Crayfish Stretch Receptor Neuron Research Articles

Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10mM NiCl2 and 10-500µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

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.

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.

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.

Characterization of a delayed rectifier potassium channel in the slowly adapting stretch receptor neuron of crayfish

Single channel recordings were performed on enzyme-cleaned slowly adapting sensory neurons of crayfish, in cell-attached configuration, with a physiological K + gradient across the neuronal membrane. An outward rectifying, voltage-gated K + channel with a slope conductance of 13 pS and a K + ion permeability of P K=6.5×10 −14 cm 3/s was characterized. This 13 pS K + channel started to be activated at around 20 mV depolarization. Its open probability increased upon depolarization with V 0.5=−25.3 mV and P max=0.83. The averaged currents showed a delay following the onset of depolarization. The activation time constant was voltage-dependent. The maximal value was 17.0 ms at −25 mV and at +35 mV the time constant was 1.7 ms. Little inactivation was observed throughout the 80- or 1500-ms long depolarization pulses. A sum of two exponentials provided the optimal fit for open time and closed time distribution. At 80-mV depolarization, the open time constants were 0.4 and 10.4 ms; the close time constants were 0.4 and 2.3 ms. The first-latency distribution suggested that at least two closed states preceded two open states. This 13 pS delayed rectifier plays a minor role in the maintenance of the resting membrane potential but contributes to the action potential repolarization. It may also modify the stretch-induced receptor potential and affect the adaptation behaviours in this neuron.

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.

The mechanotransduction of the crayfish stretch receptor neurone can be differentially activated or inactivated by local anaesthetics.

The effect of the local anaesthetics lidocaine, its meta-isomer, LL33, bupivacaine, tetracaine and procaine on the transducer properties of the stretch receptor neurone of the crayfish Pacifastacus leniusculus was investigated using a two microelectrode voltage clamp. Lidocaine increased the receptor current whereas LL33, bupivacaine and tetracaine reduced the receptor current in a reversible dose-dependent way. Procaine did not affect the receptor responses. The onset of the effect was generally slow in the order of minutes. Lidocaine increased the conductance of the mechanotransducer 50 +/- 7% (mean +/- SD, n = 4) and changed the reversal potential -8 +/- 1 mV (mean +/- SEM, n = 8), which indicates a major K+ conductance increase through the mechanosensitive channels. The other local anaesthetics increase the K+ conductance of the mechanotransducer without increasing the total conductance, which suggests that only P(Na)/P(K) is changed. These substances seem to have a Ca2+ dependent effect on the gating properties of the mechanosensitive channels in addition to their effect on the permeability through the channels as compared with lidocaine. All local anaesthetics investigated decreased the leak conductance of the receptor neurone. The effects of local anaesthetics on the mechanosensitive channels whether activating or blocking is correlated to the oil:water distribution coefficients and their relative hydrophobicity/hydrophilicity ratio. The results are consistent with the hypothesis that the local anaesthetic effect is mediated by changes in the lipid phase of the membrane.

Single neuron response to pulse-periodic laser microirradiation. Action spectra and two-photon effect

Cytoplasm regions of crayfish stretch receptor neurons were irradiated by the microbeam of a pulse-periodic dye laser (diameter 5 μm, pulse duration 15 nsec, pulse frequency 400 Hz). Irradiation wavelengths varied from 434 to 600 nm, and beam intensity (at 441.6 nm) from 0.25 to 5 × 10 3 W cm −2 (mean), or from 0.4 to 8 × 10 8 W cm −2 (peak). Under irradiation neuron firing typically accelerated and then inhibited. In some cases it accelerated again and then abruptly and irreversibly ceased. Action spectra of parameters describing different response phases had the flavin-like maximum at 460 nm. This suggests flavins as a primary intracellular photoreceptor for blue laser light. It was also found that firing acceleration rate quadratically depended on peak beam intensity. This finding indicates the participation of two-photon processes in neuronal membrane alteration leading to firing acceleration. Parameters describing inhibition phase depended on beam intensity linearly. That is characteristic for usual single-photon process.

Local anaesthetics potentiate GABA-mediated Cl- currents by inhibiting GABA uptake.

A two-electrode voltage clamp was used to examine the effects of the local anaesthetics (LAs) lidocaine, tetracaine and bupivacaine, and the meta-isomer of lidocaine (LL33), on the gamma-aminobutyric acid (GABA)-gated Cl- channel in the crayfish stretch receptor neurone. The voltage-induced current changes were recorded while exposing the neurone to GABA in saline containing different LAs. All LAs enhanced the voltage-induced Cl- current equivalent to a shift of the conductance vs GABA curve towards lower concentrations. The mechanism of the facilitation of GABA-induced conductance was found to be mainly a block of the GABA uptake in the stretch receptor neurone. Tetracaine, and to a lesser extent LL33, also seem to affect directly the GABA/receptor Cl-/channel complex resulting in an increased conductance of the channel.

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.

Extracellular Ca2+ and its effect on acid extrusion in the crayfish stretch receptor neurone

1. In the stretch receptor neurones of the crayfish Astacus astacus, the intracellular pH (pHi), the intracellular Na+ concentration ([Na+]i) and the membrane potential (Em) were measured simultaneously using ion-selective and conventional microelectrodes. Normal Astacus saline (NAS), and salines containing varying amounts of Ca2+ (Ca2+-NAS) but of constant ionic strength, with Na+, Mg2+ or Ba2+ as substituting ions, were used to investigate the effects of extracellular Ca2+ concentration ([Ca2+]o) on pHi and pHi regulation, on [Na+]i and on Em. The maximum rate of pHi recovery was used as a measure of pHi regulation. Acid loads were imposed using the NH4+/NH3 rebound technique. 2. [Ca2+]o affected pHi, pHi regulation, [Na+]i and Em. The magnitudes of the effects were inversely related to [Ca2+]o and were specific to the ion used for [Ca2+]o substitution. 3. Compared with controls, increasing [Ca2+]o threefold (in exchange for Na+) elicited some alkalization, a 7 % faster maximum rate of pHi recovery and generally lower values of [Na+]i. 4. In low-Ca2+ or Ca2+-free NAS (substitutions by Na+ or Mg2+), pHi became more acid, the maximum rate of pHi recovery was reduced by up to 50 % and [Na+]i was generally higher. The effects were faster and larger at lower [Ca2+]o, and stronger with Na+ than with Mg2+ as the substituting ion. 5. In Ca2+-free NAS (Ca2+ substituted for by Ba2+), the effects on pHi, on the maximum rate of pHi recovery and on [Na+]i were generally small. In this respect, Ba2+ had similar physiological properties to Ca2+ and was almost equally effective. 6. Changes in Em, including rapid depolarizations and occasional burst activity in Ca2+-free NAS, indicate that alterations in the properties of the membrane, such as a change in its permeability or selectivity, are occurring. Measurements of [Na+]i support this view. In addition, Ba2+ per se induced a (small) depolarization, as shown when Ba2+ was present in NAS or in low-Ca2+ NAS. 7. Changes in [Ca2+]o affected [Na+]i. *[Na+]i is defined as [Na+]i determined at the onset of the maximum rate of pHi recovery, and the ratio *[Na+]i/[Na+]o at that instant was calculated. A linear relationship between the maximum rate of pHi recovery and the *[Na+]i/[Na+]o ratio was found, irrespective of the amount and of the ion species used for [Ca2+]o substitution. This is strong evidence that pHi and pHi regulation were indirectly affected by [Ca2+]o, which altered membrane properties and thus caused a change in [Na+]i. We could find no evidence for a direct contribution of [Ca2+]o to acid extrusion or to a direct modulatory action on the transport protein of the Na+/H+ antiporter.

A light emitting diode microspectrophotometer: intracellular Ca 2+ measurements in isolated stretch receptor

A microspectrophotometer was designed to measure absorbance changes in single cells. The device utilizes sequentially activated light emitting diodes (LED) to provide different wave lengths of light. The instrument has the advantage of relative simplicity and less cost compared to other devices. The spectrophotometer was tested by measuring absorbance changes of the metallochromic Ca2+ indicator Arsenazo III (AIII) injected into the crayfish (Astacus astacus) stretch receptor. Under the conditions described the detection limit of the concentration of AIII was 0.05 mM and absorbance changes of 0.0005 can be reliably determined which correspond to a detection limit of 10-20 nM for free Ca2+ changes assuming a light path length of 0.003 cm and an apparent dissociation constant (KD) of 2 microM for the Ca(2+)-AIII complex. The upper frequency limit of the device is 3000 Hz. The absorbance measurements of AIII injected into the crayfish stretch receptor neurons revealed a Ca(i) of 375 +/- 177 nM (mean +/- SD: 14 cells). LiCl substituted for NaCl increased Ca(i) 45-100 nM in different cells, suggesting that a Na+ gradient is necessary for Ca2+ homeostasis in this receptor.

Contribution of the Na+/H+ Antiporter to the Regulation of Intracellular pH in a Crayfish Stretch Receptor Neurone

ABSTRACT Regulation of intracellular pH (pHi) following acidosis induced by NH4+/NH3 exposures was re-investigated in a crayfish stretch receptor neurone using H+- and Na+-selective microelectrodes. All experiments were performed in nominally HCO3−/CO2 -free salines. From studies in Na+-free saline and from electrochemical calculations, we concluded that pHi regulation was dependent on extracellular Na+ concentration ([Na+]o ). The half-maximal rate of pHi recovery had an apparent Michaelis–Menten constant of 21mmol l− 1 [Na+]o. The use of this experimental approach and an improved technique enabled us to observe pHi regulation even in Cl− -free saline, in contrast to earlier findings. In addition, amiloride (2mmol l− 1) inhibited the maximum rate of pHi recovery by about 80%, SITS (1mmol l− 1) by about 20%. The results strongly suggest the operation of two separate pHi-regulating mechanisms, a Na+-dependent HCO3− /Cl− antiporter (probably the Na+/H+/HCO3− /Cl− antiporter described earlier) and a Na+/H+ antiporter. Both mechanisms have been described in crayfish ganglion cells and muscle fibres, but the individual contribution to pHi regulation varies considerably in these preparations. Functional aspects of the pHi-regulating mechanisms in relation to ionic changes during the moulting cycle are discussed.

Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine, tetraethylammonium chloride and some other chemical substances.

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (Kd) of 2.3 +/- 0.2 mM (mean +/- SEM) in the slowly and 1.4 +/- 0.2 mM in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1:1 stoichiometry and the apparent Kd for TEA was 9.6 +/- 3.4 mM with a cooperativity factor n = 0.43 +/- 0.03 in the slowly adapting SRN and 34.5 +/- 9.2 mM and 0.37 +/- 0.03 respectively in the rapidly adapting SRN. Low Ca2+, apamin and charybdotoxin, which are known to block Ca(2+)-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (Kd = 0.8-1.6 mM) for TEA and the other a low affinity (Kd = 173-213 mM) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca(2+)-dependent K+ current in the SRNs.

Inward current caused by sodium-dependent uptake of GABA in the crayfish stretch receptor neurone.

A two-microelectrode current-voltage clamp and Cl(-)-selective microelectrodes were used to examine the effects of gamma-aminobutyric acid (GABA) on membrane potential, current and intracellular Cl- activity (aiCl) in the crayfish stretch receptor neurone. All experimental solutions were CO2-HCO3- free. 2. GABA (500 microM) produced a mono- or biphasic depolarization (amplitude < or = 10 mV), often with a prominent initial depolarizing component followed by a transient shift to a more negative level. In some neurones, an additional depolarizing phase was seen upon washout of GABA. Receptor desensitization, being absent, played no role in the multiphasic actions of GABA. 3. The pronounced increase in membrane conductance evoked by GABA (500 microM) was associated with an increase in aiCl which indicates that the depolarizing action was not due to a current carried by Cl- ions. 4. The currents activated by GABA under voltage clamp conditions were inwardly directed when recorded at the level of the resting membrane potential, and they often revealed a biphasic character. The reversal potential of peak currents activated by pulses of 500 microM-GABA (EGABA) was 9-12 mV more positive than the reversal potential of the simultaneously measured net Cl- flux (ECl). ECl was 2-7 mV more negative than the resting membrane potential. 5. EGABA (measured using pulses of 500 microM-GABA) was about 10 mV more positive than the reversal potential of the current activated by 500 microM-muscimol, a GABA agonist that is a poor substrate of the Na(+)-dependent GABA uptake system. 6. In the absence of Na+, the depolarization and inward current caused by 500 microM-GABA were converted to a hyperpolarization and to an outward current. Muscimol produced an immediate outward current both in the presence and absence of Na+. 7. Following block of the inhibitory channels by picrotoxin (100-200 microM), the depolarizing effect of 500 microM-GABA was enhanced and the transient hyperpolarizing shifts were abolished. 8. In the presence of picrotoxin, GABA (> or = 2 microM) produced a concentration-dependent monophasic inward current which had a reversal potential of +30 to +60 mV. This current was inhibited in the absence of Na+ and by the GABA uptake blocker, nipecotic acid. Unlike the channel-mediated current, the picrotoxin-insensitive current was activated without delay also at low (2-10 microM) concentrations of GABA.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Effect of γ-Aminobutyric Acid on Intracellular pH in the Crayfish Stretch-Receptor Neurone

The effect of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) was examined in the crayfish stretch-receptor neurone using H(+)-selective microelectrodes and a two-microelectrode voltage clamp. In the presence of 30 mmol l-1 HCO3- (pH 7.4), application of GABA (0.5 mmol l-1) produced a mean fall in pHi of 0.26 units. The initial rate of fall of pHi was attributable to a net influx of acid equivalents of 6.3 mmol l-1 min-1. In the nominal absence of HCO3-, GABA had little effect on pHi. The HCO3(-)-dependent acidosis caused by GABA was inhibited by picrotoxin (0.1 mmol l-1) but not by depletion of extracellular and intracellular Cl-. Acetazolamide (0.1 mmol l-1) decreased the rate of fall of pHi caused by a step increase in CO2 partial pressure as well as by GABA, which indicates that the neurone contains carbonic anhydrase. In the presence of both Cl- and HCO3-, the reversal potential of the GABA-activated current was more positive than under nominally HCO3(-)-free conditions. In line with this, GABA induced a marked HCO3(-)-dependent depolarization, and this depolarizing action was enhanced in the absence of Cl- so as to lead to triggering of action potentials. All these observations support the conclusion that the GABA-induced fall in pHi is due to a net efflux of HCO3- through the inhibitory anion channels.