Acutely dissociated cell bodies of mouse Purkinje neurons spontaneously fired action potentials at approximately 50 Hz (25 degrees C). To directly measure the ionic currents underlying spontaneous activity, we voltage-clamped the cells using prerecorded spontaneous action potentials (spike trains) as voltage commands and used ionic substitution and selective blockers to isolate individual currents. The largest current flowing during the interspike interval was tetrodotoxin-sensitive sodium current (approximately -50 pA between -65 and -60 mV). Although the neurons had large voltage-dependent calcium currents, the net current blocked by cobalt substitution for calcium was outward at all times during spike trains. Thus, the electrical effect of calcium current is apparently dominated by rapidly activated calcium-dependent potassium currents. Under current clamp, all cells continued firing spontaneously (though approximately 30% more slowly) after block of T-type calcium current by mibefradil, and most cells continued to fire after block of all calcium current by cobalt substitution. Although the neurons possessed hyperpolarization-activated cation current (Ih), little current flowed during spike trains, and block by 1 mM cesium had no effect on firing frequency. The outward potassium currents underlying the repolarization of the spikes were completely blocked by 1 mM TEA. These currents deactivated quickly (<1 msec) after each spike. We conclude that the spontaneous firing of Purkinje neuron cell bodies depends mainly on tetrodotoxin-sensitive sodium current flowing between spikes. The high firing rate is promoted by large potassium currents that repolarize the cell rapidly and deactivate quickly, thus preventing strong hyperpolarization and restoring a high input resistance for subsequent depolarization. (+info)
Activation of human D3 dopamine receptor inhibits P/Q-type calcium channels and secretory activity in AtT-20 cells.
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The D3 dopamine receptor is postulated to play an important role in the regulation of neurotransmitter secretion at both pre- and postsynaptic terminals. However, this hypothesis and the underlying mechanisms remain untested because of the lack of D3-selective ligands, paucity of appropriate model secretory systems, and the weak and inconsistent coupling of D3 receptors to classical signal transduction pathways. The absence of ligands that selectively discriminate between D3 and D2 receptors in vivo precludes the study of D3 receptor function in the brain and necessitates the use of heterologous expression systems. In this report we demonstrate that activation of the human D3 dopamine receptor expressed in the AtT-20 neuroendocrine cell line causes robust inhibition of P/Q-type calcium channels via pertussis toxin-sensitive G-proteins. In addition, using the vesicle trafficking dye FM1-43, we show that D3 receptor activation significantly inhibits spontaneous secretory activity in these cells. Our results not only support the hypothesis that the D3 receptor can regulate secretory activity but also provide insight into the underlying signaling mechanisms. We propose a functional model in which the D3 receptor tightly regulates neurotransmitter release at a synapse by only allowing the propagation of spikes above a certain frequency or burst-duration threshold. (+info)
Luteinizing hormone inhibits conversion of pregnenolone to progesterone in luteal cells from rats on day 19 of pregnancy.
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We have previously reported that intrabursal ovarian administration of LH at the end of pregnancy in rats induces a decrease in luteal progesterone (P4) synthesis and an increase in P4 metabolism. However, whether this local luteolytic effect of LH is exerted directly on luteal cells or on other structures, such as follicular or stromal cells, to modify luteal function is unknown. The aim of the present study was to determine the effect of LH on isolated luteal cells obtained on Day 19 of pregnancy. Incubation of luteal cells with 1, 10, 100, or 1000 ng/ml of ovine LH (oLH) for 6 h did not modify basal P4 production. The addition to the culture medium of 22(R)-hydroxycholesterol (22R-HC, 10 microgram/ml), a membrane-permeable P4 precursor, or pregnenolone (10(-2) microM) induced a significant increase in P4 accumulation in the medium in relation to the control value. When luteal cells were preincubated for 2 h with oLH, a significant (p < 0.01) reduction in the 22R-HC- or pregnenolone-stimulated P4 accumulation was observed. Incubation of luteal cells with dibutyryl cAMP (1 mM, a cAMP analogue) plus isobutylmethylxanthine (1 mM, a phosphodiesterase inhibitor) also inhibited pregnenolone-stimulated P4 accumulation. Incubation with an inositol triphosphate synthesis inhibitor, neomycin (1 mM), or an inhibitor of intracellular Ca2+ mobilization, (8,9-N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate (1 mM), did not prevent the decrease in pregnenolone-stimulated P4 secretion induced by oLH. It was concluded that the luteolytic action of LH in late pregnancy is due, at least in part, to a direct action on the luteal cells and that an increase in intracellular cAMP level might mediate this effect. (+info)
Drug-protein binding and blood-brain barrier permeability.
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The permeability surface area (PS) product, an index of permeability of the blood-brain barrier (BBB), was measured by using the in situ perfusion method. In the cerebral circulation, the fraction of drug that permeates into the brain through the BBB is not only the unbound fraction but also the fraction dissociated from the protein in the perfusate. The sum of these two fractions, the apparent exchangeable fraction, was estimated by fitting the parameters of the BBB permeability under the condition of varying BSA concentrations in the perfusate. The unbound fraction of drugs in a buffer containing 0.5 mM BSA was measured by using the ultrafiltration method in vitro, and the apparent exchangeable fraction was measured in vivo by using the intracarotid artery injection method. The apparent exchange fraction was 100% for S-8510, 96.5% for diazepam, 90.9% for caffeine, 38.3% for S-312-d, 33.1% for propranolol, and 6.68% for (+)-S-145 Na, and each of these was higher than the corresponding unbound fraction in vitro in all drugs. The apparent exchangeable fractions, for example, were 8 times higher for diazepam and 38 times for S-312-d than the unbound fractions in vitro. The apparent exchangeable fraction of drugs was also estimated from the parameters obtained with the perfusion method. Because drugs can be infused for an arbitrary length of time in the perfusion method, substances with low permeability can be measured. The apparent exchangeable fractions obtained with this method were almost the same as those obtained with the intracarotid artery injection method. (+info)
Glomerular size-selective dysfunction in NIDDM is not ameliorated by ACE inhibition or by calcium channel blockade.
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BACKGROUND: In patients with insulin-dependent diabetes mellitus (IDDM) and overt nephropathy glomerular barrier size-selectivity progressively deteriorates with time and is effectively improved by angiotensin converting enzyme (ACE) inhibition. Whether similar glomerular functional changes develop in proteinuric patients with non-insulin-dependent diabetes mellitus (NIDDM), and whether antihypertensive agents can favorably affect glomerular filtration of macromolecules in these patients, has not been documented yet. METHODS: We investigated renal hemodynamics and fractional clearance of neutral dextrans of graded sizes, in nine proteinuric patients with NIDDM and renal biopsy findings of typical diabetic glomerulopathy. Six healthy volunteers served as controls. We also investigated the effects of an ACE inhibitor and of a calcium channel blocker, both given in doses targeted to achieve a comparable level of systemic blood pressure control, on glomerular hemodynamics and sieving function. Theoretical analysis of glomerular macromolecule transport was adopted to evaluate intrinsic glomerular membrane permeability properties. RESULTS: Fractional clearance of large macromolecules (42 to 66 A in radius) was significantly higher in diabetic patients than in controls, and the distribution of membrane pore radii was calculated to be shifted towards larger pore sizes in diabetics (mean radius increased from 55 to 60 A). Despite effective blood pressure control, neither antihypertensive affected glomerular hemodynamics to any significant extent. Fractional clearance of dextrans, as well as of albumin and IgG, and total urinary proteins were not modified by either treatments. CONCLUSIONS: These data indicate that patients with NIDDM and overt nephropathy develop abnormalities in size-selective function of the glomerular barrier and, at variance to IDDM, such changes were not ameliorated either by ACE inhibition or calcium channel blockade. (+info)
N-type voltage-dependent calcium channels mediate the nicotinic enhancement of GABA release in chick brain.
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The role of voltage-dependent calcium channels (VDCCs) in the nicotinic acetylcholine receptor (nAChR)-mediated enhancement of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) was investigated in chick brain slices. Whole cell recordings of neurons in the lateral spiriform (SpL) and ventral lateral geniculate (LGNv) nuclei showed that cadmium chloride (CdCl2) blocked the carbachol-induced increase of spontaneous GABAergic IPSCs, indicating that VDCCs might be involved. To conclusively show a role for VDCCs, the presynaptic effect of carbachol on SpL and LGNv neurons was examined in the presence of selective blockers of VDCC subtypes. omega-Conotoxin GVIA, a selective antagonist of N-type channels, significantly reduced the nAChR-mediated enhancement of gamma-aminobutyric acid (GABA) release in the SpL by 78% compared with control responses. Nifedipine, an L-type channel blocker, and omega-Agatoxin-TK, a P/Q-type channel blocker, did not inhibit the enhancement of GABAergic IPSCs. In the LGNv, omega-Conotoxin GVIA also significantly reduced the nAChR-mediated enhancement of GABA release by 71% from control values. Although omega-Agatoxin-TK did not block the nicotinic enhancement, L-type channel blockers showed complex effects on the nAChR-mediated enhancement. These results indicate that the nAChR-mediated enhancement of spontaneous GABAergic IPSCs requires activation of N-type channels in both the SpL and LGNv. (+info)
Light-induced calcium influx into retinal axons is regulated by presynaptic nicotinic acetylcholine receptor activity in vivo.
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Visual activity is thought to be a critical factor in controlling the development of central retinal projections. Neuronal activity increases cytosolic calcium, which was hypothesized to regulate process outgrowth in neurons. We performed an in vivo imaging study in the retinotectal system of albino Xenopus laevis tadpoles with the fluorescent calcium indicator calcium green 1 dextran (CaGD) to test the role of calcium in regulating axon arbor development. We find that visual stimulus to the retina increased CaGD fluorescence intensity in retinal ganglion cell (RGC) axon arbors within the optic tectum and that branch additions to retinotectal axon arbors correlated with a local rise in calcium in the parent branch. We find three types of responses to visual stimulus, which roughly correlate with the ON, OFF, and SUSTAINED response types of RGC reported by physiological criteria. Imaging in bandscan mode indicated that patterns of calcium transients were nonuniform throughout the axons. We tested whether the increase in calcium in the retinotectal axons required synaptic activity in the retina; intraocular application of tetrodotoxin (10 microM) or nifedipine (1 and 10 microM) blocked the stimulus-induced increase in RGC axonal fluorescence. A second series of pharmacological investigations was designed to determine the mechanism of the calcium elevation in the axon terminals within the optic tectum. Injection of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) (20 mM) into the tectal ventricle reduced axonal calcium levels, supporting the idea that visual stimulation increases axonal calcium. Injection of BAPTA (20 mM) into the tectal ventricle to chelate extracellular calcium also attenuated the calcium response to visual stimulation, indicating that calcium enters the axon from the extracellular medium. Caffeine (10 mM) caused a large increase in axonal calcium, indicating that intracellular stores contribute to the calcium signal. Presynaptic nicotinic acetylcholine receptors (nAChRs) may play a role in axon arbor development and the formation of the topographic retinotectal projection. Injection of nicotine (10 microM) into the tectal ventricle significantly elevated RGC axonal calcium levels, whereas application of the nAChR antagonist alphaBTX (100 nM) reduced the stimulus-evoked rise in RGC calcium fluorescence. These data suggest that light stimulus to the retina increases calcium in the axon terminal arbors through a mechanism that includes influx through nAChRs and amplification by calcium-induced calcium release from intracellular calcium stores. Such a mechanism may contribute to developmental plasticity of the retinotectal system by influencing both axon arbor elaboration and the strength of synaptic transmission. (+info)
Retinal input induces three firing patterns in neurons of the superficial superior colliculus of neonatal rats.
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By using an in vitro isolated brain stem preparation, we recorded extracellular responses to electrical stimulation of the optic tract (OT) from 71 neurons in the superficial superior colliculus (SC) of neonatal rats (P1-13). At postnatal day 1 (P1), all tested neurons (n = 10) already received excitatory input from the retina. Sixty-nine (97%) superficial SC neurons of neonatal rats showed three response patterns to OT stimulation, which depended on stimulus intensity. A weak stimulus evoked only one spike that was caused by activation of non-N-methyl-D-aspartate (NMDA) glutamate receptors. A moderate stimulus elicited a short train (<250 ms) of spikes, which was induced by activation of both NMDA and non-NMDA receptors. A strong stimulus gave rise to a long train (>300 ms) of spikes, which was associated with additional activation of L-type high-threshold calcium channels. The long train firing pattern could also be induced either by temporal summation of retinal inputs or by blocking gamma-aminobutyric acid-A receptors. Because retinal ganglion cells show synchronous bursting activity before eye opening at P14, the retinotectal inputs appear to be sufficient to activate L-type calcium channels in the absence of pattern vision. Therefore activation of L-type calcium channels is likely to be an important source for calcium influx into SC neurons in neonatal rats. (+info)