Intracellular calcium stores modulate miniature GABA-mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature gamma-aminobutyric acidA (GABAA) receptor-mediated currents (in tetrodotoxin, 1 microM and kynurenic acid 1 mM) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6-9) old rats. Switching from a Ca2+-containing to a nominally Ca2+-free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 microM EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 microM over control) was 1.8+/-0.5, (n = 9). In nominally Ca2+-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mM), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mM) reversibly reduced the amplitude of the current induced by GABA (100 microM) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 microM), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine-ryanodine-sensitive stores facilitates GABA release.     

Presynaptic GABAB autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons

GABA is the primary transmitter released by neurons of the suprachiasmatic nucleus (SCN), the circadian clock in the brain. Whereas GABAB receptor agonists exert a significant effect on circadian rhythms, the underlying mechanism by which GABAB receptors act in the SCN has remained a mystery. We found no GABAB receptor-mediated effect on slow potassium conductance, membrane potential, or input resistance in SCN neurons in vitro using whole-cell patch-clamp recording. In contrast, the GABAB receptor agonist baclofen (1-100 microM) exerted a large and dose-dependent inhibition (up to 100%) of evoked IPSCs. Baclofen reduced the frequency of spontaneous IPSCs but showed little effect on the frequency or amplitude of miniature IPSCs in the presence of tetrodotoxin. The activation of GABAB receptors did not modulate postsynaptic GABAA receptor responses. The depression of GABA release by GABAB autoreceptors appeared to be mediated primarily through a modulation of presynaptic calcium channels. The baclofen inhibition of both calcium currents and evoked IPSCs was greatly reduced (up to 100%) by the P/Q-type calcium channel blocker agatoxin IVB, suggesting that P/Q-type calcium channels are the major targets involved in the modulation of GABA release. To a lesser degree, N-type calcium channels were also involved. The inhibition of GABA release by baclofen was abolished by a pretreatment with pertussis toxin (PTX), whereas the inhibition of whole-cell calcium currents by baclofen was only partially depressed by PTX, suggesting that G-protein mechanisms involved in GABAB receptor modulation at the soma and axon terminal may not be identical. We conclude that GABAB receptor activation exerts a strong presynaptic inhibition of GABA release in SCN neurons, primarily by modulating P/Q-type calcium channels at axon terminals.     

Tracer and electrical coupling of rat suprachiasmatic nucleus neurons

Whole-cell recording from single neurons of the suprachiasmatic nucleus with an electrode containing the tracer neurobiotin resulted in the staining of multiple neurons in 30% of the cases. Typically, one neuron was darkly stained with dendritic processes and an axon clearly visible while other neurons were lightly stained. The darkly-stained cells were identified as the recorded neuron and tracer-coupled to one to five lightly stained neurons. The resting membrane potential, input membrane conductance, membrane capacitance, the decay time constant and the maximum H-current amplitude of the recorded neurons with tracer-coupled cells were not significantly different from those of neurons not showing tracer coupling. Stimulation of the preoptic area activated an antidromic action potential or an all-or-none small slow inward current in some neurons when the synaptic transmission was blocked by a calcium-free/Mn2+ solution. The small slow inward current did not "collide" with an orthodromically activated action spike suggesting that the current represents the signal from an electrotonically-coupled neuron. In addition, the frequency of biphasic field currents from a neighbouring cell firing were increased by depolarization and decreased by hyperpolarization of the recorded cell. These data demonstrate a chemical and electrical low-resistance coupling of suprachiasmatic nucleus neurons, which could be important in synthesizing the suprachiasmatic nucleus circadian rhythm.     

The role of G proteins in the activity and mercury modulation of GABA-induced currents in rat neurons

The role of protein kinase A (PKA) and protein kinase C (PKC) in the function and modulation by mercury chloride of the GABA(A) receptor-chloride channel complex was studied with rat dorsal root ganglion cells using the whole-cell patch clamp technique. When added to the internal pipette solutions, both KT 5720, a selective PKA inhibitor, and calphostin C, a selective PKC inhibitor, increased the maximal current and shifted the EC50 for GABA in the direction of higher GABA concentrations. GABA-activated currents were decreased by the addition of 5 mM cAMP to the internal pipette solution, and by external perfusion of 100 nM phorbol 13-myristate 13-acetate. Mercury chloride potentiation of GABA-activated currents was blocked by internal application of 5 mM cAMP. PKA in the recording pipette abolished the mercury chloride potentiation of GABA-activated currents. In contrast, 0.56 microM KT 5720, but not calphostin C, in the internal pipette solution enhanced the effect of mercury chloride. In conclusion, both PKA and PKC negatively regulate the activity of the GABA(A) receptor-channel complex probably through phosphorylation of the receptor, and the PKA system underlies the mechanism of mercury chloride potentiation of GABA-activated currents.     

GABAergic modulation of optic nerve-evoked field potentials in the rat suprachiasmatic nucleus

The suprachiasmatic nuclei (SCN) at the base of the hypothalamus are known to be the site of the endogenous circadian pacemaker in mammals. The SCN are innervated by the retinohypothalamic tract, which conveys photic information to the SCN. GABA is one of the most abundant neurotransmitters in the SCN, and has been implicated in the modulation of photic responses of the SCN circadian pacemaker. This study sought to examine the effect of GABAergic compounds on optic nerve-evoked SCN field potentials recorded in rat horizontal hypothalamic slices. The GABAA agonist muscimol (10 microM) potentiated SCN field potentials by 23%, while application of the GABAA antagonist bicuculline (10 microM) inhibited SCN field potentials by a similar amount, (22%). Conversely, the GABA, agonist baclofen (1.0 microM) inhibited SCN field potentials by 48%, while the GABAB antagonist phaclofen (0.5 mM) augmented SCN field potentials by 62%. Recordings performed at both day and night times indicate that there were no qualitative day-night differences in GABAergic activity on SCN field potentials. This study concludes that, in general, GABAA activity tends to increase, and GABAB activity tends to decrease the response of SCN neurons to optic nerve stimulation.     

L-arginine potentiates GABA-mediated synaptic transmission by a nitric oxide-independent mechanism in rat dopamine neurons

Effects of L-arginine in the nervous system are often attributed to nitric oxide. Using whole-cell patch pipettes to record membrane currents in voltage-clamp from dopamine neurons in the rat midbrain slice, the present studies found that L-arginine potentiates GABA-dependent membrane currents via a nitric oxide-independent mechanism. L-Arginine (0.3-10 mM) increased the peak amplitude, half-width duration and time constant of decay of GABA(B) receptor-mediated inhibitory postsynaptic currents in a concentration-dependent manner. In the presence of CGP 35348 (300 microM), a GABA(B) receptor antagonist, L-arginine also prolonged the duration of inhibitory postsynaptic currents mediated by GABA(A) receptors, but their amplitudes were reduced. L-Arginine (10 mM) also evoked 17+/-3 pA of outward current (at -60 mV) which was significantly increased in the presence of exogenous GABA (100 microM). Pressure-ejection of GABA from micropipettes produced outward currents mediated by GABA(B) receptors (recorded in bicuculline) or GABA(A) receptors (recorded in CGP 35348); both types of receptor-mediated currents were increased by L-arginine (10 mM). In contrast, outward currents evoked by baclofen, a GABA(B) receptor agonist, were not potentiated by L-arginine. The GABA transport inhibitors NO 711 (1 microM) and nipecotic acid (1 mM) significantly increased the half-width duration and time-constant of decay of GABA(B)-mediated inhibitory postsynaptic currents, thus mimicking effects of L-arginine. However, nitric oxide donors failed to mimic effects of L-arginine on GABA(B) inhibitory postsynaptic currents, and inhibitors of nitric oxide synthesis failed to selectively block the action of L-arginine. These findings suggest that L-arginine potentiates GABA synaptic transmission by a nitric oxide-independent mechanism. Similarities between effects of L-arginine, NO 711 and nipecotic acid suggest that L-arginine inhibits a GABA transporter.     

Zinc and zolpidem modulate mIPSCs in rat neocortical pyramidal neurons

Pharmacological modulation of gamma-aminobutyric acid-A (GABAA) receptors can provide important information on the types of subunits composing these receptors. In recombinant studies, zinc more potently inhibits alphabeta subunits compared with the alphabetagamma combination, whereas modulation by nanomolar concentrations of the benzodiazepine type 1-selective agonist zolpidem is conferred by the alpha1betagamma2 subunit combination. We examined four properties of miniature inhibitory postsynaptic currents (mIPSCs) from identified necortical pyramidal cells in rat brain slices: decay time constant, peak amplitude, rate of rise, and interevent interval. Exposure to 50 microM zinc reduced the decay time constant, peak amplitude, and rate of rise with no effect on interevent interval. Zolpidem enhanced mIPSCs in a concentration-dependent manner. Both 20 and 100 nM zolpidem increased the decay time constants of mIPSCs. In some cells, both peak amplitude and rate of rise were also enhanced. All cells treated with zinc were also responsive to zolpidem. These results show that neocortical pyramidal cells have a population of GABAA receptors sensitive to both zinc and zolpidem.     

Direct retinal projections to GRP neurons in the suprachiasmatic nucleus of the rat

The retinal projections to gastrin-releasing peptide (GRP)-expressing neurons in the rat suprachiasmatic nucleus (SCN) were investigated by double immunofluorescence and immunoelectron microscopy. Optic nerve terminals labeled by cholera toxin B subunit (CTb) which was transported from the retinal ganglion cells were intermingled with GRP-immunoreactive cell bodies and processes in the ventrolateral portion of the SCN. Ultrastructural analysis revealed that CTb-immunoreactive retinal terminals made synaptic contacts with GRP-immunoreactive dendritic processes. These results demonstrated that photic information is directly input from the optic nerve to GRP neurons in the SCN and these GRP neurons may be involved in circadian entrainment by light.     

On the mechanism of GABA-induced currents in cultured rat cortical neurons

We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by gamma-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl- concentration ([Cl-]i). In current-clamp experiments in the presence of 26 mM HCO3- the application of 50 microM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50-200 microM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO3-. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO3--free saline, indicating a contribution from HCO3-. The reversal potential of the GABA-induced currents (EGABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO3- was -73 mV, while it was shifted to -59 mV in the presence of HCO3-. Combined patch-clamp and microfluorimetric measurements using the Cl--sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl-]i in 54% (n=13) of the neurons. We conclude that this increase of [Cl-]i in combination with the efflux of HCO3- results in a shift of EGABA above the resting membrane potential that gives rise to GABA-mediated depolarizations.     

Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15-30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.     

Calcium currents in acutely isolated stellate and pyramidal neurons of rat entorhinal cortex

Calcium currents were studied in morphologically identified pyramidal and stellate neurons acutely isolated from layer II/III of rat entorhinal cortex, using the whole-cell patch-clamp configuration. The peak amplitude of high-voltage activated current (HVA) measured at +10 mV was not different in both neuron populations with 0.94+/-0.08 nA for pyramidal and 1.03+/-0.08 nA for stellate cells. Stellate neurons had a larger capacitance (14.4+/-1. 1 pF) than pyramidal neurons (9.6+/-0.8 pF), indicating a 50% larger cell surface. Most striking was the difference between the current density in stellate (79+/-8 pA/pF) versus pyramidal neurons (113+/-13 pA/pF). The potential of half maximal inactivation was not different: -37+/-2 mV (pyramidals) and -37+/-3 mV (stellates). Half of the cells contained a low-voltage activated calcium current (LVA) with a peak amplitude that was twice as large in stellate as in pyramidal neurons (0.21+/-0.04 nA resp. 0.11+/-0.03 nA; at -50 mV). In contrast to the HVA component, the current density of the LVA component was not different between cell types (13+/-3 pA/pF vs. 13+/-2 pA/pF). This implies that the relative abundance of LVA and HVA currents in stellate and pyramidal neurons is different which could result in different firing characteristics. The potential of half maximal LVA inactivation was -88+/-4 mV (pyramidals) and -85+/-3 mV (stellates). The slope of the voltage dependent steady state inactivation was steeper in stellate (7+/-1 mV) than in pyramidal cells (10+/-2 mV).     

Differential effects of angiotensin II on the activities of suprachiasmatic neurons in rat brain slices

The effects of angiotensin II (AII) on the firing rates of suprachiasmatic neurons were determined in rat brain slices. AII in pmol ranges stimulated 25% and inhibited another 25% of 52 irregular firing neurons, while it stimulated 23% and inhibited 4% of 30 regular firing neurons. Three "oscillating" neurons whose firing rates oscillated with rather constant amplitudes and periods were recorded. AII induced the occurrence of oscillation in one unit and modulated the oscillation amplitude of the other two. Pretreatment with saralasin, an AII antagonist, effectively blocked (100%) the actions of AII (n = 5). The present findings suggest that AII may act as an important mediator in the suprachiasmatic nucleus and its mode of action may be variable in different neurons.     

Modulation of voltage-activated calcium currents by mechanical stimulation in rat sensory neurons

We examined the effects of mechanical stress, induced by a stream of bath solution, on evoked action potentials, electrical excitability, and Ca2+ currents in rat dorsal root ganglion neurons in culture with the use of the whole cell patch-clamp technique. Action-potential duration was altered reversibly by flow in 39% of the 51 neurons tested, but membrane potential and excitability were unaffected. The flow-induced increases and decreases in action-potential duration were consistent with the different effects of flow on two types of Ca2+ channel, determined by voltage-clamp recordings of Ba2+ currents. Current through omega-conotoxin-sensitive (N-type) Ca2+ channels increased by an estimated 74% with flow, corresponding to 23% increase in the total high voltage-activated current, whereas current through low-threshold voltage-activated (T-type) channels decreased by 14%. We conclude that modulation of voltage-activated Ca2+ currents constitutes a route by which mechanical events can regulate Ca2+ influx in sensory neurons.     

异丙酚对大鼠海马神经元低电压激活钙电流的抑制作用

目的:观察异丙酚对大鼠海马锥体神经元低电压激活钙电流[low-voltage-activated calcium currents,ICa(LVA)]的影响.方法:培养Wistar大鼠海马锥体神经元,采用全细胞膜片钳技术测定ICa(LVA).加用不同浓度(3、10、30、100、300μmol/L)异丙酚后,计算ICa(LVA)抑制率,建立异丙酚的浓度-效应曲线,选择20μmol/L异丙酚作ICa(LVA)的激活及失活曲线.结果:3 μmol/L的异丙酚对ICa(LVA)的电流幅度无影响;10、30、100、300 μmol/L的异丙酚对ICa(LVA)的电流幅度抑制率分别为(12.6±4.1)%、(29.2±5.7)%、(36.6±5.3)%、(31.6±2.6)%.拟合后的浓度-效应曲线的IC50为16.8 μmol/L,Hill系数为0.15.激活曲线的半数最大激活膜电位(V1/2)由(-10±1)mV移动到(-11±2)mV;K分别为12±1和8±1;失活曲线的V1/2分别为(-25±1)mV和(-25±5)mV,K分别为15±1和16±3.20 μmol/L异丙酚均未使ICa(LVA)的激活曲线及稳态失活曲线发生明显移动.结论:异丙酚对ICa(LVA)通道有抑制作用,异丙酚对中枢神经系统的麻醉作用可能与ICa(LVA)抑制有关.     

Riluzole interacts with voltage-activated sodium and potassium currents in cultured rat cortical neurons

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.     

Nitric oxide and excitatory postsynaptic currents in immature rat sympathetic preganglionic neurons in vitro

PURPOSE: A disadvantage of ovoid shields in a Fletcher-type applicator is that these shields cause artifacts on postimplant CT images. CT images, however, make it possible to calculate the dose distribution in the rectum and the bladder. To be able to estimate the possible advantage of having CT information over the use of ovoid shields without having CT information, we investigated the influence of shielding segments in a Fletcher-type Selectron-LDR applicator on the dose distribution in rectum and bladder. METHODS AND MATERIALS: Contours of rectum and bladder were delineated on transaxial CT slices of 15 unshielded applications. Of the volumes contained within these structures dose-volume histograms (DVHs) were calculated. In a similar way, DVHs of simulated shielded applications were calculated. The reduction, due to shielding, of the dose to the 2 cm3 (D2) and 5 cm3 (D5) volume of the cumulative DVHs of rectum and bladder, were determined. An isodose pattern in the sagittal plane through the center of each applicator was plotted to compare the location of the shielded area with the location of maximum dose in rectum and bladder in the unshielded situation. In two cases local dose reductions to the rectal wall were determined by calculating the dose in points at 10-mm intervals on the rectal contours. RESULTS: For the rectum, the reduction of D2 ranged from 0 to 11.1%, with an average of 5.0%; the reduction of D5 ranged from 2.3 to 12.1%, with an average of 6.4%. The reduction of D2 and D5 for the bladder ranged from 0 to 11.9% and from 0 to 11.6%, with average values of 2.2 and 2.6%, respectively. In 8 out of 15 cases the rectal maximum dose was located inferior to the shielded area. In all cases except one the bladder maximum dose was located superior to the shielded area. Local dose reductions on the rectal wall can be as high as 30% or more in an optimally shielded area. CONCLUSIONS: Reductions of D2 and D5 to rectum and bladder due to shielding are rather small, because the shielded area does usually not coincide with the high dose region and even if it does, the shielded area is too small to result in large reductions of these values. Because local dose reductions vary largely, one should proceed with caution when calculating the dose in just one rectal or bladder reference point. Because large overall dose reductions cannot be achieved with shielding, it is safe to use an unshielded applicator when post implant CT images are used to realize optimized dose distributions.     

Dopaminergic modulation of NMDA-induced whole cell currents in neostriatal neurons in slices: contribution of calcium conductances

The present experiments were designed to examine dopamine (DA) modulation of whole cell currents mediated by activation of N-methyl-D-aspartate (NMDA) receptors in visualized neostriatal neurons in slices. First, we assessed the ability of DA, D1 and D2 receptor agonists to modulate membrane currents induced by activation of NMDA receptors. The results of these experiments demonstrated that DA potentiated NMDA-induced currents in medium-sized neostriatal neurons. Potentiation of NMDA currents occurred at three different holding potentials, although it was more pronounced at -30 mV. It was mediated by D1 receptors, because it was mimicked by D1 agonists and blocked by exposure to a D1 antagonist. Activation of D2 receptors produced inconsistent effects on NMDA-induced membrane currents. Either decreases, increases, or no effects on NMDA currents occurred. Second, we examined the contributions of intrinsic, voltage-dependent conductances to DA potentiation of NMDA currents. Blockade of K+ conductances did not prevent DA enhancement of NMDA currents. However, voltage-activated Ca2+ conductances provided a major contribution to DA modulation. The dihydropyridine L-type Ca2+ channel blockers, nifedipine, and methoxyverapamil (D-600), markedly reduced but did not totally eliminate the ability of DA to modulate NMDA currents. The D1 receptor agonist SKF 38393 also enhanced Ba2+ currents in neostriatal neurons. Together, these findings provide evidence for a complex interplay between DA, NMDA receptor activation and dihydropyridine-sensitive Ca2+ conductances in controlling responsiveness of neostriatal medium-sized neurons.     

Facilitation of miniature GABAergic currents by ruthenium red in neonatal rat hippocampal neurons

The whole cell configuration of the patch-clamp technique was used to study the modulation gamma-aminobutyric acid (GABA)-mediated postsynaptic currents by ruthenium red in CA3 hippocampal neurons in slices obtained from postnatal (P) days P6-P10 old rats. In the presence of kynurenic acid (1 mM), ruthenium red (100 microM) completely blocked stimulus-elicited GABA-mediated postsynaptic currents and reduced by 50% the amplitude of the spontaneous ones. Ruthenium red (100 microM) increased the frequency but not the amplitude of miniature GABAergic currents recorded in the presence of tetrodotoxin (1 microM) and kynurenic acid (1 mM), an effect that was prevented by heparin (100 microM). Ruthenium red did not modify the kinetics of miniature postsynaptic currents and the currents induced by exogenous application of GABA (10 microM) in the presence of tetrodotoxin, suggesting that its action was presynaptic in origin. The effects of ruthenium red on quantal GABA release was independent of external calcium. In a nominally Ca2+-free solution the potentiating effect induced by this polyvalent cation on miniature postsynaptic currents was still present. Intracellular calcium stores were not involved in ruthenium red action, because this polyvalent cation was able to facilitate miniature currents also in the presence of thapsigargin (10-20 microM). These results indicate that ruthenium red has a dual action on GABA release from GABAergic interneurons: it reduces the amplitude of spontaneous events and increases the frequency of miniature currents. The former effect is calcium-dependent, whereas the latter is calcium independent.     

Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons

Cell swelling induced by activation of excitatory amino acid receptors is presumably the first step in a toxic cascade that may ultimately lead to cell death. Previously we showed that bath application of N-methyl-D-aspartate (NMDA) or kainate (KA) produces swelling of neostriatal cells. The present experiments examined modulation of NMDA and KA-induced cell swelling by dopamine (DA) and its receptor agonists. Nomarski optics and infra-red videomicroscopy were utilized to visualize neostriatal medium-sized neurons in thick slices from rat pups (12-18 postnatal days). Increase in somatic cross-sectional area served as the indicator of swelling induced by bath application of glutamate receptor agonists. NMDA induced cell swelling in a dose-dependent manner. Activation of DA receptors in the absence of NMDA did not produce swelling. DA and the D1 receptor agonist SKF 38393, increased the magnitude of swelling produced by NMDA. This effect was reduced in the presence of the D1 receptor antagonist, SCH 23390. In contrast, activation of D2 receptors by quinpirole decreased the magnitude of NMDA-induced cell swelling. DA slightly attenuated cell swelling induced by activation of KA receptors. Quinpirole produced a significant concentration-dependent reduction in KA-induced swelling while SKF38393 increased KA-induced swelling, but only at a low concentration of KA. Together, these results provide additional support for the hypothesis that the direction of DA modulation depends on the glutamate receptor subtype, as well as the DA receptor subtype activated. One possible consequence of these observations is that endogenous DA may be an important contributing factor in the mechanisms of cell death in Huntington's disease.     

M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons

Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-response relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 microM ACh was approximately 75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (/=30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinic receptor-mediated inhibition. Both dihydropyridine- and omega-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.