Modulation of high-voltage-activated calcium channels in dentate granule cells by topiramate.

PURPOSE: In this study, we assessed the effects of topiramate (TPM) on high-voltage-activated calcium channel (HVACC) currents in vitro. METHODS: HVACC currents were recorded from rat dentate gyrus granule cells by using whole-cell patch-clamp techniques. The biophysical properties of HVACCs were used to separate voltage-activated Ca2+ currents into different subtypes. Three concentrations of TPM were tested: 1, 10, and 50 microM. RESULTS: TPM inhibited L-type currents and was more effective at 10 microM than at 50 microM, suggesting that there may be an optimal concentration at which TPM decreases L-type currents. Non-L-type currents were transiently increased by TPM at a high concentration (50 microM). CONCLUSIONS: Because the location of L-type calcium channels on soma and proximal dendrites gives these channels a crucial role in controlling dendritic excitability and in providing calcium for intracellular effectors, the decrease in the L-type HVA Ca2+ currents may be an important anticonvulsant mechanism of TPM.

Differential control of three after-hyperpolarizations in rat hippocampal neurones by intracellular calcium buffering.

1. The whole-cell recording technique, combined with internal perfusion, was used to study the effects of intracellular Ca2+ buffering on fast, medium and slow after-hyperpolarizations (fAHP, mAHP and sAHP) in hippocampal CA1 pyramidal neurones in rat brain slices at room temperature. 2. The action potentials and the fAHP were unaffected by 100 microM to 3 mM concentrations of the internally applied fast Ca2+ chelator BAPTA. At higher (10-15 mM) concentrations, BAPTA inhibited the fAHP and prolonged the decay of the action potential, suggesting that the corresponding large-conductance Ca2+-activated K+ channels are located close to the sites of Ca2+ entry during an action potential. Addition of Ca2+ to the BAPTA-containing solution (at a ratio of 4.5 [Ca2+] : 10 [BAPTA]) to maintain the control level of [Ca2+]i did not prevent the effects of high concentrations of BAPTA. 3. The mAHP, activated by a train of action potentials, was inhibited by internally applied BAPTA within the range of concentrations used (100 microM to 15 mM), and this effect could not be reversed or prevented by addition of Ca2+ to the BAPTA-containing solution. The inhibition of the mAHP by BAPTA could also be observed after blockade of the hyperpolarization-activated IQ type mixed Na+-K+ current (also known as Ih) component of the mAHP by bath-applied 3-5 mM Cs+, suggesting that the inhibition of the mAHP by BAPTA is due to inhibition of the depolarization-activated IM (muscarinic) type K+ current. 4. The sAHP, activated by a train of action potentials, was potentiated by 100-300 microM internally applied BAPTA, both with and without added Ca2+. At 1-2 mM or higher concentrations, the potentiation of the sAHP by BAPTA without added Ca2+ was transient and was followed by a fast decrease. With added Ca2+, however, BAPTA caused a persistent potentiation of the sAHP with more than a 10-fold increase in duration for periods exceeding 1 h even at concentrations of the buffer as high as 10-15 mM. Earlier reports showing a blockade of the sAHP by BAPTA, based on experiments without added Ca2+, were apparently due to a sharp reduction in intracellular free [Ca2+] and to a high intracellular concentration of the free buffer. 5. Internally applied BAPTA caused a prolongation of the spike discharge during an 800 ms-long depolarizing current step. At 100-300 microM BAPTA, but not at 1-2 mM or higher concentrations, this effect could be reversed by addition of Ca2+. The effects of BAPTA on the spike discharge occurred in parallel with the changes in the sAHP time course, which was more prolonged at higher concentrations of the buffer. 6. The concentration-dependent differential control of the three types of AHP in hippocampal neurones by BAPTA is related to modulation of intracellular Ca2+ diffusion by a fast acting mobile Ca2+ buffer.

A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.

The inability to determine the precise intracellular location of non-fluorescent organic calcium chelators such as BAPTA is a persistent problem which has precluded much detailed analysis of the chelators' spatial or temporal dynamics in live cells. Similarly, following physiological experiments with fluorescent indicators like Fura-2, it has often been desirable to maintain the dye within the cell for later analysis by additional histological techniques. Based on chemical considerations, and its prior use in tissue fixation, we examined the water soluble reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a potential fixative for diverse calcium chelators. The utility of EDC, but not other common fixatives, was confirmed through electrophysiological means, through a novel ELISA, which exploits anti-BAPTA antibodies to assess the extent and kinetics of fixation; by autoradiography of neurons loaded with [14C]-BAPTA, and by immunocytochemistry and imaging of intracellular BAPTA or Calcium Green in neurons. At concentrations > 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of > 95% of BAPTA free acid. Fixation of intracellular BAPTA was confirmed in hippocampal brain slices loaded with BAPTA/AM ester, and showed biphasic kinetics consistent with rapid loading and subsequent extrusion of the chelator. Immunocytochemistry on neurons microinjected with BAPTA free acid and the dye Lucifer Yellow showed BAPTA-specific staining which was distributed in the cell similarly to that of the accompanying marker dye. Application of EDC also efficiently fixed in situ analogs of BAPTA such as Calcium Green (a fluorescent Ca2+ indicator) as shown by confocal imaging of EDC-fixed brain slices loaded with this indicator. Taken together, these data show that EDC is an effective, inexpensive and versatile fixative for calcium chelators in diverse cells. The availability of a suitable fixative now makes it possible to determine the distributions of such chelators at both the light and, possibly, the electron microscope level. Two important features of EDC, arise from its specificity for free carboxyl groups. First, the ability to fix, selectively, the chelators but not their AM esters; and, second, its enormous potential as a fixative for the numerous other carboxyl-containing chelators, dyes and pH indicators currently available.

Reversible inhibition of IK, IAHP, Ih and ICa currents by internally applied gluconate in rat hippocampal pyramidal neurones.

Previously, we reported that the spike frequency adaptation and slow afterhyperpolarizations (sAHP) in hippocampal pyramidal neurones are best preserved during whole-cell recording with a methylsulfate (MeSO4-)- based internal solution, but undergo a fast rundown when gluconate- (Gluc-)- based internal solution is used. Here we show, with internal perfusion of patch pipettes, the reversibility of the inhibitory effects of Gluc- on spike frequency adaptation and sAHP, and extend these observations to fast and medium-duration AHPs. Contrary to what might be expected based on Gluc- binding of Ca2+, the sAHP and its underlying current could be temporarily enhanced by adding 1-3 mM of the calcium chelator BAPTA to the internal solution in the presence of Gluc-. Replacement of internal MeSO4- with Gluc- did not affect the membrane resting potential or the amplitude and duration of action potentials, but reversibly increased the cell input resistance and decreased the threshold current for spike generation. Gluc- reversibly inhibited the hyperpolarization-activated non-selective cationic current (Ih), the depolarization-activated delayed rectifier K+ current (IK), the high-voltage-activated Ca2+ current and the Ca2+-activated K+ current that underlies the sAHP. The combination of these effects of Gluc- significantly alters the electrophysiological "fingerprint" of the neurone.

Development of astrocytes and neurons in cultured brain slices from mice lacking connexin43.

Astrocyte and neuronal development was investigated in organotypic brain slice cultures from mouse fetuses with a null mutation in the connexin43 gene. Astrocyte morphology and electrical properties were indistinguishable in null mutant slices and control slices but at 18 days in vitro astrocyte density in the central regions of the null mutant slices was significantly higher than in control slices. Neuronal development assessed morphologically and electrophysiologically appeared normal in the mutant slices. These results suggest that intercellular communication mediated through connexin43 is not essential for the development of astrocytes and neurons but may play a role in regulating astrocytic migration.

Enhanced LTP in mice deficient in the AMPA receptor GluR2.

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.

Modulation of hippocampal synaptic transmission by low concentrations of cell-permeant Ca2+ chelators: effects of Ca2+ affinity, chelator structure and binding kinetics.

Calcium chelators are commonly used for fluorescence and electrophysiological studies of neuronal Ca2+ signalling. Recently, they have also been used as neuroprotectants. Since they buffer calcium ions, these agents also modify the same signals which are being studied. These properties may be used to modulate Ca2+ signals such as those involved in synaptic transmission, and may explain their neuroprotective mechanism. To define factors which govern the modulation of synaptic transmission by Ca2+ chelators, we examined their actions on synaptic responses evoked in CA1 neurons of rat hippocampal slices. We used a spectrum of cell-permeant Ca2+ chelators having different structures, Ca(2+)-binding kinetics and Ca2+ affinities, as well as an impermeant, intracellularly perfused chelator salt. Application of the cell-permeant 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate acetoxymethyl ester (50 microM) markedly attenuated evoked synaptic responses. This application produced an intracellular chelator accumulation of 79-125 microM, as estimated using 14C-labelled chelator. The actions of a Ca2+ chelator on synaptic responses were dependent on the chelator's Ca2+ affinity, Ca(2+)-binding rate and Ca2+ selectivity, because 1,2-bis(2-amino-5-nitrophenoxy)ethane-N,N,N',N'-tetra-acetate acetoxymethyl ester (a low Ca2+ affinity analogue), ethyleneglycolbis(beta-aminoethyl ether)-N,N,N',N'-tetra-acetate acetoxymethyl ester (a slow buffer with similar Ca2+ affinity to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate) and the selective Zn2+ chelator, tetrakis(2-pyridylmethyl)ethylenediamine, were ineffective. The intrinsic cell membrane properties, including the post-spike train afterhyperpolarization, were not significantly affected by any of the Ca2+ chelators used in this study. Intracellular perfusion of 100-200 microM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetate salt through patch pipettes into postsynaptic cells did not affect synaptic potentials, suggesting a presynaptic action of cell-permeant Ca2+ chelators on transmitter release. Other cell-permeant, fast Ca(2+)-binding chelators reduced synaptic responses according to their Ca2+ affinities, and not their chemical structure: those chelators with Kd values < or = 25 microM attenuated synaptic responses, whereas chelators of lesser affinity did not. These data support the ideas that [Ca2+]i rises to high (micromolar) levels during transmitter release, and that Ca2+ chelators may be used to attenuate excitotoxicity by attenuating excitatory neurotransmission without affecting Ca2+ signalling in the submicromolar [Ca2+]i range.

Potentiation of a slow Ca(2+)-dependent K+ current by intracellular Ca2+ chelators in hippocampal CA1 neurons of rat brain slices.

1. In hippocampal CA1 neurons of rat brain slices, a Ca(2+)-dependent slow afterhyperpolarization (sAHP) and underlying K+ current (IsAHP) are activated by Ca2+ influx and presumably reflect the time course of the intracellular Ca2+ signal produced by neuronal stimulation. We tested the hypothesis that when exogenous Ca2+ chelators become the predominant mobile Ca2+ buffer in the neuron, they alter the shape of intracellular Ca2+ signals responsible for IsAHP. The nature of this alteration provides insight into the mechanism of IsAHP generation. 2. Derivatives of 1,2-bis-[2-amino phenoxy] ethane N,N,N',N' tetraacetic acid (BAPTA) with different dissociation constants (KDS) for Ca2+ ranging from 0.15 to 7,000 microM were used to test this hypothesis. We also examined the effects of ethylene glycolbis (beta-aminoethyl either)-N,N,N',N'-tetraacetic acid (EGTA), which has a KD similar to that of BAPTA for Ca2+, but which binds and releases Ca2+ 100 times more slowly. When delivered to the cytoplasm by dialysis from a patch pipette, these chelators potentiated, inhibited, or had no effect on IsAHP depending on their concentration, affinity, and binding kinetics. 3. IsAHP decayed exponentially over much of its time course, with a half-decay time of 0.9 +/- 0.1 s (mean +/- SE, n = 22). Immediately after breakthrough into the whole cell configuration, there was an initial period of approximately 5 min during which IsAHP magnitude increased approximately 3.5-fold with no change in time course. Thereafter, the time course and amplitude of IsAHP were stable for > 45 min. 4. Addition of 1 mM of the high-affinity chelators 5,5'-dimethyl BAPTA or BAPTA to the pipette solution first increased the decay time of IsAHP 1.5-fold. However, within 10-15 min after break-through, the current was abolished. Addition of Ca2+ (0.1-1.0 mM) to the patch pipette containing the BAPTA derivatives reduced the ability of a given concentration of high-affinity chelator to inhibit IsAHP and also prolonged the period of IsAHP enhancement. A similar prolongation of the period of enhancement with even less attenuation of IsAHP was apparent with 0.1 mM 5,5'-dimethyl BAPTA and 0.1 mM Ca2+. 5. The intermediate-affinity chelator 4.4'-difluoro BAPTA (1 mM) prolonged the decay phase of the sAHP/IsAHP without attenuating the current. A twofold prolongation of IsAHP also was observed in neurons dialyzed with internal solution containing 3 mM EGTA and 0.3 mM Ca2+. Dialysis with 1 mM of the low-affinity chelators 2-amino-5-fluorophenol-N,N,O-triacetic acid (5-fluoro APTRA) or 5,5'-dinitro BAPTA had no apparent effect on IsAHP. All of the chelators that prolonged the decay phase of IsAHP also induced a rising phase such that a well-defined peak of IsAHP could be discerned at approximately 0.6 s after the end of the stimulus used to evoke the current. 6. Weak stimulation of muscarinic receptors selectively inhibits IsAHP. Thus the uncontaminated time course of IsAHP can be deduced by subtracting currents recorded before and after such muscarinic stimulation. With minimal exogenous buffer in the pipette (0.1 mM EGTA), the muscarinic-receptor-sensitive current exhibited a rising phase lasting approximately 300 ms and then decayed with a half-time of approximately 1 s. Both the rising and decay phases of the muscarinic-receptor-sensitive current were prolonged at least twofold by dialysis with BAPTA or 4,4'-difluoro BAPTA. Thus the effect of the chelators on the time course of IsAHP is not simply and artifact of inhibition of early components of the outward current. 7. The effects of BAPTA analogues on the time course of IsAHP are not due to changes in mobilization of intracellular Ca2+. External application of caffeine (10 mM), ryanodine (20 microM), dantrolene (20 microM), or thapsigargin (100 microM) had no effect on IsAHP recorded with the standard pipette solution or

Adapters for combined intrapipette pressure pulses and patch pipette step movements during 'blind' cell search in brain slices.

A procedure is described for 'blind' cell search in brain slices based on pressure pulses instead of steady-state pressure applied to the patch pipette during its stepwise movements. For reproducibility of the pressure/movement pattern during the cell search, we have developed two adapters, one for electrically and the other for hydraulically driven micromanipulators which generate pressure pulses synchronized with patch-pipette step movements. Both adapters increase the intrapipette pressure prior to a step movement of the pipette, maintain the pressure during the pipette movement, and release it between steps, thus minimizing the possibility of 'blowing-away' the cells during the search. The hydraulic micromanipulator adapter converts this into a stepping one. Both adapters also allow simultaneous recording of pipette step movements and of intrapipette pressure. The use of these adapters allows standardization of the 'blind' cell search and greatly increases the success rate of cells detection.

Whole-cell recording of the Ca(2+)-dependent slow afterhyperpolarization in hippocampal neurones: effects of internally applied anions.

Using the whole-cell recording technique, we have examined the slow Ca(2+)-activated afterhyperpolarization (AHP) and its underlying current (IAHP) in hippocampal CA1 neurones of brain slices obtained from mature rats. Specifically we have studied the effects of the anion component of various K+ salts commonly used to make the pipette filling solution that dialyses neurones during whole-cell recordings. Among the K+ salts examined which included potassium methylsulfate, potassium methanesulfonate, potassium gluconate, potassium chloride, potassium citrate and potassium glutamate, stable AHPs/IAHP and strong spike firing adaptation could only be observed in neurones recorded with the patch pipette solution containing potassium methylsulfate. These AHPs and firing patterns closely mimicked those recorded with sharp electrodes. Similarly, the sustained component of voltage-activated Ca2+ currents was more stable in neurones dialysed with cesium methanesulfonate than in those dialysed with cesium gluconate or cesium chloride. Although the mechanisms underlying the interaction(s) between internally applied anions and ionic channels need further investigation, the present experiments illustrate that in mammalian brain neurones at 33 degrees C, the Ca(2+)-activated IAHP is dramatically altered by internal anions. We suggest that among anions commonly used in electrode filling solutions for whole-cell recordings, methylsulfate is the least disruptive to intracellular structures or Ca2+ homeostasis and permits stable whole-cell recording of the IAHP and Ca2+ currents in mammalian CNS neurones.

A simple method for internal perfusion of mammalian central nervous system neurones in brain slices with multiple solution changes.

A simple system for internal perfusion of whole-cell patch-clamped neurones in brain slices allowing multiple, fast solution exchanges is described. With this system, perfusing solutions can be loaded immediately before use during the recording. At perfusion rates of 5-10 microliters/min, complete replacement of solutions in the patch pipette tip, as determined by changes in the pipette resistance and liquid junction potential, was achieved in less than 20 s. This occurred after a 1-min latency that was due to solution flow through the infusion tube. The effectiveness of the system was tested on rat hippocampal CA1 neurones in the slice preparation. The effects of replacement of internal 150 mM K+ by 150 mM Cs+ ions on voltage-activated K+ currents and of changing internal [Cl-] between 20 mM and 150 mM on evoked GABAA-mediated inhibitory postsynaptic currents (IPSCs) were studied. The blockade of K+ currents by Cs+ ions and the changes of IPSCs by altered internal [Cl-] ion concentration were achieved within 3.2 and 1.5 min, respectively, including the 'flow latency' of about 1 min, and recovery following solution change occurred within 5.2 and 1.5 min, respectively. More than 10 effective internal solution replacements could be performed within 1 h in a single neurone without affecting the recording stability.

The role of calcium homeostasis and calcium currents in ethanol actions on central mammalian neurons.

The ubiquitous role of calcium in ethanol actions measured electrophysiologically in central neurons is discussed. Acute ethanol administration to rat hippocampal neurons in vitro causes a hyperpolarization, increased AHPs, increased EPSPs and IPSPs, decreased modelled electronic interneuronal coupling, decreased high threshold Ca2+ currents, increased Ik, and increased synaptic GABAA currents. Alcohol withdrawal reverses some of these actions. Ca2+ is implicated in all of the above ethanol mediated effects.

[The demonstration of recurrent motor axon collaterals in the chick embryo by using horseradish peroxidase axonal transport]. / Demonstratsiia vozratnykh kollateralei motornykh aksonov u kurinogo émbriona s pomoshch'iu aksonogo transporta peroksidazy khrena.

Motoneurons were labelled by retrograde axonal transport of HRP applied to transected spinal nerves in 9-11-day chick embryos in the in vitro spinal cord preparation. Recurrent motor axon collaterals were revealed in 17 of 48 motor axons which could be followed in the edge regions of labelled motoneuronal pools. The results, coupled with author's earlier electrophysiological data, provide further evidence for the presence of the Renshaw inhibition in the avian spinal cord.

[The formation of the segmental projections of the primary afferents of the wing and leg in the chick embryo spinal cord]. / Formirovanie segmentarnykh proektsii pervichnykh afferentov kryla i nogi v spinnom mozge kurinogo émbriona.

A comparative HRP study of formation of connections between primary sensory nerve fibers and motoneurones in brachial and lumbosacral cord segments has been made on chick embryos between the 6.5th and 10th days of incubation. HRP was applied to the cut ends of the appropriate nerves via suction pipettes on isolated superfused spinal cord preparation. The first contacts between primary sensory collaterals and motoneuronal dendrites were found to appear both in lumbosacral and branchial cord segments at the same stage, i.e. at the 7.5-8th days of development. This observation does not confirm the widely accepted belief on rostrocaudal sequence of development of the spinal cord, indicating that exceptions from this developmental gradient are quite possible.

Direct evidence for postsynaptic inhibition in the embryonic chick spinal cord.

Motoneurons were recorded intracellularly in the isolated perfused spinal cord of 10 - 16-day chick embryos. Inhibitory postsynaptic potentials (IPSPs) were present in motoneurones of all ages studied and could be evoked by both ventral white column and dorsal root stimulation. IPSPs produced by orthodromic stimulation displayed many features of mature vertebrate motoneuronal IPSPs including the chloride dependence and sensitivity to currents passed through the cell membrane. Strychnine and chloride-free solution produced marked disinhibitory effects in the spinal cord indicating the presence of inhibitory synapses in interneuronal circuits of at least 11-day and older embryos. Possible sources of descending inhibitory influences on motoneurones and some functional aspects are discussed. The results support the hypothesis that the inhibition starts in the embryonic chick spinal cord rather early, before the 10th day of development.

Intracellular recordings from embryonic chick motoneurones in the isolated perfused spinal cord.

Potentials of 13- to 18-day chick embryo motoneurones were recorded intracellularly. Action potentials could be evoked by antidromic or direct stimulation of the cells, but the amplitudes were relatively low (less than 40-45 mV) and no overshoot or marked afterpotentials were observed. Pronounced evoked and spontaneous postsynaptic potentials were observed. Both irregular miniature and bursting high-voltage spontaneous postsynaptic depolarizations were present. The association of spontaneous action potentials with the latter type of spontaneous synaptic activity suggests its possible involvement in mechanisms of spontaneous embryonic motility.