Neurosteroid-induced enhancement of glutamate transmission in rat hippocampal slices.

Pregnenolone sulfate, one of the most abundantly produced neurosteroids in the hippocampus, has well characterized effects at postsynaptic receptors including the N-methyl-D-asparate type of glutamate receptor. Little is known, however, about the mechanism of action of neurosteroids on the release of glutamate. In this study we describe a robust effect of pregnenolone sulfate at glutamatergic synapses in the CA1 region of the hippocampus. In particular, we found that pregnenolone sulfate enhances paired-pulse facilitation of EPSPs at the two major classes of ionotropic glutamate receptors with an EC(50)<1 microM. Thus, we propose a novel mechanism of action of neurosteroids in hippocampal neurons that involves the modulation of glutamate release.

Block of hippocampal CAN channels by flufenamate.

Ca(2+)-activated non-selective cation (CAN) channels are activated by cytoplasmic Ca(2+) and I(CAN) underlies many slow depolarizing processes in neurons including a putative role in excitotoxicity. CAN channels in many non-neuronal cells are blocked by non-steroidal antiinflammatory drugs that are derivatives of diphenylamine-2-carboxylate (DPC). The DPC derivative flufenamate (FFA) has a complex effect on certain neurons, whereby it blocks CAN channels and increases [Ca(2+)](i). We report here that FFA, but not the parent compound, DPC, blocks CAN channels in hippocampal CA1 neurons. As was the case in other neurons, the effects of FFA are complex and include a maintained rise in [Ca(2+)](i). Furthermore, the CAN channel blocking ability of FFA persists even when the channels have been potentiated by a Ca(2+)-dependent process. The use of a CAN channel-blocking drug is important for delineating CAN channel-dependent processes and may provide a basis for therapy for CAN channel-dependent events in ischemia.

Coupling of AMPA receptors with the Na(+)/Ca(2+) exchanger in cultured rat astrocytes.

Astrocytes exhibit three transmembrane Ca(2+) influx pathways: voltage-gated Ca(2+) channels (VGCCs), the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na(+)/Ca(2+) exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca(2+) concentration ([Ca(2+)](i)); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca(2+)](i) is not known. We demonstrate here that 100 microM AMPA in the presence of 100 microM cyclothiazide (CTZ) causes an increase in [Ca(2+)](i) in cultured cerebral astrocytes that requires transmembrane Ca(2+) influx. This increase of [Ca(2+)](i) is blocked by 100 microM benzamil or 0.5 microM U-73122, which inhibit reverse-mode operation of the Na(+)/Ca(2+) exchanger by independent mechanisms. This response does not require Ca(2+) influx through VGCCs, nor does it depend upon a significant Ca(2+) influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca(2+) stores, although depletion of these Ca(2+) stores does not decrease the peak [Ca(2+)](i) response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca(2+)](i) to increase through the reverse mode operation of the Na(+)/Ca(2+) exchanger with an associated release of Ca(2+) from intracellular stores. This proposed mechanism requires neither Ca(2+)-permeant AMPARs nor the activation of VGCCs to be effective.

Ca2+ store-dependent potentiation of Ca2+-activated non-selective cation channels in rat hippocampal neurones in vitro.

1. Potentiation of calcium-activated non-selective cation (CAN) channels was studied in rat hippocampal neurones. CAN channels were activated by IP3-dependent Ca2+ release following metabotropic glutamate receptor (mGluR) stimulation either by Schaffer collateral input to CA1 neurones in brain slices in which ionotropic glutamate and GABAA receptors, K+ channels, and the Na+-Ca2+ exchanger were blocked or by application of the mGluR antagonist ACPD in cultured hippocampal neurones. 2. The CAN channel-dependent depolarization (DeltaVCAN) was potentiated when [Ca2+]i was increased in neurones impaled with Ca2+-containing microelectrodes. 3. Fura-2 measurements revealed a biphasic increase in [Ca2+]i when 200 microM ACPD was bath applied to cultured hippocampal neurones. This increase was greatly attenuated in the presence of Cd2+. 4. Thapsigargin (1 microM) caused marked potentiation of DeltaVCAN in CA1 neurones in the slices and of the CAN current (ICAN) measured in whole cell-clamped cultured hippocampal neurones. 5. Ryanodine (20 microM) also led to a potentiation of DeltaVCAN while neurones pretreated with 100 microM dantrolene failed to show potentiation of DeltaVCAN when impaled with Ca2+-containing microelectrodes. 6. The mitochondrial oxidative phosphorylation uncoupler carbonyl cyanide m-chlorophenyl hydrazone (2 microM) also caused a potentiation of DeltaVCAN. 7. CAN channels are subject to considerable potentiation following an increase in [Ca2+]i due to Ca2+ release from IP3-sensitive, Ca2+-sensitive, or mitochondrial Ca2+ stores. This ICAN potentiation may play a crucial role in the 'amplification' phase of excitotoxicity.

Hydroxylamine blocks pre- but not postsynaptic adenosine A(1) receptor-mediated actions in rat hippocampus.

The commonly used nitric oxide donor, hydroxylamine (NH(2)OH), can block or reverse the inhibition of glutamatergic transmission by adenosine or an adenosine A(1) agonist in rat hippocampal slice. In these experiments, hydroxylamine did not affect the adenosine A(1) receptor-mediated depression of postsynaptic excitability. We conclude that hydroxylamine acts presynaptically to counter adenosine A(1) receptor-mediated inhibition of synaptic transmission.

Hydroxylamine blocks adenosine A1 receptor-mediated inhibition of synaptic transmission in rat hippocampus.

The nitric oxide donor hydroxylamine (NH2OH) induced a transient depression of the evoked synaptic potential recorded in the rat hippocampal CA1 region. This depression was abolished with an adenosine A1 antagonist, 8-cyclopentyltheophylline. In addition, hydroxylamine reversed adenosine A1 receptor-mediated inhibition of the evoked population spike, the fEPSP and the intracellularly recorded EPSP. The inhibitory modulation of adenosine A1 receptor activation by hydroxylamine suggests the presence of a potent endogenous regulatory site.

Dissociated dopaminergic neurons from substantia nigra zona compacta in young rats lack functional NMDA receptors.

Glutamate-mediated excitotoxicity plays an important role in the degeneration of nigrostriatal dopamine (DA) neurons induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), although the role of the N-methyl D-aspartate (NMDA) receptor subtype in this process is still uncertain. We studied glutamate receptor subtype agonist-induced ionic currents in acutely dissociated DAergic neurons from the rat substantia nigra zona compacta (SNc) using the nystatin-perforated patch-clamp whole-cell recording technique. The results fall into four main categories. First, single neurons, freshly isolated from SNc, exhibited a large soma and multipolar morphology, responded to DA, and stained positively for tyrosine hydroxylase (TH). Second, rapid application of L-glutamate (> 10(-5) M) induced an inward current with minimal desensitization at a clamp voltage of -60 mV. Third, kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-isoxazole (AMPA) induced an inward current that was similar to the glutamate-induced current while, in the same neuron, NMDA (10(-4) M) failed to induce any current response in Mg2+-free solution that contained 10(-5) M glycine at a clamp voltage of -60 mV. Under the same experimental conditions, NMDA induced a clear current response in isolated substantia nigra reticulata (SNr) neurons. Fourth, the specific NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV, 10(-4) M) failed to block 10(-4) M glutamate-induced inward current, while the specific KA/AMPA receptor antagonist 6-cyano-7-nitroguinoxaline-2, 3-dione (CNQX, 10(-5) M) completely blocked the glutamate-induced current. These results indicate that in single SNc DAergic neurons of 2-week-old rats, L-glutamate-induced inward current is mediated by non-NMDA receptors rather than by NMDA receptors.

Mechanism of action of the non-steroidal anti-inflammatory drug flufenamate on [Ca2+]i and Ca(2+)-activated currents in neurons.

We have shown previously that the non-steroidal anti-inflammatory drug flufenamate (FFA) causes a maintained increase in [Ca2+]i and transient increases in a Ca(2+)-activated nonselective cation current (ICAN) and a Ca(2+)-activated slow, outward Cl- current (lo-slow) in molluscan neurons [Shaw T., Lee R.J., Partridge L.D. Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca2+]i and Ca(2+)-activated channels in neurons. Neurosci Lett 1995; 190:121-124]. Here we demonstrate that pretreatment of neurons with 10 microM thapsigargin eliminates the FFA-induced increase in [Ca2+]i and substantially reduces both ICAN and Io-slow supporting the hypothesis that the FFA-induced increase in [Ca2+]i results primarily from Ca2+ release from a thapsigargin-sensitive intracellular store. The [Ca2+]i response appears to be sustained, not by influx of extracellular Ca2+, but by inhibitory effects of FFA on Ca2+ removal from the cytosol. Inhibition of Ca2+ efflux may be an important component of the FFA-induced activation of both ICAN and Io-slow, as Ca2+ release by thapsigargin alone is not sufficient to activate either current. Our data also demonstrate that the effects of FFA on [Ca2+]i, ICAN and Io-slow are reversible and suggest that protein phosphorylation as well as an increase in [Ca2+]i are involved in the FFA-induced activation of Io-slow. Effects on neuronal Ca2+ handling as well as activation of ICAN or Io-slow may partially explain the analgesic effects of FFA.

Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca2+]i and Ca(2+)-activated channels in neurons.

Ca(2+)-activated channels, including Ca(2+)-activated non-selective (CAN) channels and Ca(2+)-activated Cl- channels play important roles in regulating the electrical activity of neurons. No blockers of neuronal CAN channels have been previously reported. We used 2-electrode voltage clamping to measure membrane currents and fura-2 fluorescence imaging to measure [Ca2+]i in molluscan neurons. We show that the diphenylamine carboxylate derivative flufenamate (FFA), but not mefenamate or the parent compound, cause a transient increase in ICAN and a slow outward current, and a maintained increase in [Ca2+]i. We interpret this as a FFA-dependent release of Ca2+ from intracellular stores and Ca2+ influx, [Ca2+]i-dependent activation of the CAN and slow outward currents, and slow FFA-dependent channel block.

Intracellular calcium signaling induced by thapsigargin in excitable and inexcitable cells.

Signaling between intracellular Ca2+ stores and cell membrane channels or transporters is important to Ca(2+)-based second messenger systems. Two hypotheses, the capacitative and the Ca(2+)-induced Ca(2+)-influx models have been proposed to explain aspects of this signaling. In this study, we examined the applicability of these models in neuroendocrine (PC12), neuronal (dorsal root ganglion), immune (spleen), and fibroblast (3T3) cells. We used thapsigargin (TPG) to deplete specific intracellular Ca2+ stores and to increase the cytoplasmic Ca2+ concentration ([Ca2+]), and Ca2+ free medium to prevent Ca2+ influx and lower cytoplasmic [Ca2+]. We demonstrate that, although TPG causes an increase of [Ca2+]i in all cells examined, the subsequent stimulation of Ca2+ influx varies from high in spleen, to moderate in 3T3 and PC12, to undetectable in DRG cells. All cell types exhibited Ca2+ influx when Ca2+ was added to the medium following an exposure to Ca(2+)-free medium. Without added provisions, the two aforementioned hypotheses are inadequate in explaining the TPG-induced Ca(2+)-influx in all cell types. These results support the hypothesis of the existence of unique Ca2+ channels or transporters in spleen cells that operate subsequent to TPG treatment and are distinct from the voltage-gated Ca2+ channels and Ca(2+)-activated non-selective cation channels present in excitable cells.

Calcium-activated non-selective channels in the nervous system.

In the decade, since the first description of calcium-activated non-selective (CAN) channels in cardiac myocytes, pancreatic acini and neuroblastoma, this type of channel has been shown to have a ubiquitous distribution across a variety of tissues. Recently, their role in the function of cells of the nervous system has become better delineated. Because CAN channels pass depolarizing current, respond to cytoplasmic Ca2+ activity and do not inactivate, they are capable of producing maintained depolarization of neurons. This property endows upon CAN channels an important role in both physiological functions and pathological processes of the nervous system.

Cytoplasmic Ca2+ activity regulation as measured by a calcium-activated current.

Calcium-activated non-selective cation (CAN) currents were activated by quantitative injections of Ca2+ into voltage clamped bursting neurons of the snails Helix aspersa or Helix pomatia. Membrane potential was held at the potassium equilibrium potential and CAN currents were fit with a rising and falling exponential function. Ca2+ transporters and pumps of the cell membrane, endoplasmic reticulum, and mitochondria were selectively blocked with pharmacological agents. Bath solutions containing 0 Na Ringers, chlorpromazine, Na3VO4, or thapsigargin did not significantly change the CAN current decay constants from those measured in Ringers. External 2,4-dinitrophenol or internal ruthenium red, however, significantly lengthened the CAN current decay constant. It is concluded that mitochondria are the most important sink for sub-membrane Ca2+ activity in the range necessary to effectively activate CAN currents.

Calcium buffering in bursting Helix pacemaker neurons.

Bursting pacemaker neurons of the snail Helix pomatia were voltage-clamped and Ca currents in response to depolarizing steps were recorded. Simultaneously, changes in intracellular Ca concentrations were measured using the fluorescent dye fura-2 and a highly sensitive digital camera. Ca influx through voltage-gated channels induced a spatially non-uniform increase in intracellular Ca. The Ca signals decayed with a time constant of about 5 s. By increasing the concentration of the indicator dye, its Ca-buffering capacity was enhanced and Ca transients in response to depolarization were diminished. Thereby, the endogenous Ca buffer capacity could be determined and was calculated to be about 480 buffered ions for every free Ca ion. The buffer capacity did not vary significantly with the amount of Ca influx within the range tested, suggesting that the buffer is not saturated at Ca concentrations of up to 1 microM.

Activation and modulation of calcium-activated non-selective cation channels from embryonic chick sensory neurons.

We have shown that calcium-activated non-selective (CAN) channels from embryonic chick sensory neurons are permeable to both Na+ and K+ and are not blocked by TTX, TEA, or 4-AP. These neuronal CAN channels are activated by sub-micromolar cytoplasmic Ca2+ with negative cooperativity. The effect of Ca2+ is to decrease the closed times of the channel with little effect on the time the channel remains open. Isolated neuronal CAN channels can be phosphorylated by cAMP-dependent protein kinase (PKA). The effect of phosphorylation is to shorten channel open time and to minimize the effect of Ca2+ on channel closed time.

Modulation of calcium-activated non-specific cation currents by cyclic AMP-dependent phosphorylation in neurones of Helix.

1. Currents through calcium-activated non-specific cation (CAN) channels were studied in the fast burster neurone of Helix aspersa and Helix pomatia. CAN currents were activated by reproducible intracellular injections of small quantities of Ca2+ utilizing a fast, quantitative pressure injection technique. 2. External application of forskolin (10-25 microM), an activator of adenylate cyclase, caused the endogenous bursting activity of the cells to be replaced by beating activity. These same concentrations of forskolin reduced CAN currents reversibly to about 50%. 3. External application of IBMX (3-isobutyl-1-methylxanthine, 100 microM), an inhibitor of phosphodiesterase, the enzyme which breaks down cyclic AMP, reduced CAN currents reversibly to about 40%. 4. External application of the membrane-permeable cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl-cyclic AMP (100 microM) caused almost complete block of the CAN current. A marked reduction in the CAN current was also observed following quantitative injections of cyclic AMP (internal concentrations up to 50 microM) directly into the cells from a second pressure injection pipette. 5. Similar results were obtained with quantitative injections of the catalytic subunit (C-subunit) of the cyclic AMP-dependent protein kinase (internal concentrations 10(-4) units of enzyme) directly into the cells from a second pressure injection pipette. 6. Injection of the non-hydrolysable GTP analogue, GTP-gamma-S (internal concentrations 100 microM), which stimulates G-proteins, produced a prolonged increase in CAN current amplitude by as much as 300%. 7. External application of serotonin (100-200 microM) caused a transition from bursting to beating activity of the neurones and mimicked cyclic AMP's effects on CAN currents. Two other neurotransmitters, dopamine and acetylcholine, were not significantly effective in reducing CAN currents. 8. Injection of a peptide inhibitor of cyclic AMP-dependent protein kinase suppressed serotonin's action on bursting and on CAN current. 9. Our results indicate that CAN currents in Helix burster neurones are modulated by cyclic AMP-dependent membrane phosphorylation. They suggest that the physiological transmitter that induces this second messenger action is serotonin. The dual control of CAN channels by two second messengers, namely Ca2+ and cyclic AMP, has important functional implications. While Ca2+ activates these channels which generate the pacemaker current in these neurones, cyclic AMP-dependent phosphorylation down-regulates them, thereby resulting in modulation of neuronal bursting activity.

The sequential-interval state space: a means of displaying temporal information in neuron firing.

Variability in neuronal firing exhibits sufficient uncertainty so that a simple average firing frequency code is probably inadequate for most nervous system signalling. Temporal patterns certainly play an important role in neuronal coding. We have used interval histogram and 3-dimensional sequential interval state space plots to investigate various common patterns of firing in neurons of the land snail, Helix aspersa. Typical firing patterns included random, highly regular, doublet, and burst firing. Individual neurons could be made to change their temporal firing pattern in response to changes in transmembrane currents, or temperature, or the application of convulsant drugs. In every instance, the sequential interval state space plot provided a more distinctive display of temporal pattern than did the more common interval histogram. State space plots were also investigated for evidence of a predicted chaotic attractor. In no instance was this type of state space plot observed.

Single Ca-activated cation channels in bursting neurons of Helix.

The depolarizing drive that maintains bursting in Helix neurons is carried by a long-lasting calcium-activated inward current. This current was studied using cell-attached and inside-out patches from the right parietal fast burster neuron of Helix pomatia. One population of unitary currents was inward at -50 mV and showed an increased probability of opening when Ca2+ was injected or when excised patches were bathed in solutions with 10(-7) to 10(-5) M free Ca2+ levels. Cell-attached patches (patch electrodes filled with 10(-7) M Ca2+ Ringer) had single channel conductances near 30 pS with reversal potentials near -20 mV; excised patches had similar conductances in symmetrical Na+ solutions and reversal potentials within a few millivolts of zero. Calculations, assuming a simple spherical cell, yield a channel density of only about 1/6 micron2. The increased channel opening probability characteristically persisted well beyond the duration of transient whole-cell inward current. We conclude from this that the later phase of Ca-activated inward currents is normally masked by outward currents.