, antiapoptotic and antinecrotic processes in the remote satellite glia surrounding the axotomized mechanoreceptor neuron.

Epigenetic regulation of death of crayfish glial cells but not neurons induced by photodynamic impact

Epigenetic processes are involved in regulation of cell functions and survival, but their role in responses of neurons and glial cells to oxidative injury is insufficiently explored. Here, we studied the role of DNA methylation and histone deacetylation in reactions of neurons and surrounding glial cells to photodynamic treatment that induces oxidative stress and cell death. Isolated crayfish stretch receptor consisting of a single mechanoreceptor neuron surrounded by glial cells was photosensitized with aluminum phthalocyanine Photosens that induced neuron inactivation, necrosis of the neuron and glia, and glial apoptosis. Inhibitors of DNA methylation 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) reduced the level of PDT-induced necrosis of glial cells but not neurons by 1.3 and 2.0 times, respectively, and did not significantly influence apoptosis of glial cells. Histone deacetylase inhibitors valproic acid and trichostatin A inhibited PDT-induced both necrosis and apoptosis of satellite glial cells but not neurons by 1.6-2.7 times. Thus, in the crayfish stretch receptor DNA methylation and histone deacetylation are involved in epigenetic control of glial but not neuronal necrosis. Histone deacetylation also participates in glial apoptosis.

Elevated activity of the crayfish stretch receptor neuron increases resistance of surrounding glial cells to apoptosis induced by photodynamic treatment

Neuroglial interaction is very important for functioning and survival of nerve and glial cells. In the present work, we studied the influence of the intense neuronal activity on survival of the isolated crayfish stretch receptor neuron and surrounding glial cells subjected to photodynamic treatment, which induces intense oxidative stress. In the experimental group, neurons were stimulated by multiple extensions of the receptor muscle for 1 h so that the firing rate did not fall below 10–15 Hz, whereas in the control group, the receptor muscles were relaxed and neurons were silent. After stimulation, the preparations were photosensitized with alumophthalocyanine Photosens and irradiated by 670 nm laser diode. The isolated stretch receptors were stained with propidium iodide and Hoechst 33342, which reveal the nuclei of the necrotic and the apoptotic cells, respectively. The level of apoptosis of photosensitized glial cells was significantly lower in the experimental group compared to the resting control. Necrosis of neurons and glial cells was not significantly influenced. Therefore, elevated neuronal activity increased the resistance of the surrounding glial cells to photoinduced apoptosis. This could be attributed to the depletion of the energetic resources, which are transferred from glia into the neuron to support its firing, or to the neurotrophic neuron-to-glia signaling.

1017 Impact of sequence variants in CYP2C8 on paclitaxel clearance in ovarian cancer patients

Epigenetic processes are involved in regulation of cell functions and survival, but their role in responses of neurons and glial cells to oxidative injury is insufficiently explored. Here, we studied the role of DNA methylation and histone deacetylation in reactions of neurons and surrounding glial cells to photodynamic treatment that induces oxidative stress and cell death. Isolated crayfish stretch receptor consisting of a single mechanoreceptor neuron surrounded by glial cells was photosensitized with aluminum phthalocyanine Photosens that induced neuron inactivation, necrosis of the neuron and glia, and glial apoptosis. Inhibitors of DNA methylation 5-azacytidine and 5-aza-2′-deoxycytidine (decitabine) reduced the level of PDT-induced necrosis of glial cells but not neurons by 1.3 and 2.0 times, respectively, and did not significantly influence apoptosis of glial cells. Histone deacetylase inhibitors valproic acid and trichostatin A inhibited PDT-induced both necrosis and apoptosis of satellite glial cells but not neurons by 1.6–2.7 times. Thus, in the crayfish stretch receptor DNA methylation and histone deacetylation are involved in epigenetic control of glial but not neuronal necrosis. Histone deacetylation also participates in glial apoptosis.

Intracellular regulation of oxygen consumption in the isolated crayfish stretch receptor neuron

Morphological correlations of the functional regulation of oxygen consumption have been studied on single isolated crayfish mechanoreceptor neurons. An enhancement of oxygen consumption is promoted by the following: (1) redistribution of mitochondria and an increase in cytochrome oxidase (CO) activity in mitochondria near the plasmatic membrane, (2) coordination of mitochondria aggregation rhythms with pO2 rhythms in the medium external for a cell, (3) a decrease in the area of high CO activity and mitochondria and a shortening of the oxygen diffusion pathway, (4) an increase of the CO activity gradient from the neuron body periphery to its center, (5) a transfer of oxygen with the water flow during neuron body hydration and cytoplasm dilution during the transfer of a portion of the gel into sol, (6) cyclic changes in the ratio of the neuron body and hillock sizes at which there is a transfer of oxygen with the water flow into the neuron body, its mitochondrial uptake in the neuron body, and transfer of the oxygen-free water from the neuron body into the axonal hillock and further into the external medium.

Dynamics of ultrastructural changes in the isolated crayfish mechanoreceptor neuron under photodynamic impact

Photodynamic therapy (PDT) is used for treatment of cancer, including brain tumors. To explore the mechanism of photodynamic injury of neurons, we studied the PDT effect of 10(-7) M Photosens on ultrastructure of isolated crayfish stretch receptor neuron that was used as a model object. After a 5-min treatment that only slightly changed neuron activity, the initial injury (alteration of some mitochondria, vacuolization of the cytoplasm) was observed in parallel with compensatory changes (chromatin decondensation, elongation and aggregation of mitochondria, formation of lysosomes and autophagosomes). Longer photosensitization (30 min) abolished firing, destroyed mitochondria and Golgi apparatus, depleted energy sources (glycogen granules), and impaired granular endoplasmic reticulum and polysomes involved in protein synthesis. Therefore, mitochondria and Golgi apparatus were the primary targets for Photosens-mediated PDT in a single neuron. Their alteration might underlie functional shifts. These structural changes continued to develop after abolishment of neuronal activity and led to necrosis.

Crayfish mechanoreceptor neuron prevents photoinduced apoptosis of satellite glial cells

Interactions between neurons and glia play a key role in the development, functioning and survival of the nervous system. However, the influence of neurons on glial cells has received less attention than the role of glia in supporting neural functions. We here investigated the role of isolated crayfish stretch receptor neuron in the death of satellite glial cells under photodynamic impact. After staining with aluminum phthalocyanine photosens, the neuronal cell body was locally irradiated with a focused beam of He–Ne (633 nm, 200 W/cm 2) or semiconductor laser (650 nm, 50 W/cm 2). This rapidly abolished neuronal activity. The whole preparation was then subjected to total laser irradiation with lower intensity (633 nm, 0.3 W/cm 2), which induced death of glial cells. Double staining of the preparation with propidium iodide and Hoechst 33342 in the following 6–7 h allowed the visualization of necrotic, apoptotic and alive cells. Previous neuron inactivation with the focused laser beam was found to increase photodynamically-induced apoptosis but not necrosis of satellite glial cells enwrapping the axon. Therefore, the intact neuronal cell body protected satellite glial cells against photoinduced apoptosis. Altogether the data indicate that mechanoreceptor neurons release some signaling molecules involved in the prevention of glial apoptosis. This may provide integrity of the stretch receptor organ and its resistance to injurious factors.

PDT‐induced death of sensory neurons and glial cells in the isolated crayfish stretch receptor after proteolytic treatment

To study the involvement of neuroglial interactions in photodynamic damage of crayfish stretch receptor, which consists of only two neurons surrounded by satellite glial cells (SGCs), we attempted to proteolytically uncouple neurons and glia and then compare the responses of these cells to photosensitization when intercellular communications were intact or impaired. After incubation of isolated stretch receptors with pronase or collagenase they were photosensitized with Photosens, a mixture of sulfonated alumophthalocyanines AlPcS(n) (n = 2, 3, and 4; mean n = 3.1). In the next 6 hr the preparations were double fluorochromed with propidium iodide and Hoechst-33342 to visualize necrotic and apoptotic cells. Proteolytic treatment shortened bioelectric neuron response and precipitated its functional inactivation; however, it did not significantly impair neuron morphology and did not induce its necrosis either in the darkness or under photosensitization. Photodynamic treatment induced necrosis of neurons and SGC and apoptosis of glial cells. Pronase but not collagenase increased percent of necrotic and apoptotic SGCs in the darkness and thus reduced the number of glial cells around the neuron; however, both pronase and collagenase prevented photodynamically induced apoptosis of glial cells. The involvement of neuron-to-glia signaling interactions in this phenomenon is suggested.

PDT effect of different photo sensitizers on a single nerve cell: electrophysiological and pharmacological study

The study of the single neuron response to photodynamic effect provides information on the dynamics of cytotoxic events leading to cell death and allows comparison of the phototoxicity of different photosensitizers. Isolated crayfish stretch receptor neurons were incubated 30 min with different concentrations of various photosensitizers and then irradiated with a He-Ne laser (632.8 nm; 0.3 W/cm/sup 2/) until irreversible firing abolition. The dynamics of neuron impulse response was continuously recorded throughout the experiment. The following photosensitizers were studied: hematoporphyrin derivatives Photoheme and Photofrin II, 6 deuteroporphyrin IX derivatives and sulphonated aluminum and zinc phthalocyanines. Nerve cells were found to be insensitive to either He-Ne laser irradiation or photosensitization alone, but very vulnerable to photodynamic effect: neurons changed their firing rate and irreversibly ceased their impulse activity at nanomolar (phthalocyanines and hematoporphyrin derivatives) and even pikomolar (deuteroporphyrin derivatives) concentrations of photosensitizers. The dynamics of the neuron responses to photodynamic effects included stages of firing activation and/or inhibition followed by irreversible firing abolition. It depended on the photosensitizer type and concentration. Decrease of extracellular pH or action of pharmacological agents increasing intracellular Ca/sup 2+/ concentration or inhibiting ATP synthesis exacerbated photodynamic neuron injury. In contrast, agents decreasing intracellular calcium concentration or bioenergetic substrates protected the neuron against photodynamic injury. The dependencies of neuron lifetimes on the photosensitizer concentration provide comparison of photodynamic efficiencies of different photosensitizers.

Photodynamic Inactivation of the Single Crayfish Nerve Cell: Dynamics of Electrophysiological Responses and Comparison of Photosensitisers

. The study of single neuron response to photodynamic effect provides a means for the study of the dynamics of cytotoxic events leading to cell death and allows comparison of the phototoxicity of different photosensitisers. Isolated crayfish stretch receptor neurons were photosensitised for 30 min, then irradiated with a He-Ne laser (632.8 nm; 0.3 W/cm2) until irreversible firing cessation. The dynamics of neuron firing frequency were continuously recorded throughout. The following photosensitisers were studied: methylene blue, janus green B, protoporphyrin IX, chlorins e 6 and p 6, haematoporphyrin derivative (Photoheme) and sulphonated aluminium phthalocyanine (Photosens). Nerve cells were found to be insensitive to either He-Ne laser irradiation or photosensitisation alone, but very vulnerable to the photodynamic effect: neurons changed firing rate and died at nanomolar concentrations of photosensitisers. The dynamics of neuron responses was found to depend on photosensitiser type and concentration. The current approach provides a means of evaluation of initial threshold cell membrane alteration and cytotoxic events leading to cell death. The dependence of firing acceleration rate and neuron lifetime on photosensitiser concentration additionally allowed comparison of efficiencies of different photosensitisers. Photosens, Photoheme and chlorin p 6 were found to be the most potent photosensitisers: neurons responded to their photodynamic effects at concentrations as low as 1–5 nM.

Effect of Weak Extremely Low Frequency Magnetic Field on Isolated Crayfish Stretch Receptor Neuron: Nonlinear Dependence on Field Amplitude and Frequency

Responses of single mechanoreceptor crayfish neurons to weak extremely low frequency magnetic fields were studied over a wide range of frequencies from 0.001 to 100 Hz, and magnetic fields from 1 to 400 fiT. Observed shifts of neuron impulse activity were weak and variable. They were usually characterized by a slow increase or decrease of spike frequency developing during tens of minutes with markedly variable latencies. Frequency and amplitude “action spectra” were complex, nonlinear, and included several bands (frequency and amplitude “windows”). Neuron response probabilities (if response type and field amplitudes were excluded from consideration) were maximal at 0.001, 0.3, 3, and 60 Hz. They were also maximal at 5, 20, 50, and 300 jaT if field frequencies were not taken into account. Minimal neuron response probabilities were observed at 0.03, 0.8-2, 11-15, and 100 Hz and 1, 10, 30, and 100 /iT, respectively.

Transients in the inhibitory driving of neurons and their postsynaptic consequences

The presynaptic fiber at an inhibitory synapse on a pacemaker neuron was forced to generate transients, defined here as spike trains with a trend, unceasingly accelerating or slowing. Experiments were on isolated crayfish stretch receptor organs. Spike train analyses used tools and notions from conventional point processes and from non-linear dynamics. Pre- and postsynaptic discharges contrasted clearly in terms of rates and interspike intervals. The inhibitory train evolved monotonically and smoothly, following tightly the simple prescribed curves; it was uniform, exhibiting throughout a single and simple discharge form (i.e. interval patterning). The inhibited postsynaptic train alternately accelerated and slowed, not following tightly any simple curve; it was heterogeneous, exhibiting in succession several different and often complex discharge forms, and switching abruptly from one to another. The inhibited trains depended on the inhibitory transient's span, range and average slope. Accordingly, transients separated (not cuttingly) into categories with prolonged spans (over 1 s) and slow slopes (around 1/s 2) and those with short spans (under 1 s) and fast slopes (around 30/s 2). Special transients elicited postsynaptic discharges that reproduced it faithfully, e.g. accelerated with the transient and proportionately; no transient elicited postsynaptic discharges faithful to its mirror image. Crayfish synapses are prototypes, so these findings should be expected in any other junction, as working hypotheses at least. Implications involve the operation of neural networks, including the role of distortions and their compensation, and the underlying mechanisms. Transients have received little attention, most work on synaptic coding concentrating on stationary discharges. Transients are inherent to the changing situations that pervade everyday life, however, and their biological importance is self-evident. The different discharges encountered during a transient had strong similarities to the stationary forms reported for different pacemaker drivings that are called locking, intermittency, erratic and stammering; they were, in fact, trendy versions of these. Such forms appear with several synaptic drivings in the same order along the presynaptic rate scale; they may constitute basic building blocks for synaptic operation. In terms of non-linear science, it is as if the attractors postulated for stationary drivings remained strongly influential during the transients, though affected by the rate of change.

GABA: history and perspectives

In 1957, factor I, a brain agent I had discovered earlier, was chemically identified as GABA in a collaboration between myself and Alva Bazemore at the Montréal Neurological Institute (MNI) in the Neurochemistry Laboratory then headed by K. A. C. Elliott. A personally biased excursion into the history of neurobiology illuminates the development of methods and concepts that led to this event, and recounts the early days at the MNI, when Hugh McLennan and I applied factor I to the exposed surface of the spinal cord and to sympathetic ganglia of cats and rabbits. It also tells of earlier studies at Graz, Naples, and elsewhere that prompted the experiments at the California Institute of Technology in which factor I was discovered as the agent in nerve extracts causing inhibition of isolated crayfish stretch receptor neurons, and in which it was found that this inhibition could be prevented by picrotoxin. There was justified doubt that GABA is indeed the transmitter substance of inhibitory neurones. Later studies, however, resolved the controversy. The functional role of GABA in brain and spinal cord and its mechanism of action are still far from being fully understood. Special problems are the extent and significance of spontaneous quantal and nonquantal release, the functional role and the mechanism of excitatory actions of GABA, its release from glial cells, and the energetics of its metabolic turnover.

Phenylpyrazoles, a new class of pesticides: An electrophysiological investigation into basic effects

Phenylpyrazoles constitute a newly developed class of pesticides that may exert direct excitatory effects on the nervous system. Basic electrophysiological effects were investigated in vitro in the sciatic nerve and the lateral-line sense organ of the clawed frog, Xenopus laevis, by recording nerve activity extracellularly. In the slowly adapting stretch receptor organ of the crayfish, Astacus sp., effects on the membrane potential of the neurone were recorded using intracellular microelectrodes. Both in the sciatic nerve and in the lateral-line sense organ the main effect of phenylpyrazoles was to decrease the survival time of the isolated preparation. In the isolated crayfish stretch receptor organ a similar decrease of the survival time was accompanied by a depolarization of the stretch receptor neurone membrane. The depolarizing effect of the phenylpyrazoles is not caused by a modification of the tetrodotoxin-sensitive voltage-dependent Na + channels, nor by an effect on the picrotoxin-sensitive neurotransmission mediated by γ-aminobutyric acid. The phenylpyrazoles neither act by inhibiting the electrogenic Na/K pump. Although the cause of the phenylpyrazole-induced membrane depolarization remains to be elucidated, it is concluded that the underlying mechanism is distinct from that of the pyrethroid insecticides.

Anesthetics produce differential actions on membrane responses of the crayfish stretch receptor neuron

The actions of ethanol, halothane and pentobarbital on the membrane electrical properties and synaptic transmission of isolated crayfish stretch receptor neurons were studied to determine possible sites of action contributing to differential effects previously described on physiological discharge activity. The three agents depressed GABA-mediated transmission and altered postsynaptic membrane electrical properties. Both pre- and postsynaptic sites of action appeared to contribute to the anesthetic-induced alteration of neuronal function. The agents studied produced different, concentration-dependent, membrane effects which included biphasic actions on membrane resistance and spike threshold. The results suggest that multiple-sites of action are involved and different anesthetics may not act via the same mechanism(s) at these sites.

An in vitro study of copper and copper chelators on the crayfish stretch receptor

This study demonstrates the effects of micromolar concentrations of copper on the discharge firing activity of the isolated crayfish stretch receptor. Different pharmacological profiles of action were obtained with the divalent (cupric) and monovalent (cuprous) salt solutions. Cupric solutions produced a concentration-dependent triphasic effect (depression-excitation-depression); in contrast, a monophasic effect (depression) was produced by cuprous solutions. The effectiveness of copper chelators, in reversing these effects, were compared with normal physiological solution. Only the first phase of the effects of cupric were partially reversed with physiological solution or ethylenediamine tetraammonium (EDTA). In the presence of d-penicillamine and triethylene tetramine (trien), all phases of the effects of both cuprous and cupric salts were completely restored to control values. In addition, copper was shown to be the most neurotoxic divalent cation examined. The results demonstrate that effects of copper on neuronal activity are dependent on time, concentration and valency state.

Ammonium action on post-synaptic inhibition in crayfish neurones: implications for the mechanism of chloride extrusion.

1. The reversal potential of the Cl(-)-dependent, inhibitory post-synaptic potential (E(i.p.s.p.)) was measured in the isolated crayfish stretch receptor neurone using two intracellular micro-electrodes. The difference between E(i.p.s.p.) and the resting membrane potential (E(m)), the i.p.s.p. driving force, was reversibly decreased by addition of NH(3)/NH(4) (+), and the mechanism of this decrease was investigated.2. The NH(3)/NH(4) (+)-induced decrease in i.p.s.p. driving force was dose-dependent with an onset at about 0.2 mM. E(i.p.s.p.) always remained more negative than E(m) or, when the neurone was spontaneously firing, the threshold potential. E(m) and resting membrane resistance (R(m)) also decreased in a dose-dependent fashion. Synaptic conductance (g(s)) increased with low doses, but decreased on application of 20 mM-NH(3)/NH(4) (+). All the effects were fully reversible on return to normal Ringer solution.3. Intracellular acidification (substitution of 50% Cl(-) by acetate compared with isethionate) considerably reduced the i.p.s.p. driving force. Simultaneous application of NH(3)/NH(4) (+) and acetate-substituted Ringer solution caused a similar decrease in the driving force to application of the same concentration of NH(3)/NH(4) (+) under normal conditions. Increasing the extracellular pH at which a given concentration of NH(3)/NH(4) (+) was applied caused a smaller decline in the i.p.s.p. driving force. These results suggest that intracellular acidification decreases the i.p.s.p. driving force and that the NH(3)/NH(4) (+)-induced decline is caused by an action of the ammonium ion.4. Elevation of extracellular K(+) (K(+) (0)) decreased the i.p.s.p. driving force, E(m) and R(m), and increased g(s). Reduction of K(+) (0) had the converse effects on all parameters.5. Application of Rb(+) or Cs(+) mimicked the effects of NH(3)/NH(4) (+). Substitution of K(+) (0) by Rb(+), Cs(+) or NH(3)/NH(4) (+) opposed or even reversed the increase in i.p.s.p. driving force when Na(+) was used as the substitute. The effectiveness of the various cations in decreasing the driving force was in the following order: Rb(+) > NH(4) (+) > K(+) > Cs(+).6. Inhibition of the Na pump by ouabain or K(+)-free Ringer solution caused a gradual reduction in the i.p.s.p. driving force. Since the driving force also decreased when the Na(+) gradient probably was increased (elevated K(+) (0)), this suggests a dependence on the K(+) gradient rather than the Na(+) gradient or the Na pump itself.7. Frusemide (6 x 10(-4) M) reversibly decreased the i.p.s.p. driving force and E(m), and increased g(s). R(m) was not significantly affected. Application of frusemide in the presence of 5 mM-Rb(+) and vice versa, caused a further reduction in the driving force. The recovery of the driving force on removal of either agent was slowed by the presence of the other.8. Application of 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS; 10(-4) M) caused spontaneous firing and reduced E(i.p.s.p.) to the threshold potential. R(m) and g(s) increased. The effects were slowly reversible on removal of the drug.9. It is proposed that the i.p.s.p. driving force is maintained by a K(+)-Cl(-) co-transport mechanism, driven by the K(+) gradient. The K(+) site exhibits the binding selectivity: Rb(+) > NH(4) (+) > K(+) > Cs(+) and the mechanism is inhibited partially by frusemide and completely by DIDS.