Calbindin28kDa is specifically associated with extranuclear constituents of the dense particulate fraction.

Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.

Group I metabotropic glutamate receptors mediate slow inhibition of calcium current in neocortical neurons.

Metabotropic glutamate receptor (mGluR)-mediated inhibition of high-voltage-activated Ca2+ currents was investigated in pyramidal neurons acutely isolated from rat dorsal frontoparietal neocortex. Whole cell recordings were made at 30-32 degrees C, with Ca2+ as the charge carrier. Selective agonists were used to classify the subgroup of mGluRs mediating the response. Ca2+ currents were inhibited by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD) and by the group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) but not by the group II agonist (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl)glycine (DCG-IV) or the group III agonist (+)-2-amino-4-phosphonobutryic acid (-AP4). (2S,1'S, 2'S)-2-(carboxycyclopropyl)glycine (-CCG-I) was effective at 10 and 100 microM but not at 1 microM, consistent with involvement of group I mGluRs. Variable results were obtained with the putative mGluR5-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) and the putative mGluR1-selective antagonist (S)-4-carboxyphenylglycine [(S)-4CPG], indicating that the group I mGluR subtypes may vary between cells or that these compounds were activating other receptors. The actions of (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG] were consistent with it being a low-potency antagonist. Several features of the Ca2+ current inhibition evoked by DHPG distinguished it from the rapid modulation typical of a direct action of G proteins on Ca2+ channels; the inhibition was slow to reach maximum (tens of seconds), current activation was not slowed or shifted in the positive voltage direction, and the inhibition was not relieved by positive prepulses. Nimodipine and omega-conotoxin GVIA blocked fractions of the current and also reduced the magnitude of the responses to DHPG, indicating that both L- and N-type Ca2+ channels were regulated. These results further differentiate the slow modulatory pathway observed in neocortical neurons when Ca2+ is used as the charge carrier from the rapid voltage-dependent mechanism reported to inhibit Ba2+ currents under Ca2+-free conditions.

Intracellular QX-314 inhibits calcium currents in hippocampal CA1 pyramidal neurons.

1. The effects of intracellular QX-314 on Ca2+ currents were examined in CA1 pyramidal cells acutely isolated from rat hippocampus. In neurons dialyzed with 10 mM QX-314 (bromide salt), the amplitude of the high-threshold Ca2+ current was on average 20% of that in control cells and the current-voltage relationships (I-Vs) were shifted in the positive voltage direction. 2. The positive shift in the I-Vs was due to the presence of intracellular Br-, because it was reproduced by 10 mM NaBr and was not present when the chloride salt of QX-314 was used. 3. Low-threshold (T-type) Ca2+ currents, at test voltages of -50 and -40 mV, were on average < 45% of control amplitude in cells containing 10 mM QX-314 (chloride salt) and < 10% of control amplitude in cells with 10 mM QX-314 (bromide salt). 4. In neurons dialyzed with 1 mM QX-314, high-threshold Ca2+ currents were still significantly different from control and Na+ currents were not completely blocked. 5. The proportions of high-threshold Ca2+ current blocked by omega-conotoxin GVIA, omega-agatoxin IVA, and nimodipine were similar in cells dialyzed with 10 mM QX-314 and control cells, indicating that the drug does not selectively inhibit any of the Ca2+ channel subtypes distinguished by these antagonists.

P-type calcium channels in rat neocortical neurones.

1. The high threshold, voltage-activated (HVA) calcium current was recorded from acutely isolated rat neocortical pyramidal neurones using the whole-cell patch technique to examine the effect of agents that block P-type calcium channels and to compare their effects to those of omega-conotoxin GVIA (omega-CgTX) and nifedipine. 2. When applied at a saturating concentration (100 nM) the peptide toxins omega-Aga-IVA and synthetic omega-Aga-IVA blocked 31.5 and 33.0% of the HVA current respectively. 3. A saturating concentration of nifedipine (10 microM) inhibited 48.2% of the omega-Aga-IVA-sensitive current, whereas saturating concentrations of both omega-Aga-IVA (100 nM) and omega-CgTX (10 microM) blocked separate specific components of the HVA current. 4. Partially purified funnel web spider toxin (FTX) at a dilution of 1:1000 blocked 81.4% of the HVA current and occluded the inhibitory effect of omega-Aga-IVA. Synthetic FTX 3.3 arginine polyamine (sFTX) at a concentration of 1 mM blocked 61.2% of the HVA current rapidly and reversibly. The effects of sFTX were partially occluded by pre-application of omega-Aga-IVA. We conclude that neither FTX nor sFTX blocked a specific component of the HVA current in these cells. 5. In view of the specificity of omega-Aga-IVA for P-type calcium channels in other preparations and for a specific component of the HVA current in dissociated neocortical neurones we conclude that about 30% of the HVA current in these neurones flow through P-channels.

Calcium currents in acutely isolated human neocortical neurons.

1. Ca2+ currents were investigated in neurons acutely isolated from adult human temporal neocortex. The aim was to compare the basic characteristics of the currents with those previously described in animals and to examine the effects of dihydropyridine Ca2+ antagonists and antiepileptic drugs. The tissue, obtained from patients undergoing temporal lobe surgery for medically intractable epilepsy, was sliced, incubated in papain, and triturated. 2. Most of the isolated neurons (34 of 36) were judged to be pyramidal cells by their morphology. Whole-cell voltage-clamp recordings revealed two components of Ca2+ current: 1) a low-threshold (T-type) current that was transient, small in amplitude, and required hyperpolarization more negative than -70 mV for removal of inactivation and 2) a high-threshold current that was slowly inactivating and was available for activation from more positive potentials. The characteristics of the Ca2+ currents were very similar to those in the neocortical neurons of young rats, although the low-threshold current was less prominent in the human cells. 3. Subcomponents of the high-threshold current were identified by pharmacology. About 20% of the peak current was blocked by omega-conotoxin GVIA (presumed N current) and 40-50% of the peak current was blocked by micromolar concentrations of the dihydropyridine Ca2+ antagonists nifedipine and nimodipine (presumed L current). In two neurons tested with a range of nimodipine concentrations, the threshold for suppression of the high-threshold current was approximately 10 nM. 4. The antiepileptic agents ethosuximide, carbamazepine, and valproate did not affect the Ca2+ currents at therapeutically relevant concentrations. Phenytoin marginally reduced the low- and high-threshold Ca2+ currents at 8 microM (a concentration corresponding to the upper therapeutic range). The results do not support the hypothesis that inhibition of Ca2+ currents in neocortical pyramidal neurons is a major action of these drugs.

Metabotropic glutamate receptor-mediated suppression of L-type calcium current in acutely isolated neocortical neurons.

1. The effects of metabotropic glutamate receptor (mGluR) stimulation on whole-cell Ca2+ currents were studied in pyramidal neurons isolated from the dorsal frontoparietal neocortex of rat. The selective mGluR agonist cis-(+/-)-1-aminocyclopentane-1,3-dicarboxylic acid [trans-ACPD (100 microM)] suppressed the peak high-threshold Ca2+ current by 21 +/- 1.7% (mean +/- SE) in 40 of 43 cells from 10- to 21-day-old rats. Consistent with previous findings for mGluR, glutamate, quisqualate, and ibotenate [but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)] reduced the Ca2+ currents, and the responses were not blocked by the ionotropic glutamate receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovaleric acid (APV). EC50S for Ca2+ current suppression were 29 nM for quisqualate, 2.3 microM for glutamate, and 13 microM for trans-ACPD. 2. The low-threshold Ca2+ current was not modulated by trans-ACPD. The component of the high-threshold CA2+ current suppressed by mGluR was determined by pharmacology; the responses were not affected by omega-conotoxin GVIA but were occluded by the dihydropyridine Ca2+ antagonist nifedipine. Ca2+ tail currents prolonged by the dihydropyridine Ca2+ agonist (+)-SDZ 202-79] were suppressed by mGluR stimulation in parallel with the peak current. These findings strongly suggest that L-type Ca2+ channels are modulated by mGluR. 3. In neurons dialyzed with 100 microM guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S), Ca2+ current suppression was elicited by the first application of trans-ACPD (in 5 of 6 cells), but not by subsequent applications. Responses in neurons dialyzed with 2 mM guanosine 5'-(beta-thio)diphosphate (GDP-beta-S) were significantly smaller than controls. The results are consistent with mGluR acting via linkage to a G protein. 4. The responses to mGluR agonists were smaller when the external Ca2+ was replaced by Ba2+, indicating that some part of the mechanism underlying the current suppression is Ca2+ dependent. Because mGluR stimulates phosphoinositide turnover and release of Ca2+ from intracellular stores in other types of neurons, the possibility of released Ca2+ mediating inactivation of Ca2+ channels was considered. However, the Ca2+ current suppression was not attenuated by strong intracellular Ca2+ buffering [20 mM bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)], by dialysis with 100 microM inositol-1,4,5-triphosphate (IP3), or by external application of 1 microM thapsigargin. 5. We conclude that in neocortical neurons, one action of mGluR is to suppress the component of high-threshold Ca2+ current conducted by L-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

High- and low-threshold calcium currents in neurons acutely isolated from rat sensorimotor cortex.

Neurons were isolated by papain treatment and trituration of the frontoparietal cortex of 14 to 28-day-old rats. Whole cell voltage clamp revealed a slowly inactivating high-threshold Ca2+ current, activated positive to -45 mV, and a transient low-threshold Ca2+ current, activated positive to -65 mV. The high-threshold current was more sensitive to block by Cd2+ and the low-threshold current was more sensitive to block by Ni2+. Replacement of Ca2+ by Ba2+ increased the high-threshold current and reduced the low-threshold current. The high-threshold current was enhanced by Bay K 8644 and reduced by nimodipine and omega-conotoxin. The low-threshold current was also reduced by nimodipine but was insensitive to Bay K 8644 and omega-conotoxin. The properties of the currents were consistent with different underlying Ca2+ channel types.

Evaluation of long-term potentiation of small compound and unitary EPSPs at the hippocampal CA3-CA1 synapse.

Long-term potentiation (LTP) was evaluated for small monosynaptic CA3-mediated EPSPs in CA1 neurons in the guinea pig hippocampal slice. Small EPSPs included those elicited by stimulation of Schaffer axon collaterals of several CA3 neurons (160-480 microV amplitude, n = 40 EPSPs in 40 neurons) and those elicited by stimulation of an individual CA3 neuron (89-563 microV amplitude, n = 14 EPSPs in 11 neurons). Various protocols were employed to induce LTP and were deemed successful as evaluated by recording sustained enhancement of the mean peak amplitude of conventionally elicited large compound EPSPs and extracellular field potentials. However, in 47 of 54 cases, tetanization did not lead to a potentiation of the small or unitary EPSPs. In 9 cases, it was possible to directly evaluate the compound EPSP (elicited by stimulating a group of CA3 neuron's axons) and the unitary EPSP (elicited by stimulating a single CA3 neuron) in the same CA1 neuron. The tetanization protocol was successful in inducing LTP in 7 of 9 of these CA1 neurons as evaluated by the compound EPSP but resulted in LTP for only 1 of 9 of the unitary EPSPs for the same neurons. One explanation for these results is a threshold mechanism controlling the expression of LTP. Although LTP induction occurred in most cases, it is proposed that a critical level of depolarization (achieved by the test activation of a sufficient number of CA3 neurons) is necessary so that the enhancement at the modified synapse is expressed.

The time course and amplitude of EPSPs evoked at synapses between pairs of CA3/CA1 neurons in the hippocampal slice.

Unitary EPSPs were evoked in CA1 pyramidal neurons by activation of single CA3 pyramidal neurons. Seventy-one EPSPs were recorded. The peak amplitudes of these EPSPs ranged from 30 to 665 microV with a mean of 131 microV. Rise times and half-widths were measured, the means +/- SD being 3.9 +/- 1.8 and 19.5 +/- 8.0 msec, respectively. The time courses of these EPSPs were consistent with a brief synaptic current at a localized electrotonic region of the dendritic tree followed by passive spread of current to the soma. EPSPs varied in amplitude from trial to trial. Sufficient records were collected for 12 EPSPs to demonstrate that this variation was greater than could be accounted for by baseline noise. The amplitude variations of one EPSP were reliably resolved from the background noise, and this EPSP fluctuated between 4 discrete amplitudes (including failures) separated by a quantal increment of 278 microV.

Responses of monkey precentral neurones to passive movements and phasic muscle stretch: relevance to man.

Single cell recordings were made from movement-related neurones from the precentral cortex of two monkeys, trained to perform a simple lever-pulling task. They were also trained to remain relaxed while the arm was explored with passive movements at different joints, cutaneous stimuli and during the application of two types of phasic muscle stretch: percutaneous vibration and percussion of muscle tendons. Recordings were made of the responses of cortical neurones both to the 'natural' stimuli and to vibration of specific muscle tendons or percussion of the triceps tendon. Both tendon percussion and vibration excited neurones within area 4 with an average latency for tendon percussion of 21.0 msec. There was a high degree of consistency in the effects on single neurones of tendon percussion and vibration at the same site. Although long-term facilitation was not seen. vibration-induced discharge in the motor cortex should be considered as a potential mechanism of its effects in intact man. In contrast to the similarity of the effect of the two forms of phasic stretch, the relationship between a single neurone's response to either tendon percussion or vibration and to passive movement was complex. The dissociation seen between the effects of phasic muscle stretch and that of passive movement may underlie the failure, in man, to find uniformly increased long-latency stretch reflexes in clinical states of extrapyramidal rigidity.

Amplitude fluctuations in small EPSPs recorded from CA1 pyramidal cells in the guinea pig hippocampal slice.

EPSPs have been evoked in CA1 pyramidal cells by (1) activation of single CA3 neurons (unitary EPSPs), and (2) low-intensity stimuli to the CA1 stratum radiatum. Five unitary EPSPs were obtained; their mean peak amplitudes ranged from 85 to 275 microV and 3 of the 5 showed fluctuations in amplitude that were too great to be attributed to baseline noise. After subtraction of the variance due to the noise, these EPSPs had coefficients of variation much higher than those reported for variability in the quantal EPSP in other preparations. These results suggest that intermittent transmitter release is a major cause of EPSP amplitude fluctuation at this synapse. A noise deconvolution technique based on a nonrestrictive model of transmitter release was applied to the EPSPs obtained in this study. For 2 of the EPSPs evoked by stratum radiatum stimulation, the amplitudes fluctuated between discrete values that were sufficiently separated with respect to the noise to be resolved by the deconvolution procedure. Quantal increments of 224 and 193 microV were determined for the 2 EPSPs.

Rat organum vasculosum laminae terminalis in vitro: responses to transmitters.

Spontaneous single-action potentials (units) were recorded extracellularly from explants of the rat organum vasculosum laminae terminalis in vitro. Increasing the osmotic pressure of the bathing solution by 15% by adding NaCl or mannitol increased frequency, whereas reducing the osmotic pressure by 15% by omitting NaCl reduced frequency. The mean frequency ratio (test/control) for 6 (of 13) units responding to a 15% increase was 2.2 +/- 0.5 (SE), and for 8 (of 11) units responding to a 15% decrease it was 0.6 +/- 0.1. These responding units all lay within 55 microns of the ventricular surface. Reduction of the calcium concentration from 2 to 0.75 mmol X l-1 increased the mean frequency of units by 55%. Putative transmitters were added to the bathing solution at 0.1 mol X l-1. The results were as follows: carbamylcholine (26 units), 27% excited and 19% inhibited; luteinizing hormone-releasing hormone (34 units), 38% excited and 12% inhibited; angiotensin II (ANG II) (34 units), 26% excited, and 6% inhibited; somatostatin (14 units), 36% excited and 43% inhibited; serotonin (15 units), 67% excited and none inhibited; and dopamine (13 units), 46% excited and none inhibited. Of the units tested, 86% lay less than 100 microns from the ventricular surface. Units responsive to ANG II lay deeper than units responding to serotonin (P less than 0.005), osmotic changes (P less than 0.001), or carbamylcholine (P less than 0.02).