Presynaptic Modulation of Glutamate Release Targets Different Calcium Channels in Rat Cerebrocortical Nerve Terminals

We have studied which type/s of Ca²⁺ -channel/s support glutamate exocytosis and its modulation by presynaptic receptors in cerebrocortical nerve terminals. Depolarization of nerve terminals with 30 mM KCI induced a Ca²⁺ -dependent release of 3.64 ± 0.25 nmol/mg of protein. The addition of either 2 μM ω-conotoxin-GVIA or 200 nM ω-agatoxin-IVA reduced the KCI-evoked release by 47.7 ± 3.5% and 70.4 ± 8.9% respectively, and by 85.7 ± 4.1% when both toxins were co-applied. The activation of adenosine A₁ receptors with N ⁶-cyclohexyladenosine or the activation of rnetabotropìc glutamate receptors with L(+)-2-amino-4-phosphonobutyrate inhibited the KCI-evoked release by 41.0 ± 5.9 and 54.3 ± 10% respectively. The extent of these inhibitions was not altered by the prior addition of 2 μM ω-conotoxin-GVIA but they were significantly enhanced when ω-agatoxin-IVA was added together with the adenosine A₁ receptor agonist or the metabotropic glutamate receptor agonist, suggesting that ω-conotoxin-GVIA-sensitive and not ω-agatoxin-IVA-sensitive Ca²⁺-channels are ínvolved in the action of these inhibitory receptors. By contrast, the facilitation of glutamate release that follows the activation of the protein kinase C, either with phorbol esters or with the stimulation of phospholipase C-linked metabotropic receptors, was expressed by both ω-conotoxin-GVIA-sensitive and ω-agatoxin-sensitive Ca²⁺-channels. It is concluded that different Ca²⁺-channels support the modulation of glutamate release by presynaptic receptors.     

Comparative Patch-clamp Studies with Freshly Dissociated Rat Hippocampal and Striatal Neurons on the NMDA Receptor Antagonistic Effects of Amantadine and Memantine

Patch- and concentration-clamp techniques were used to compare the effects of the uncompetitive N -methyl-D-aspartate (NMDA) receptor antagonists (+)-MK-801 (dizocilpine, (+)-5-methyl-10, 11-dihydro-5H-dibenzo-cyclohepten-5, 10-imine maleate), ketamine, memantine (1-amino-3, 5-dimethyladamantane) and amantadine (1-amino-adamantane) on agonist-induced inward currents in freshly dissociated rat hippocampal and striatal neurons. In hippocampal neurons, ketamine (5 μM), memantine (10 μM) and amantadine (100 μM) selectively antagonized inward current responses to NMDA (500 μM plus glycine 5 μM) in a voltage-dependent manner without affecting responses to ( s )-α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (100 μM) or γ-aminobutyric acid (10 μM). The NMDA receptor antagonistic effect of all four agents was typical of open channel blockade. The kinetics of blockade/unblockade was inversely related to antagonist affinity. In hippocampal neurons amantadine was the least potent NMDA receptor antagonist (IC₅₀ 18.6 ± 0.9 μM) and showed the fastest blocking kinetics, whereas (+)-MK-801 was the most potent (IC₅₀ 0.12 ± 0.01 μM) and showed the slowest blocking kinetics. Memantine (IC₅₀ 1.04 ± 0.26 μM) and ketamine (IC₅₀ 0.43 ± 0.10 μM) were almost equipotent and had similar, intermediate blocking kinetics. In striatal neurons recorded under identical conditions (+)-MK-801, ketamine and memantine were 3- to 4-fold less potent whereas amantadine was somewhat more potent than on hippocampal neurons. This could offer an explanation for the better clinical profile of amantadine in Parkinson's disease, as therapeutically relevant concentrations of amantadine are likely to be more active in the striatum whereas memantine is likely to be more active in other structures.     

Exocytosis and Selective Neurite Calcium Responses in Rat Cerebellar Granule Cells During Field Stimulation

The free calcium concentration, [Ca²⁺]_c, in fura-2-loaded rat cerebellar granule cells was investigated by digital imaging during trains of uniform field stimuli in order to compare the ability of calcium channels in somata and neurites to respond to brief, physiologically relevant depolarizations. Very few somata responded to 20 Hz trains of 1 ms pulses, while virtually all neurites showed an extensive increase which was rapidly reversed when stimulation was terminated. In contrast, both somata and neurites responded when cells were depolarized with 50 mM KCl. The field stimuli evoked a tetrodotoxin-sensitive increase in Na⁺ concentration in both somata and neurites. When 4-aminopyridine, which inhibits delayed K⁺ currents in these cells, was present during the field stimulus both somata and neurites increased their [Ca²⁺]_c, suggesting that prolongation of the duration of depolarization is required for somatic Ca²⁺ channel activation. The neurite response did not depend on the orientation of the neurite relative to the applied field. The neurite response was insensitive to nifedipine (1 μM) and ω-agatoxin-IVA (30 nM) but was uniformly inhibited by ω-conotoxin-GVIA (30% inhibition at 1 μM) and ω-conotoxin-MVIIC (44% inhibition at 5 μM). The two inhibitors were not additive. The neurite [Ca²⁺]_c response was insensitive to the combination of ionotropic glutamate receptor antagonists. Field stimulation caused the exocytosis of the fluorescent probe FM1-43 previously loaded during KCl depolarization, suggesting that presynaptic Ca²⁺ channels contribute to the field-evoked neurite response.     

Lamotrigine Reduces Voltage-Gated Sodium Currents in Rat Central Neurons in Culture

Summary: Purpose : To study the mechanism or mechanisms of action of lamotrigine (LTG) and, in particular, to establish its effects on the function of NA⁺ channels in mammalian central neurons.
Methods : Rat cerebellar granule cells in culture were subjected to the whole-cell mode of voltage clamping under experimental conditions designed to study voltage-gated Na⁺ currents.
Results : Extracellular application of LTG (10–500 μ M , n = 21) decreased in a dose-related manner a tetrodotoxin-sensitive inward current that was elicited by depolarizing commands (from −80 to +20mV). The peak amplitude of this Na⁺-mediated current was diminished by 38.8 ± 12.2% (mean ± SD, n = 6) during application of 100 μ M LTG, and the dose-response curve of this effect indicated an IC₅₀ 145 μM. The reduction in the inward currents produced by LTG was not associate with any signficant change in the current decay, whereas the voltage dependency of the steady-state inactivation shifted toward more negative values (midpoint of the inactivation curve: –47.5 and –59.0 mV under control conditions and during application of 100 μM LTG, respectively, n = 4).
Conclusions : Our findings indicate that LTG reduces the amplitude of voltage-gated Na⁺ inward current in rat cerebellar granule cells and induces a negative shift of the steady-state inactivation curve. Both mechanisms may be instrumental in controlling the repetitive firing of action potentials (AP) that occurs in neuronal networks during seizure activity.     

Cholinergic Modulation of the Action Potential in Rat Hippocampal Neurons

The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 μM) caused an increase in spike duration; high doses (10–40 μM) or long-lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the muscarinic antagonist atropine, and were prevented by Ca²⁺-free medium and by Ca²⁺-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca²⁺-dependent K⁺ currents I_c or I_AHP, suggesting that these K⁺ currents are not involved in the modulation.     

Cyclic Nucleotide-gated Channels in Identified Human Olfactory Receptor Neurons

Patch-clamp recordings revealed the presence of a non-desensitizing cyclic nucleotide-gated channel on human olfactory receptor neurons and a fast-desensitizing non-specific cation channel activated by nucleotides on human supporting cells. Cyclic nucleotide-gated channels on olfactory receptor neurons showed selective channel activation by cAMP (K_1/2= 5 μM) and cGMP (K_1/2= 2 μM), a unitary conductance of ∼20 pS, a reversal potential of single-channel currents close to 0 mV, a linear current-voltage relationship over the range of −80 to 80 mV and a strong extracellular but a weaker intracellular blocking effect of Ca²⁺. The channel activity outlasted the cyclic nucleotide pulses for hundreds of milliseconds when higher agonist concentrations (>50 μM cAMP) were applied. The duration of the response was longer than in cyclic nucleotide-gated channels from other species studied so far. The plateau duration and the decay remained constant for pulses with a length of 50−150 ms, whereas pulses shorter than 50 ms successively reduced the time required by shortening the plateau phase. A larger difference for the K_1/2 values of cAMP (K_1/2= 22 μM) and cGMP (K_1/2= 2.5 μM) were found for a small group (n = 3) of cyclic nucleotide-gated channels, pointing to the selective expression of the a-subunit in a small subgroup of olfactory receptor neurons.     

Effects of P-Aminophenyl Dichloroarsine on Reduced High-affinity [3H]Nicotine Binding Sites from Chick Brain: A Covalent,Yet Reversible,Agent for Neuronal Nicotinic Receptors

Neuronal nicotinic acetylcholine receptor (nAChR) α-subunits contain a conserved disulphide that is essential for function. Here, we have examined the effects of sulphydryl redox reagents on [³H]nicotine binding to chick brain nAChR immunoisolated with the monoclonal antibody mAb35. The disulphide reducing agent, dithiothreitol (DTT), inhibited [³H]nicotine binding [50% inhibitory concentration (IC₅₀) = 146 μM] but this effect was reversed (93±1.5%) by subsequent reoxidation with 1 mM dithio-bis(nitrobenzoic acid) (DTNB). The trivalent arsenical, p -aminophenyl dichloroarsine (APA), which reacts with pairs of spatially close sulphydryls, was a potent inhibitor of reoxidation by DTNB (IC₅₀= 35 nM). However, application of the 'anti-arsenical', 2,3-dimercaptopropane sulphonic acid (DMPS), restored agonist binding after APA treatment (50% effective concentration = 120 μM). Paradoxically, DMPS was also found to be a potent oxidizing agent of these receptors. Affinity alkylation of reduced nAChRs with bromoacetylcholine (BAC; 100 μM) irreversibly blocked nicotine binding (>90%). We propose (but have not proven) that APA interacts with the cysteines homologous to Cys192–193 in Torpedo AChRs, since APA pretreatment of reduced neuronal receptors protected against irreversible BAC alkylation, as shown by subsequent reversal of DMPS (2 mM; 20 min). This study illustrates the potent and reversible nature of the arsenical's covalent interaction with an isolated nAChR and suggests that modified arsenicals could be useful nAChR probes.     

The Effect of Barium on [3H]Noradrenalin Release from the Human Neuroblastoma SH-SY5Y

Replacement of Ca²⁺ with Ba²⁺ in HEPES-buffered saline stimulated [³H]noradrenalin release in the human neuroblastoma clone SH-SY5Y by up to 20% of the cell content in the absence of other secretory stimuli. The Ba²⁺-evoked release was inhibited by 85% by 3 μM tetrodotoxin and 95% by 5 μM nifedipine. Ba²⁺ also increased the potency of K⁺-evoked release of [³H]noradrenalin, as maximal release was observed with 60 mM K⁺ compared with the 100 mM K⁺ necessary to achieve maximal release in the presence of Ca²⁺. In contrast, replacing Ca²⁺ with Ba²⁺ had little effect on carbachol- and bradykinin-evoked release of [³H]noradrenalin. No evidence was obtained from studies on changes in [Ca²⁺]_i (in response to 100 pM carbachol) using fura-2 that Ba²⁺ could enter intracellular stores in SH-SY5Y cells. Whole-cell patch-clamp studies showed that Ba²⁺ depolarizes SH-SY5Y cells as well as enhancing inward Ca²⁺ channel currents and shifting their voltage dependence to more negative values. These results are discussed in terms of the hypothesis that Ba²⁺ blocks K⁺ channels, leading to depolarization followed by opening of voltage-sensitive Na⁺ channels. This in turn opens voltage-sensitive L-type Ca²⁺ channels, which are coupled to the release of [³H]noradrenalin in SH-SY5Y cells.     

Electrophysiological Interactions Between 5-Hydroxytryptamine and Thyrotropin Releasing Hormone on Rat Hippocampal CA1 Neurons

Intracellular recording from CA1 neurons of the rat hippocampal slice preparation was used to examine the possibility of functional interactions between 5-hydroxytryptamine (5-HT) and thyrotropin releasing hormone (TRH), which act as cotransmitters in other areas of the central nervous system. 5-HT (30 μM) elicited complex effects consisting of biphasic changes in membrane potential and a strong depression of the afterhyperpolarization (AHP) following a spike burst. TRH (10 μM) did not alter membrane potential or input conductance but it produced a partial block of the AHP. Under single-electrode voltage clamp, 5-HT and TRH both reduced the amplitude of voltage-activated total K⁺ currents. When the two substances were co-applied, their actions were occluded. The voltage-activated K⁺ current remaining in Ca²⁺-free solution lost its sensitivity to 5-HT and TRH, suggesting that the K⁺ current modulated by TRH and 5-HT was Ca²⁺-dependent, although TRH itself did not depress high-threshold voltage-activated Ca²⁺ currents. When a relatively small concentration (5 μM) of 5-HT was co-applied with an equimolar amount of TRH, the degree of block of the spike AHP was the sum of the two individual effects of these drugs. It is suggested that in hippocampal pyramidal cells 5-HT and TRH influenced neuronal excitability by depressing a Ca²⁺-dependent K⁺ current, a phenomenon perhaps mediated through a common intracellular second messenger pathway.     

Effects of a Cognition-enhancer, Linopirdine (DuP 996), on M-type Potassium Currents (IK(M)) Some Other Voltage- and Ligand-gated Membrane Currents in Rat Sympathetic Neurons

Linopirdine is a cognition enhancer which augments depolarization-induced transmitter release in the cortex and which is under consideration for potential treatment of Alzheimer's disease. It has previously been reported to inhibit M-type K⁺ currents in rat hippocampal neurons. In the present experiments we have tested its effect on whole-cell M-currents and single M-channels, and on a range of other membrane currents, in dissociated rat superior cervical sympathetic ganglion cells. Linopirdine inhibited the whole-cell M-current with an IC50 of 3.4 μM and blocked M-channels recorded in excised outside-out membrane patches but not in inside-out patches. This suggests that linopirdine directly blocks M-channels from the outside. It was much less effective in inhibiting other voltage-gated potassium currents [delayed rectifier (I_K(v)), IC50 63 μM; transient (I_A) current, IC50 69 μM] and produced no detectable inhibition of the fast and slow Ca²⁺-activated K⁺ currents I_c and I_AHP or of a hyperpolarization-activated cation current (I_Q/I_h), at 10–30 μM. However, it reduced acetylcholine-activated nicotinic currents and GABA-activated Cl^- currents with IC50 values of 7.6 and 26 μM respectively. It is concluded that linopirdine shows some 20–fold selectivity for M-channels among different K⁺ channels but can also block some transmitter-gated channels. The relationship between M-channel block and the central actions of linopirdine are discussed.     

PACAP and VIP Stimulate Ca2+ Oscillations in Rat Gonadotrophs through the PACAP/VIP Type 1 Receptor (PVR1) Linked to a Pertussis Toxin-Insensitive G-Protein and the Activation of Phospholipase C-β

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are hypothalamic factors that play roles in the regulation of anterior pituitary cell activity. PACAP exists in 2 forms physiologically, a 38 amino acid form (PACAP38) and a form possessing the N-terminal 27 amino acids of PACAP38 (PACAP27). We have previously shown that PACAP38 stimulates an increase in [Ca²⁺]_i in rat gonadotrophs. In an attempt to identify the PACAP receptor type underlying this effect, we compared the potency of PACAP38, PACAP27 and VIP to stimulate Ca²⁺ changes in identified single rat gonadotrophs. All 3 peptides at 100  nM were capable of stimulating high amplitude Ca²⁺ oscillations, which were also observed in the absence of extracellular Ca²⁺. The order of potency of these peptides was PACAP38>PACAP27>VIP, and a potent antagonist of the PACAP/VIP type II binding site ([4-Cl-D-Phe⁶, Leu¹⁷]-VIP) failed to block these responses, suggesting that these effects are mediated through a PACAP/VIP type 1 receptor (PVR1). The Ca²⁺ responses to PACAP38 and VIP were unaffected by overnight treatment of the cells with pertussis toxin (PTX; 250  ng/ml) indicating that these responses are mediated by a PTX-insensitive G-protein. Finally, the Ca²⁺ responses stimulated by PACAP38 and VIP were blocked by the phospholipase C- β blocker U73122 (5  μM). In summary, PACAP stimulates Ca²⁺ oscillations in rat gonadotrophs through the activation of the PVR1 linked to a PTX-insensitive G-protein and the activation of phospholipase C- β . VIP can stimulate the same pathway in rat gonadotrophs, although it is at least 100 fold less potent than PACAP38.     

Effects of Phenytoin, Carbamazepine, and Gabapentin on Calcium Channels in Hippocampal Granule Cells from Patients with Temporal Lobe Epilepsy

Summary: Purpose: The anticonvulsants phenytoin (PHT), carbamazepine (CBZ), and gabapentin (GBP) are commonly used in the treatment of temporal lobe epilepsy. Ca²⁺ current modulation has been proposed to contribute to the antiepileptic activity of these drugs. The purpose of this study was to determine the effects of these anticonvulsants on voltage-dependent calcium channels in pathologically altered neurons from patients with chronic temporal lobe epilepsy.
Methods : Acutely isolated human hippocampal granule cells were examined by using the whole-cell configuration of the patch-clamp technique.
Results : PHT and CBZ produced a reversible, concentrationdependent inhibition of high-voltagectivated (HVA) Ca²⁺ currents without affecting voltage-dependent activation. The concentration-response curves of PHT and CBZ indicated maximal inhibition of 35 and 65%, respectively, with halfmaximal inhibition being obtained at 89 and 244 μ M , respectively. At therapeutic cerebrospinal fluid (CSF) concentrations, HVA currents were not significantly altered by PHT and CBZ. However, PHT but not CBZ showed a reduction of HVA currents of 16% at a therapeutic whole-brain concentration of 80 μ M . In ontrast to CBZ, PHT produced a small hyperpolarizing shift in the voltage dependence of steady-state inactivation. PHT, 80 μ4, shifted the potential of half-maximal inactivation by -3.1 ± 0.5 mV (p < 0.05). GBP, which was recently found to bind to the ಠsubunit of a neuronal Ca²⁺ channel, showed no modulation of Ca²⁺ conductances.
Conclusions : These results suggest that, in contrast to GBP and CBZ, modulation of postsynaptic Ca2+ channels can contribute to the anticonvulsant action of PHT in human hippocampal granule cells.     

Skin Sympathetic Response In Subclinical Diabetic Neuropathy

Hyperglycemia and its associated Na⁺/K⁺ pump activity has been implicated in the development of diabetic neuropathy. We recently reported that high glucose in the presence of ouabain induced a progressive increase in the delayed K⁺ current which was suppressed by a blocker of Ca²⁺-activated K⁺ channels and blockers of Ca²⁺ channels in rat single myelinated nerve fibers, suggesting an increase of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). However, the influences of high glucose with ouabain on [Ca²⁺]_i in sensory neurons remain to be elucidated. The present study was undertaken to examine the modulation of depolarization-induced Ca²⁺ transients by high glucose and ouabain in isolated adult rat dorsal root ganglion (DRG) neurons using the fluorescent Ca²⁺ indicator fura-2. Bath application of KCl (50 mM) evoked a rapid increase in [Ca²⁺]_i through voltage dependent Ca²⁺ channels ([Ca²⁺]_i: 154.2 ± 22.5 nM). This increase was enhanced under high glucose (30 mM D-glucose) in the presence of ouabain (100 M) ([Ca²⁺]_i: 764.8 ± 210.1 nM). We conclude that a combination of high glucose and decreased Na⁺/K⁺ pump activity leads to an increase in [Ca²⁺]_i in rat DRG neurons, thereby resulting in nerve dysfunction.     

GABAergic signaling induces divergent neuronal Ca2+ responses in the suprachiasmatic nucleus network

Intercellular communication between γ-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca²⁺ imaging techniques to record changes in the intracellular calcium concentration ([Ca²⁺]_i) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca²⁺ responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABA_A and GABA_B receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca²⁺]_i. Application of the GABA_A receptor antagonist gabazine induced changes in baseline [Ca²⁺]_i in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca²⁺ response. GABA application induced Ca²⁺ responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca²⁺]_i. These results suggest that physiological GABA induces opposing effects on [Ca²⁺]_i based on the chloride equilibrium potential, and may play an important role in neuronal Ca²⁺ balance, synchronization and modulation of light input signaling in the SCN network.     

Responses to Metabotropic Glutamate Receptor Activation in Cerebellar Purkinje Cells: Induction of an Inward Current

The responses to activation of metabotropic glutamate receptors (mGluRs) of Purkinje cells in rat cerebellar slice cultures were investigated using intracellular recordings in single-electrode voltage-clamp mode combined with microfluorometric measurements of cytosolic free calcium using fura-2. Purkinje cells were perfused with saline containing 0.5 μM tetrodotoxin and 10 μM bicuculline and voltage-clamped at –60 mV. Bath-applied trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid ( t -ACPD, 50–100 μM), a selective agonist of mGluRs, induced a transient inward current that was followed by an outward current. The response induced by t -ACPD was not affected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, up to 40 μM). In contrast, inward currents caused by (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1–2 μM) were completely abolished, while inward currents caused by quisqualate (0.25 μM) were only partially depressed by CNQX (5–40μM). The inward current induced by t -ACPD was unaffected by external Ba²⁺ (1 mM), tetraethylammonium (10 mM) and Cs⁺ (1 mM), and was associated with an increase in apparent input conductance of the cell membrane. The extrapolated reversal potential of inward currents induced by t -ACPD was +18 mV while Cl⁻ currents induced by muscimol reversed at –66 mV. Inward currents induced by t -ACPD, but not those induced by AMPA, were associated with a rise in cytosolic Ca²⁺ concentration and suppressed by intracellular injection of a calcium chelator. Replacement of external Na⁺ by choline or Li⁺ depressed the inward current and resulted in a slower decay of the Ca²⁺ signal.     

Mechanisms for Synchronous Calcium Oscillations in Cultured Rat Cerebellar Neurons

Removal of Mg²⁺ caused oscillations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the membrane potential in cultured cerebellar granule neurons. Oscillations of [Ca²⁺]_i were synchronous in all the cells, and were restricted to the neurons (immunocytochemically identified) that responded to exogenous N -methyl-D-aspartate (NMDA). Oscillations were blocked by Ca²⁺ removal, nickel, NMDA receptor antagonists, ω-agatoxin IVA, tetrodotoxin, sodium removal and γ-aminobutyric acid, but not by dihydropyridines, ω-conotoxin M VIIA or by emptying the intracellular Ca²⁺ stores with thapsigargin or ionomycin. The upstroke of the [Ca²⁺]_i oscillations coincided in time with an increase in manganese permeability of the plasma membrane. Propagation of the [Ca²⁺]_i wave followed more than one pathway and the spatiotemporal pattern changed with time. Membrane potential oscillations consisted of transient slow depolarizations of ˜20 mV with faster phasic activity superimposed. We propose that the synchronous [Ca²⁺]_i oscillations are the expression of irradiation of random excitation through a neuronal network requiring generation of action potentials and functional glutamatergic synapses. Oscillations of [Ca²⁺]_i are due to cyclic Ca²⁺ entry through NMDA receptor channels activated by synaptic release of glutamate, which requires Ca²⁺ entry through P-type Ca²⁺ channels activated by action potentials at the presynaptic terminal.     

Copper Modulation of NMDA Responses in Mouse and Rat Cultured Hippocampal Neurons

The effect of Cu²⁺ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu²⁺ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu²⁺ directly inhibits NMDA receptors with an IC₅₀ of 0.27 μM. Cu²⁺ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu²⁺ and glycine-Cu²⁺ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu²⁺ (10–100 μM) of responses to 10 μM NMDA was essentially voltage-independent. The onset of inhibition by 100 μM Cu²⁺ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU²⁺ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu²⁺ to NMDA receptors, calculated from the rate of onset of block by Cu²⁺ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu^z+ interacts preferentially with agonist-bound receptors. Our results show that Cu²⁺ is a potent inhibitor of NMDA receptor-mediated responses.     

Voltage-dependent Currents in Microvillar Receptor Cells of the Frog Vomeronasal Organ

Vomeronasal receptor cells are differentiated bipolar neurons with a long dendrite bearing numerous microvilli. Isolated cells (with a mean dendritic length of 65 μm) and cells in mucosal slices were studied using whole-cell and Nystatin-perforated patch-clamp recordings. At rest, the membrane potential was −61 ± 13 mV (mean ± SD; n = 61). Sixty-four per cent of the cells had a resting potential in the range of –60 to –86 mV, with almost no spontaneous action potential. The input resistance was in the GΩ range and overshooting repetitive action potentials were elicited by injecting depolarizing current pulses in the range of 2 – 10 pA. Voltage-dependent currents were characterized under voltage-clamp conditions. A transient fast inward current activating near –45 mV was blocked by tetrodotoxin. In isolated cells, it was half-deactivated at a membrane potential near –75 mV. An outward K⁺ current was blocked by internal Cs⁺ ions or by external tetraethylammonium or Ba²⁺ ions. A calcium-activated voltage-dependent potassium current was blocked by external Cd²⁺ ions. A voltage-dependent Ca²⁺ current was observed in an iso-osmotic BaCl₂ solution. Finally, a hyperpolarization-activated inward current was recorded. Voltage-dependent currents in these microvillar olfactory receptor neurons appear qualitatively similar to those already described in ciliated olfactory receptor cells located in the principal olfactory epithelium.     

Glial Cells in the Mouse Hippocampus Express AMPA Receptors with an Intermediate Ca2+ Permeability

Recently, we could demonstrate that 'complex' glial cells in mouse hippocampal slices express glutamate receptor channels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate subtypes. In the present study, we further characterized this glial receptor. Since voltage-clamp control is imperfect and diffusion barriers hinder the quantitative analysis of the receptor currents in situ , the patch-clamp technique was applied to glial cells acutely isolated from the mouse hippocampal CA1 stratum radiatum subregion. A concentration-clamp technique was used which enabled very fast exchange of the extracellular solutions. Thus, it was possible to characterize the transient receptor currents with high time resolution. Application of L-glutamate, AMPA and L-homocysteate induced rapidly activating and fast desensitizing receptor currents in the suspended glial cells. In contrast, kainate induced non-desensitizing currents. The corresponding dose-response curve revealed a half-maximum of current activation at 350 μM. The current/voltage relationship of the kainate-evoked response was linear, with a reversal potential at ∼9 mV. Analysis of the reversal potential in solutions containing high concentrations of CaCl₂ confirmed earlier in situ data by demonstrating significant Ca²⁺ permeability of the glial glutamate receptor channels in the hippocampus. The kainate-induced receptor currents were markedly increased by cyclothiazide, a substance which selectively potentiates glutamate receptors of the AMPA subtype. We conclude that glial cells of the juvenile hippocampus mainly express heteromeric high-affinity AMPA receptors. Most probably, the receptor channels are assembled from the low Ca²⁺-permeable glutamate receptor-2 subunit together with Ca²⁺-permeable AMPA-preferring subunits.