AMP kinase regulates K-ATP currents evoked by NMDA receptor stimulation in rat subthalamic nucleus neurons.

Our lab recently showed that N-methyl-D-aspartate (NMDA) evokes ATP-sensitive K(+) (K-ATP) currents in subthalamic nucleus (STN) neurons in slices of the rat brain. Both K-ATP channels and 5'-adenosine monophosphate-activated protein kinase (AMPK) are considered cellular energy sensors because their activities are influenced by the phosphorylation state of adenosine nucleotides. Moreover, AMPK has been shown to regulate K-ATP function in a variety of tissues including pancreas, cardiac myocytes, and hypothalamus. We used whole-cell patch clamp recordings to study the effect of AMPK activation on K-ATP channel function in STN neurons in slices of the rat brain. We found that bath or intracellular application of the AMPK activators A769662 and PT1 augmented tolbutamide-sensitive K-ATP currents evoked by NMDA receptor stimulation. The effect of AMPK activators was blocked by the AMPK inhibitor dorsomorphin (compound C), and by STO609, an inhibitor of the upstream AMPK activator CaMKKß. AMPK augmentation of NMDA-induced K-ATP current was also blocked by intracellular BAPTA and by inhibitors of nitric oxide synthase and guanylyl cyclase. However, A769662 did not augment currents evoked by the K-ATP channel opener diazoxide. In the presence of NMDA, A769662 inhibited depolarizing plateau potentials and burst firing, both of which could be antagonized by tolbutamide or dorsomorphin. These studies show that AMPK augments NMDA-induced K-ATP currents by a Ca(2+)-dependent process that involves nitric oxide and cGMP. By augmenting K-ATP currents, AMPK activation would be expected to dampen the excitatory effect of glutamate-mediated transmission in the STN.

5-HT inhibits synaptic transmission in rat subthalamic nucleus neurons in vitro.

The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 microM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35+/-4%. However, a much higher concentration of 5-HT (100 microM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration-response curves showed that the 5-HT inhibitory concentration 50% (IC50) for inhibition of IPSCs (20.2 microM) was more than fivefold greater than the IC50 for inhibition of EPSCs (3.4 microM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT1B receptors.

Multiple conductances are modulated by 5-HT receptor subtypes in rat subthalamic nucleus neurons.

Firing patterns of subthalamic nucleus (STN) neurons influence normal and abnormal movements. The STN expresses multiple 5-HT receptor subtypes that may regulate neuronal excitability. We used whole-cell patch-clamp recordings to characterize 5-HT receptor-mediated effects on membrane currents in STN neurons in rat brain slices. In 80 STN neurons under voltage-clamp (-70 mV), 5-HT (30 microM) evoked inward currents in 64%, outward currents in 17%, and biphasic currents in 19%. 5-HT-induced outward current was caused by an increased K(+) conductance (1.4+/-0.2 nS) and was blocked by the 5-HT(1A) antagonist WAY 100135. The 5-HT-evoked inward current, which was blocked by antagonists at 5-HT(2C) and/or 5-HT(4) receptors, had two types of current-voltage (I-V) relations. Currents associated with the type 1 I-V relation showed negative slope conductance at potentials <-110 mV and were occluded by Ba(2+). In contrast, the type 2 I-V relation appeared linear and had positive slope conductance (0.64+/-0.11 nS). Type 2 inward currents were Ba(2+)-insensitive, and the reversal potential of -19 mV suggests a mixed cation conductance. In STN neurons in which 5-HT evoked inward currents, 5-HT potentiated burst firing induced by N-methyl-d-aspartate (NMDA). But in neurons in which 5-HT evoked outward current, 5-HT slowed NMDA-dependent burst firing. We conclude that 5-HT receptor subtypes can differentially regulate firing pattern by modulating multiple conductances in STN neurons.

Presynaptic inhibition of synaptic transmission by adenosine in rat subthalamic nucleus in vitro.

Whole-cell patch clamp recordings were made from the subthalamic nucleus in rat brain slice preparations to examine the effect of adenosine on inhibitory and excitatory synaptic transmission. Adenosine reversibly inhibited both GABA-mediated inhibitory and glutamate-mediated excitatory postsynaptic currents. Adenosine at 100 microM reduced the amplitude of inhibitory and excitatory postsynaptic currents by 42+/-5% and 34+/-6%, respectively. Reductions in the amplitude of both inhibitory and excitatory postsynaptic currents were accompanied by increases in paired-pulse ratios. In addition, adenosine decreased the frequency of spontaneous miniature excitatory postsynaptic currents but had no effect on their amplitude. These results are consistent with a presynaptic site of action. The adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine completely reversed the adenosine-induced attenuation of inhibitory and excitatory postsynaptic currents, but 8-cyclopentyl-1,3-dipropylxanthine alone had no effect on synaptic currents evoked at 0.1 Hz. However, 8-cyclopentyl-1,3-dipropylxanthine inhibited a time-dependent depression of excitatory postsynaptic currents that was normally observed in response to a 5 Hz train of stimuli, suggesting that endogenous adenosine could be released during higher frequencies of stimulation. These results suggest that adenosine inhibits synaptic release of GABA and glutamate by stimulation of presynaptic A(1) receptors in the subthalamic nucleus.

Presynaptic GABA(B) receptors inhibit synaptic inputs to rat subthalamic neurons.

Effects of baclofen on synaptic transmission were studied in rat subthalamic neurons using whole-cell patch clamp recording from brain slices. Focal electrical stimulation of the brain slice evoked GABAergic inhibitory postsynaptic currents and glutamatergic excitatory postsynaptic currents. Baclofen reduced the amplitude of evoked inhibitory postsynaptic currents in a concentration-dependent manner with an IC(50) of 0.6+/-0.2 microM. Evoked excitatory postsynaptic currents were also reduced by baclofen concentration-dependently (IC(50) of 1.6+/-0.2 microM), but baclofen was more potent at reducing the GABA(A) receptor inhibitory postsynaptic currents. The GABA(B) receptor antagonist CGP 35348 blocked these inhibitory effects of baclofen on evoked inhibitory and excitatory postsynaptic currents. Baclofen increased the paired-pulse ratios of evoked inhibitory and excitatory postsynaptic currents. Furthermore, baclofen reduced the frequency of spontaneous miniature excitatory postsynaptic currents, but had no effect on their amplitude. These results provide evidence for presence of presynaptic GABA(B) receptors that modulate both GABA and glutamate release from afferent terminals in the subthalamus.

Potentiation of GABA(A) receptor agonists by GABA uptake inhibitors in the rat ventral midbrain.

Whole-cell patch recordings were made from dopamine-containing neurons in the ventral tegmental area (VTA) and substantia nigra zona compacta (SNC). Isoguvacine evoked an outward current (at -60 mV) in a concentration-dependent manner with an EC50 of 62+/-8 microM. The gamma-aminobutyric acid (GABA) uptake inhibitor 1-(2(((diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711) (3 microM) shifted the isoguvacine concentration-response curve to the left, with a new EC50 of 22+/-4 microM. L-Arginine (3 mM) also shifted the isoguvacine concentration-response curve to the left, with a new EC50 of 29+/-5 microM. L-Arginine (3 mM) increased the currents evoked by GABA (100 microM) and muscimol (1 microM) by 208% and 261%, respectively. The GABA uptake inhibitor 4,5,6,7,-tetrahydroisoxazolo[4,5-c]-pyridin-3-ol hydrobromide (THPO) (300 microM) not only mimicked but also occluded the ability of L-arginine (3 mM) to potentiate currents evoked by isoguvacine. Equimolar replacement of Na+ with choline increased GABA-evoked currents, suggesting that a low Na+ concentration has an inhibitory effect on GABA transport. Low Na+ concentration (25 mM) inhibited isoguvacine currents but still occluded the potentiating effects of L-arginine. We conclude that GABA uptake inhibitors potentiate the actions of the GABA(A) receptor agonists, isoguvacine and muscimol, probably because they are effective substrates for GABA transporters in the ventral midbrain.

Presynaptic dopamine D2 and muscarine M3 receptors inhibit excitatory and inhibitory transmission to rat subthalamic neurones in vitro.

Whole-cell patch-clamp recordings were made from subthalamic nucleus (STN) neurones in brain slices from rats. Stimulation with bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA (IPSCs) receptors. Dopamine reversibly reduced the amplitude of GABAA IPSCs by up to 48 % with an IC50 value of 3.4 +/- 0.8 microM. The dopamine D2 receptor agonist quinpirole, but not the D1 receptor agonist SKF 82958, also inhibited GABAA IPSCs. This effect was completely reversed by the D2 receptor antagonist sulpiride but not by SCH 23390, a D1 antagonist. Muscarine reversibly reduced the amplitude of GABAA IPSCs by up to 70 % with an IC50 value of 0.6 +/- 0.1 microM. Inhibition of IPSCs by muscarine was completely blocked by scopolamine (10 microM), a muscarinic receptor antagonist. The M3 muscarinic receptor antagonist 4-DAMP effectively reversed muscarine-induced inhibition of IPSCs with an IC50 of 0.11 +/- 0.03 microM. Although the M1 receptor antagonist pirenzepine also reversed the inhibition of IPSCs by muscarine, this effect was only observed at relatively high concentrations (IC50 = 21.7 +/- 9.4 microM). Dopamine and muscarine both increased the paired-pulse ratio of GABAA IPSCs. Neither agent produced sustained changes in postsynaptic holding current. Glutamate EPSCs were also inhibited reversibly by dopamine (by up to 29%; IC50 = 16 +/- 3 microM) and muscarine (by up to 41%; IC50 = 1.0 +/- 0.4 microM). However, both agents were more potent and efficacious for reducing GABA IPSCs compared with glutamate EPSCs. These results suggest that the most significant effect of dopamine and muscarine in the STN is to reduce inhibitory synaptic input by acting at presynaptic dopamine D2 and muscarinic M3 receptors, respectively.

Presynaptic inhibition preferentially reduces in NMDA receptor-mediated component of transmission in rat midbrain dopamine neurons.

We used patch pipettes to record whole-cell currents from single dopamine neurons in slices of rat midbrain. Pharmacological methods were used to isolate EPSCs evoked by focal electrical stimulation. Baclofen was significantly more potent for inhibiting NMDA receptor-mediated EPSCs (IC50=0.24 microM) compared with inhibition of EPSCs mediated by AMPA receptors (IC50=1.72 microM). The increased potency of baclofen for inhibiting the NMDA component persisted in superfusate that contained zero Mg2+ and when postsynaptic K+ conductances were reduced by Cs+ and QX-314. Effects of baclofen on EPSCs were blocked by the GABA(B) receptor antagonist CGP-35348. Adenosine was 20 fold more potent for reducing the NMDA component of transmission (IC50=31 microM) compared with inhibition of AMPA receptor-mediated EPSCs (IC50=654 microM). Effects of adenosine on EPSCs were blocked by the A1 receptor antagonist DPCPX. Both baclofen and adenosine significantly increased the ratio of EPSCs in paired-pulse studies, suggesting presynaptic sites of action. Although adenosine (1 mM) did not reduce currents evoked by exogenous NMDA (10 microM), baclofen (1 microM) reduced NMDA currents by 29%. Neither baclofen nor adenosine altered currents evoked by exogenous AMPA (1 microM). We conclude that adenosine acts at presynaptic A1 receptors to cause a preferential reduction in the NMDA component of synaptic transmission. In contrast, baclofen preferentially reduces NMDA EPSCs by acting at both pre- and postsynaptic GABA(B) receptors. By regulating NMDA receptor function, A1 and GABA(B) receptors may play important roles in regulating the excitability of dopamine neurons.

Sodium pump evokes high density pump currents in rat midbrain dopamine neurons.

1. Patch pipettes contained various concentrations of Na+ ([Na+]pip) in order to record strophanthidin-sensitive currents under voltage clamp in dopamine neurons in slices of rat substantia nigra and ventral tegmental area. 2. When [Na+]pip was 40 mM and the external K+ concentration ([K+]o) was 2.5 mM, strophanthidin (10 microM) evoked 461 +/- 121 pA of inward current. This effect was concentration dependent, with an EC50 of 7.1 +/- 2.6 microM. At potentials of -60 to -120 mV, strophanthidin-induced currents were not associated with significant changes in chord conductance. 3. Strophanthidin (10 microM) evoked 234 +/- 43 pA of inward current when [Na+]pip was 0.6 mM, and 513 +/- 77 pA when [Na+]pip was 80 mM. Despite higher pump currents with greater [Na+]pip, the strophanthidin EC50 was not significantly different for any of six different [Na+]pip. 4. Sodium pump currents were half-maximal when the [Na+]pip was about 1.3 mM. Maximum pump current was estimated at 830 pA (29 microA cm-2) at concentrations of intracellular Na+ that were assumed to be saturating (50-100 mM). 5. Strophanthidin currents were smaller in a reduced [K+]o (EC50 = 0.2 mM). 6. These data show that intracellular Na+ loading evokes relatively large pump currents. Our results are consistent with the physiological role of the sodium pump in burst firing in midbrain dopamine neurons

Aromatic residues affecting permeation and gating in dSlo BK channels.

Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site.

Presynaptic GABAB and adenosine A1 receptors regulate synaptic transmission to rat substantia nigra reticulata neurones.

1. Patch pipettes were used to record whole-cell currents under voltage clamp in substantia nigra zona reticulata (SNR) neurones in the rat midbrain slice. Bipolar electrodes evoked synaptic currents mediated by glutamate (EPSCs) and GABAA receptors (IPSCs). 2. Baclofen reduced the amplitude of IPSCs by 48% at its IC50 value of 0.60 microM. The GABAB antagonist CGP 35348 blocked this effect with a Kd value estimated by Schild analysis of 5 microM. 3. Adenosine reduced IPSCs by 48% at its IC50 value of 56 microM. Adenosine agonists reduced IPSCs with the following rank order of potency: CPA (N6-cyclopentyladenosine) > R-PIA (R(-)N6-(2-phenylisopropyl)adenosine) > CHA (N6-cyclohexyladenosine) = NECA (5'-N-ethylcarboxamidoadenosine) > 2-CADO (2-chloroadenosine) > adenosine. Schild analysis yielded a Kd value of 0.4 nM for antagonism of CPA by the adenosine A1 receptor antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine). 4. Both baclofen and adenosine reduced the magnitude of paired-pulse depression of IPSCs, and neither blocked currents evoked by GABA, which was pressure-ejected from micropipettes. 5. Glutamate EPSCs were reduced by baclofen (IC50 = 0.78 microM) and adenosine (IC50 = 57 microM). Schild analysis yielded a Kd value of 11 microM for antagonism of baclofen-induced inhibition of EPSCs by CGP 35348. DPCPX (1 microM) completely blocked the inhibitory effects of adenosine (100 microM) and CPA (100 nM) on EPSCs. Neither adenosine nor baclofen reduced inward currents evoked by glutamate which was pressure-ejected from micropipettes. 6. These results show that presynaptic GABAB and A1 receptors reduce glutamate and GABA release from nerve terminals in the SNR.

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.

A slow excitatory postsynaptic current mediated by G-protein-coupled metabotropic glutamate receptors in rat ventral tegmental dopamine neurons.

Dopamine neurons in the substantia nigra and ventral tegmental area express metabotropic glutamate receptors, but activation of these receptors by synaptic release of neurotransmitter has not been demonstrated thus far. Patch pipettes were used to record membrane currents under voltage clamp from presumed dopamine-containing neurons in the whole-cell configuration in the rat brain slice. A short train of electrical stimuli delivered to bipolar electrodes placed in the slice evoked a slow excitatory postsynaptic current (EPSC; 50-300 pA at -70 mV) which peaked 560 ms after onset and lasted several seconds, with a decay time-constant of 630 ms. This slow EPSC was voltage-dependent, and was abolished by tetrodotoxin (0.5 microM) or by perfusate containing low calcium (0.5 mM) and high magnesium (10 mM). The metabotropic glutamate receptor antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine (MCPG; 300 microM) blocked the slow EPSC, but L(+)-2-amino-3-phosphonopropionic acid (AP3; 300 microM) had no effect. The slow EPSC was largely occluded by inward current produced by the metabotropic receptor agonist trans-(+/-)-1-amino-1, 3-cyclopentanedicarboxylic acid (t-ACPD; 300 microM), and the EPSC was reduced > 90% during acute desensitization produced by prolonged perfusion with t-ACPD. (+/-)-2-Amino-4-phosphonobutyric acid (AP4; 300 microM), another metabotropic receptor agonist, reduced the slow EPSC but had no effect on currents evoked by t-ACPD applied by pressure-ejection from micropipettes. The slow EPSC was progressively reduced in amplitude when pipettes contained the G-protein inhibitor GDP-beta-S (0.5 mM). When pipettes contained GTP-gamma-S (0.5 mM), a non-hydrolysable analogue of GTP, onset of the slow EPSC was more rapid and its decay was significantly prolonged. These results demonstrate that a slow EPSC mediated by G-protein-coupled metabotropic glutamate receptors can be evoked in dopamine neurons.

Pharmacology of ACEA-1416: a potent systemically active NMDA receptor glycine site antagonist.

Excitatory amino acid receptor antagonists show potential for the treatment of ischemic stroke and head trauma. In search of novel antagonists, a series of alkyl- and alkoxyl-substituted 1, 4-dihydro-2,3-quinoxalinediones were synthesized and assayed for inhibition of glutamate receptors. We report on the pharmacological characterization of one such compound, 7-chloro-6-methyl-5-nitro-1,4-dihydro-2, 3-quinoxalinedione (ACEA-1416). Electrophysiological assays showed that ACEA-1416 is a potent antagonist of rat brain NMDA receptors expressed in Xenopus oocytes, and NMDA receptors expressed by cultured rat cortical neurons. Antagonism is via competitive inhibition at glycine co-agonist sites (Kb = 7.9 nM in oocytes, Kb = 11 nM in neurons). ACEA-1416 also antagonizes AMPA receptors, though potency is considerably lower (Kb = 3.5 microM in oocytes, Kb = 1.6 microM in neurons). Oocyte assays indicated that ACEA-1416 is weak or inactive as an antagonist at NMDA receptor glutamate binding sites (Kb > 5.9 microM) and metabotropic glutamate receptors (Kb > 57 microM). Many NMDA receptor glycine site antagonists show poor penetration of the blood-brain barrier. Systemic bioavailability of ACEA-1416 was assessed by measuring the ability of the compound to protect against electroshock-induced seizures in mice. Protective effects of ACEA-1416 had rapid onset following i.v. administration. Peak efficacy was at approximately 2 min and the biological half-time of protection was approximately 60 min. The ED50 measured at peak efficacy was approximately 1.5 mg/kg. Our results show that ACEA-1416 is a high potency systemically active NMDA receptor glycine site antagonist and a moderate potency AMPA receptor antagonist. Separate studies indicate that ACEA-1416 is efficacious as a neuroprotectant in a rat model of focal cerebral ischemia. Taken together, our results suggest that ACEA-1416 has potential for clinical development as a neuroprotectant.

Inhibition of morphine tolerance by NMDA receptor antagonists in the formalin test.

5-Nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1328) was characterized in vitro for antagonism of excitatory amino acid receptors, and subsequently tested in vivo and compared with MK-801 for phencyclidine (PCP)-like motor stimulation, antinociception, and effects on morphine tolerance in mice. Assayed on rat cerebral cortical glutamate receptors expressed in Xenopus oocytes ACEA-1328 showed potent (Kb approximately 40 nM) antagonism at NMDA receptor/glycine sites and moderate (Kb approximately 3 microM) antagonism at non-NMDA receptors. In both cases inhibition was predominantly competitive. ACEA-1328 was weak, or inactive, at NMDA receptor glutamate recognition sites, metabotropic receptors and opioid binding sites. In the formalin and rotarod tests ACEA-1328 and MK-801 produced both antinociception and disturbances of motor coordination. MK-801 caused a PCP-like motor stimulatory effect, whereas ACEA-1328 was devoid of such an effect. In tolerance studies, ACEA-1328 and MK-801 each blocked morphine tolerance in the formalin test, the effect of ACEA-1328 was dose-dependent. Our data contribute to a growing body of evidence which suggests that activation of NMDA receptors is critical for the development of opioid tolerance, and that antagonism at NMDA receptor/glycine sites may have potential as a means of diminishing tolerance with no PCP-like motor stimulatory side effects.

3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor.

Neuroactive steroids bind to a unique site on the gamma-aminobutyric acidA (GABAA) receptor complex and allosterically modulate the binding of convulsant ([35S]t-butylbicyclophosphorothionate, [35S]TBPS), GABA ([3H]muscimol), and benzodiazepine ([3H]flunitrazepam) site ligands. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [35S]TBPS binding and enhancing [3H]flunitrazepam and [3H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [35S]TBPS (44% inhibition), [3H]flunitrazepam (41% enhancement), and [3H]muscimol (< 10% enhancement). In competition experiments, Co 2-1970 (10 microM) reduced the apparent potency of 3 alpha, 5 alpha-P by 7-17-fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABAA receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [35S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 microM, reduced the apparent potency of 3 alpha, 5 alpha-P 57-fold for inhibiting [35S]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these findings functionally, the effects of 3 alpha, 5 alpha-P and Co 2-1970 were examined electrophysiologically in Xenopus oo-cytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3 alpha, 5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha, 5 alpha-P-induced potentiation that was associated with a reduction in the apparent affinity of 3 alpha, 5 alpha-P (11-fold at 10 microM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABAA receptors.

Sulfonylurea-sensitive potassium current evoked by sodium-loading in rat midbrain dopamine neurons.

In Parkinson's disease, there is evidence of impaired mitochondrial function which reduces the capacity to synthesize ATP in dopamine neurons. This would be expected to reduce the activity of the sodium pump (Na+/K+ ATPase), causing increased intracellular levels of Na+. Patch pipettes were used to introduce Na+ (40 mM in pipette solutions) into dopamine neurons in the rat midbrain slice in order to study the electrophysiological effects of increased intracellular Na+. We found that intracellular Na+ loading evoked 100-300 pA of outward current (at -60 mV) and increased whole-cell conductance; these effects developed gradually during the first 10 min after rupture of the membrane patch. Extracellular Ba2+ reduced most of the outward current evoked by Na+ loading; this Ba(2+)-sensitive current reversed direction at the expected reversal potential for K+ (EK), and was also blocked by extracellular tetraethylammonium (30 mM) and intracellular Cs+ (which replaced K+ in pipette solutions). The sulfonylurea drugs glipizide (IC50 = 4.9 nM), tolbutamide (IC50 = 23 microM) and glibenclamide (1 microM) were as effective as 300 microM Ba2+ in reducing the K+ current evoked by Na+ loading. When recording with "control" pipettes containing 15 mM Na+, diazoxide (300 microM) increased chord conductance and evoked outward current at -60 mV, which also reversed direction near EK. Effects of diazoxide were blocked by glibenclamide (1 microM) or glipizide (300 nM). Diazoxide (300 microM) and baclofen (3 microM), which also evoked K(+)-mediated outward currents recorded with control pipettes, caused little additional increases in outward currents during Na+ loading. Raising ATP concentrations to 10 mM in pipette solutions failed to significantly reduce currents evoked by diazoxide or Na+ loading, suggesting that these currents may not be mediated by ATP-sensitive K+ channels. Finally, Na+ loading using pipettes containing Cs+ in place of K+ evoked a relatively small outward current (50-150 pA at -60 mV), which developed gradually over the first 10 min after rupturing the membrane patch. This current was reduced by dihydro-ouabain (3 microM) and a low extracellular concentration of K+ (0.5 mM instead of 2.5 mM), but was not affected by Ba2+. We conclude that intracellular Na+ loading evokes a current generated by Na+/K+ ATPase in addition to sulfonylurea-sensitive K+ current. This Na(+)-dependent K+ current is unusual in its sensitivity to sulfonylureas, and could protect dopamine neurons against toxic effects of intracellular Na+ accumulation.

Blockade of morphine tolerance by ACEA-1328, a novel NMDA receptor/glycine site antagonist.

Recent studies indicate that competitive and non-competitive NMDA receptor antagonists can block the development of morphine tolerance. Since glycine is considered to be a co-agonist for activation of NMDA receptors we examined the effect of a novel bioavailable NMDA receptor/glycine site antagonist, 5-nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1328), on the development of morphine tolerance. Administration of ACEA-1328 (20 mg/kg) completely blocked tolerance to morphine-induced antinociception in the tail flick test in CD-1 mice, without affecting the basal nociceptive response or potentiating morphine-induced antinociceptive effects. These data suggest that inhibition of NMDA receptor activity via blockade of the glycine co-agonist site is potentially viable as a therapeutic approach for preventing development of morphine tolerance.

Functional differences among alternatively spliced variants of Slowpoke, a Drosophila calcium-activated potassium channel.

The Slowpoke locus of Drosophila melanogaster encodes a family of alternatively spliced mRNAs which encode large conductance calcium-activated potassium channels. Variability residues in blocks of amino acids designated boxes A, C, E, G, and I. Oocytes were injected with cRNAs that had been chosen for direct functional comparison of single box differences. Single channel records from inside-out patches of oocyte membranes expressing A1 or A3 forms, E1 or E2 forms, and G2-G5 forms were analyzed and compared. The main functional difference between A1 and A3 was in unitary conductance, whereas the main difference in properties between E1 and E2 was in calcium sensitivity. Activation kinetics were different between G3 and G5, but not consistently in different A and E box backgrounds. The results indicate that alternative splicing of a common RNA precursor contributes to the functional diversity of the expressed channel. Our findings suggest that the variable region of the Slowpoke channel subunit comprises modular, yet interactive functional domains which influence the essential features of unit conductance, calcium sensitivity, and gating.

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation.

Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by beta functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 microM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%.(ABSTRACT TRUNCATED AT 250 WORDS)