Problems with mapping the auroral oval and magnetospheric substorms.

Accurate mapping of the auroral oval into the equatorial plane is critical for the analysis of aurora and substorm dynamics. Comparison of ion pressure values measured at low altitudes by Defense Meteorological Satellite Program (DMSP) satellites during their crossings of the auroral oval, with plasma pressure values obtained at the equatorial plane from Time History of Events and Macroscale Interactions during Substorms (THEMIS) satellite measurements, indicates that the main part of the auroral oval maps into the equatorial plane at distances between 6 and 12 Earth radii. On the nightside, this region is generally considered to be a part of the plasma sheet. However, our studies suggest that this region could form part of the plasma ring surrounding the Earth. We discuss the possibility of using the results found here to explain the ring-like shape of the auroral oval, the location of the injection boundary inside the magnetosphere near the geostationary orbit, presence of quiet auroral arcs in the auroral oval despite the constantly high level of turbulence observed in the plasma sheet, and some features of the onset of substorm expansion.

Histamine potentiates N-methyl-D-aspartate receptors by interacting with an allosteric site distinct from the polyamine binding site.

Histamine potentiates activation of native and recombinant N-methyl-d-aspartate receptors (NMDARs), but its mechanisms of action and physiological functions in the brain remain controversial. Using four different models, we have further investigated the histamine-induced potentiation of various NMDAR-mediated responses. In single cultured hippocampal neurons, histamine potentiated NMDA currents. It also potentiated the NMDA-induced increase in intracellular calcium in the absence, as well as with saturating concentrations, of exogenous d-serine, indicating both glycine-dependent and glycine-independent components of its effect. In rat hippocampal synaptosomes, histamine strongly potentiated NMDA-induced [(3)H]noradrenaline release. The profile of this response contained several signatures of the histamine-mediated effect at neuronal or recombinant NMDARs. It was NR2B-selective, being sensitive to micromolar concentrations of ifenprodil. It was reproduced by tele-methylhistamine, the metabolite of histamine in brain, and it was antagonized by impromidine, an antagonist/inverse agonist of histamine on NMDA currents. Up to now, histamine was generally considered to interact with the polyamine site of the NMDAR. However, spermine did not enhance NMDA-induced [(3)H]noradrenaline release from synaptosomes, and the potentiation of the same response by tele-methylhistamine was not antagonized by the polyamine antagonist arcaine. In hippocampal membranes, like spermine, tele-methylhistamine enhanced [(3)H]dl-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) binding to the glutamate site. In contrast, spermine increased nonequilibrium [(3)H]5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) binding, and suppressed [(3)H]ifenprodil binding, whereas histamine and tele-methylhistamine had no effect. In conclusion, the histamine-induced potentiation of NMDARs occurs in the brain under normal conditions. Histamine does not bind to the polyamine site, but to a distinct entity, the so-called histamine site of the NMDAR.

AMPA receptor properties and coexpression with sodium-calcium exchangers in rat hypothalamic neurons.

The histaminergic tuberomamillary (TM) nucleus, a center for the regulation of wakefulness, is excited by glutamatergic, aminergic and peptidergic inputs. AMPA receptor properties in relation to their expression were investigated in acutely isolated TM neurons with the help of whole-cell patch-clamp recordings combined with single-cell RT-PCR. The mRNAs encoding for the AMPA receptor GluR2 (100% of the neurons) and GluR1 (75%) were the most frequently detected, followed by the mRNA for GluR4 (56%), whereas GluR3 cDNA amplification did not yield a PCR product in any neuron. Flip splice variants prevailed over flop, in keeping with a strong glutamate-response potentiation by cyclothiazide. The expression pattern of AMPA subunits in their two splice variants was correlated with the different subtypes of Na+/Ca2+ (NCX) and Na+/Ca2+/K+ (NCKX) exchangers: glutamate receptor subunits GluR1-4 displayed no coordinated pattern with NCX. However, NCKX2 mRNA occurred only in TM cells with a fast desensitizing glutamate response, where it was coexpressed with the GluR4 subunit in the flop splice variant. NCKX3 mRNA was detected in neurons with fast or slow desensitization of glutamate responses. AMPA receptors in TM neurons were Ca2+-impermeable. As reverse Na+/Ca2+ exchange contributes to the immediate rise in intracellular calcium resulting from glutamate receptor activation, we suggest that the coordinated expression of NCKX2 with the fast desensitizing AMPA receptor-type reflects either a receptor-exchanger coupling or separate mechanisms for maintaining calcium homeostasis in neurons with fast or slow glutamate responses.

Neuroprotective effects of the antifungal drug clotrimazole.

Pretreatment with 10 microM of the antifungal drug clotrimazole potently reduced the death of cultured rat cerebellar granule cells induced by oxygen/glucose deprivation, and the excitotoxic effect of glutamate on cultured hippocampal neurons and cerebellar granule cells. In patch-clamped hippocampal pyramidal neurons, 10-50 microM clotrimazole caused a decrease in the amplitude of N-methyl-D-aspartate (NMDA) receptor-mediated currents. Glutamate induced intracellular Ca(2+) overload, as measured by Fluo-3 confocal fluorescence imaging, while clotrimazole reduced Ca(2+) overload and promoted the recovery of intracellular calcium homeostasis after glutamate treatment. Using tetramethylrhodamine ethyl ester fluorescence as a marker of mitochondrial membrane potential we found that clotrimazole prevented the glutamate-induced loss of mitochondrial membrane potential. Our data provide evidence that the protective effect of clotrimazole against oxygen/glucose deprivation and excitotoxicity is due to the ability of this drug to partially block NMDA receptor-gated channel, thus causing both reduced calcium overload and lower probability of the mitochondrial potential collapse.

Differential modulation of AMPA receptors by cyclothiazide in two types of striatal neurons.

The modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration-response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was approximately 90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 mM glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds.

Interaction between copper and zinc at GABA(A) receptors in acutely isolated cerebellar Purkinje cells of the rat.

Nanomolar concentrations of Cu(2+) induce a slowly reversible block of GABA(A) receptor-mediated currents which can be removed by chelating substances. The possible interaction of Cu(2+) with the Zn(2+) binding site on the GABA(A) receptor complex was studied in acutely isolated Purkinje cells using whole-cell recording and a fast drug application system. When Zn(2+) was applied together with 2 microM GABA, the Zn(2+)-induced block of GABA-mediated currents was not additive to the Cu(2+)-induced block. In the presence of 0.1 microM Cu(2+) in the bath solution the degree of inhibition of GABA-mediated responses by Zn(2+) was strongly attenuated. Preapplication of 100 microM Zn(2+) during 10 s, terminated 1 s before exposure to 2 microM GABA did not affect the GABA current in Cu(2+)-free solution, but relieved its block by 0.1 microM Cu(2+). This effect of Zn(2+) was concentration-dependent with an EC(50) of 72 microM. When the Cu(2+)-induced block was removed by histidine, preapplication of Zn(2+) did not increase the GABA current, indicating that the relief of Cu(2+) block by Zn(2+) is the result of its ability to actively remove Cu(2+) from the GABA receptor complex. It is proposed that the inhibitory effects of Zn(2+) and Cu(2+) on GABA-induced currents result from an action of these metal ions at distinct, but conformationally linked sites on the GABA(A) receptor protein. Under physiological conditions Zn(2+) would liberate Cu(2+) from the GABA(A) receptor, thus facilitating Cu(2+) turnover and its binding by other endogenous chelating molecules.

The open channel blocking drug, IEM-1460, reveals functionally distinct alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in rat brain neurons.

The properties of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were examined in various cell types isolated from young rat hippocampus, striatum and cerebellum using patch-clamp and fast application techniques. A dicationic adamantane derivative, IEM-1460, reversibly inhibited kainate-induced currents. In the presence of 100 microM IEM-1460, kainate currents in striatal giant cholinergic interneurons and hippocampal non-pyramidal neurons were inhibited by 95% and 81%, respectively, at Vh = - 70 mV. Striatal GABAergic principal cells, hippocampal pyramidal neurons and cerebellar Purkinje cells had low sensitivity to IEM-1460 (inhibition by 4-15%). Analysis of averaged data from the cell types studied revealed a highly significant positive correlation (r= 0.93, P < 0.01) between percentage inhibition by 100 microM IEM-1460 and relative calcium permeability of AMPA receptors, P(Ca)/P(Na). Also, within each brain structure, the sensitivity of IEM-1460 block was lower the stronger the outward rectification of kainate currents. Some hippocampal neurons exhibited intermediate sensitivity to IEM-1460. Kainate currents were suppressed by 40% in the presence of 100 microM IEM-1460. Meanwhile, AMPA receptors in this cell type had low calcium permeability (P(Ca)/P(Na) = 0.13) and demonstrated outwardly rectifying kainate currents. The interrelation of different properties of AMPA receptors considering their assembly is discussed. The data obtained suggest that IEM-1460 may be a convenient and promising marker of native AMPA receptor assembly: it selectively inhibits Ca(2+)-permeable, GluR2-lacking AMPA receptors.

Characterization of AMPA receptor populations in rat brain cells by the use of subunit-specific open channel blocking drug, IEM-1460.

Dicationic adamantane derivative, IEM-1460, which selectively blocks GluR2-lacking, Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, was used to characterize the distribution of AMPA receptors among populations of rat brain cells. IEM-1460 inhibited kainate-induced inward currents (at -80 mV) in a dose-dependent manner. IEM-1460 concentrations producing 50% inhibition of kainate-induced current amplitude (IC50) varied greatly depending on the cell type studied. Striatal giant cholinergic interneurons and putative Bergmann glial cells isolated from the cerebellum were found to be highly sensitive to IEM-1460 block (IC50=2.6 microM), indicating the expression of GluR2-lacking AMPA receptor subtype. Among hippocampal and cortical non-pyramidal neurons, there were cell-to-cell differences in the pattern of AMPA receptor subtype expression. Some cells which are known to express AMPA receptors lacking GluR2 subunit exhibited high sensitivity of IEM-1460 block (IC50 about 1 microM) but in the others, the part of AMPA receptor population seemed to be represented by GluR2-having receptor subtype. The latter subtype was mainly expressed by pyramidal neurons isolated from hippocampus (IC50=1102 microM) and sensorimotor cortex (IC50=357 microM) which showed low affinity for IEM-1460 block. In conclusion, IEM-1460 can be utilized as an indicator of the distribution of AMPA receptor subtypes among populations of rat brain cells, and pharmacological detection of the absence of GluR2 subunit in AMPA receptor assembly can provide useful information for the interpretation of physiological events.

High-affinity copper block of GABA(A) receptor-mediated currents in acutely isolated cerebellar Purkinje cells of the rat.

The actions of Cu2+ ions on GABAA receptor-mediated currents in acutely isolated Purkinje cells from rat cerebellum were studied using the whole-cell patch-clamp technique and a rapid perfusion system. Bath application of Cu2+ reduced currents induced by 2 microM gamma-aminobutyric acid (GABA) in a concentration-dependent manner with an IC50 of 35 nM. The Cu2+-induced block of GABA responses was not voltage-dependent. Increasing the GABA concentration (from 2 to 50 microM) decreased the blocking effect of Cu2+. Dose-response analysis for activation of GABAA receptors revealed a twofold decrease in apparent affinity for GABA in the presence of 0.1 microM Cu2+. Recovery from the block required several minutes after removal of Cu2+ from the medium. The block was removed by histidine, which preferentially forms complexes with Cu2+, or by other chelating substances. Application of 10 microM histidine immediately before application of 2 microM GABA completely relieved the block of GABA responses produced by 0.1 microM Cu2+. The effect of histidine was concentration-dependent with an EC50 of 0.75 microM. The results demonstrate that Cu2+ is a potent inhibitor of GABA-evoked responses in rat Purkinje cells. Copper may be an endogenous synaptic modulating factor. Cu2+ toxicity, notably in Wilson's disease, could result to some extent from chronic GABAA receptor blockade.

A simple perfusion system for patch-clamp studies.

Many systems for rapid perfusion of isolated cells or membrane patches operate with separate capillaries for each drug to be applied. We describe here a simple way by which the solutions in a single capillary can be quickly exchanged during recording. The device consists of a 100 microliters reservoir mounted 20 mm above the preparation and a capillary through which test perfusion media are driven by gravity. The application system is used for preparations attached to a recording pipette and includes two capillaries with solution exchange capabilities within a larger bath tube; it is driven by a capstan motor which is held in a neutral position by a spring. An unlimited number of solutions can be applied through each channel. The volume for washing and refilling a channel is typically 0.5 ml; it may be as low as 0.1 ml. The time interval necessary for sequential applications of different solutions through the same channel is less than 20 s. The solution flows through the capillaries and the bath tube at a speed of about 3 mm/s (30 and 600 nl/s respectively). The mechanical displacement for concentration jumps is adjustable from 0 to 0.4 mm in two directions and can be set to occur within 2-100 ms.

Abecarnil enhances GABA-induced currents in acutely isolated cerebellar Purkinje cells.

The effect of abecarnil, a beta-carboline derivative acting at central gamma-aminobutyric acid (GABAA)/benzodiazepine receptors, on the response to GABA of isolated Purkinje cells acutely dissociated from rat cerebellar slices was studied. Using a rapid superfusion system to apply drugs and whole-cell voltage-clamp recording configuration, abecarnil was found to be of similar efficacy to diazepam (DZP) in enhancing GABA-mediated responses. Abecarnil potentiated GABA-induced chloride currents maximally by 241%, while DZP showed a maximal potentiation of 217%. However, abecarnil was more potent than DZP and exhibited different potentiation kinetics. While the response to DZP was fast and reversible, abecarnil after a 1-3 sec application initially produced only a very small enhancement of the GABA response. The effect then developed gradually even after cessation of abecarnil application, and depended on both abecarnil concentration and exposure time. It is suggested that abecarnil accumulates in the lipid membrane resulting in slow effect kinetics and prolonged presence at the benzodiazepine binding site. Abecarnil is a full agonist at the GABAA/benzodiazepine receptor on Purkinje cell somatic membranes.

Subcortical modulation of synaptic plasticity in the hippocampus.

A two-stage model of memory trace formation in the hippocampus considers exploratory behavior associated with theta activity and consumatory behaviors or immobility associated with the occurrence of synchronous pyramidal cell discharges. Threshold, frequency and extent of CA3 bursts differ between differently performing strains of mice and are subject to subcortical modulation. Mechanisms for such actions are described: interference with potassium and other cation channels through second messengers and a direct action of histamine on the NMDA receptor channel. Furthermore long-lasting potentiations of synaptic transmission and excitability following brief exposures of hippocampal slices to histamine, noradrenaline, and serotonin are mediated by cyclic AMP and presumably protein kinase A.

Tetrahydroaminoacridine blocks and prolongs NMDA receptor-mediated responses in a voltage-dependent manner.

N-Methyl-D-aspartate (NMDA) receptor-mediated currents were recorded from acutely isolated rat hippocampal neurones using patch-clamp and fast perfusion techniques. Tetrahydroaminoacridine blocked NMDA receptor currents in a concentration-dependent fashion with IC50 25 +/- 6 microM and slope factor 2 +/- 0.2 at a membrane potential -80 mV. The block was voltage-dependent being greater at a hyperpolarized potential. The NMDA responses blocked by tetrahydroaminoacridine at concentrations greater than 25 microM were followed by a transient inward current hump with a decay time constant of about 200 ms at -90 mV. The tetrahydroaminoacridine-induced NMDA tail current was voltage-dependent, blocked by magnesium and tetrahydroaminoacridine itself and was not affected by NMDA and glycine recognition site antagonists. Magnesium suppressed the tail current amplitude without changing its time course whereas the tetrahydroaminoacridine block was accompanied by a dramatic prolongation. It is suggested that tetrahydroaminoacridine prevents the closing of the blocked NMDA channels thus keeping them in the activated state after the removal of agonist. The observed properties of the tetrahydroaminoacridine block could be explained in terms of a sequential model of an open channel block.

Histamine potentiates N-methyl-D-aspartate responses in acutely isolated hippocampal neurons.

N-methyl-D-aspartate (NMDA)-evoked currents were recorded from acutely isolated rat hippocampal neurons, using the whole-cell patch-clamp technique and a rapid perfusion system. Histamine, at concentrations from 0.5 to 100 microM, reversibly enhanced NMDA currents by up to 50%. The effect cannot be ascribed to activation of the known histamine receptors (H1, H2, H3) but is occluded by spermine. These results suggest an interaction of histamine with the polyamine-binding site on the NMDA receptor complex. This modulatory action could allow the histaminergic system to determine time and loci of NMDA receptor-mediated events, such as memory formation according to behavioral state.

Vibrodissociation of sliced mammalian nervous tissue.

A new simple and reliable mechanical method of the acute isolation of viable neurones from mammalian brain slices is described. The method is based on the local application of mechanical vibration directly on the chosen site of a slice and does not require the enzymatic pretreatment of the tissue. The application of the method to the isolation of the CA1 pyramidal neurones from rat hippocampal slices has been shown to be highly efficient. The characteristic morphology and sensitivity to N-methyl-D-aspartate and other substances of liberated cells are maintained. Clearly, this method should be attractive in patch-clamp studies of individual cells.