Bradykinin-induced vasodilation of human coronary arteries in vivo: role of nitric oxide and angiotensin-converting enzyme

Voltage-clamped GABA(A fast) and GABA(A slow) inhibitory postsynaptic currents (IPSCs) were selectively elicited in hippocampal area CA1 pyramidal neurons. Clinically relevant concentrations of halothane (1.2 vol.%) prolonged both GABA(A fast) and GABA(A slow) IPSC decay times approximately 2.5 fold, while having little to no effect on current amplitudes or rise times. Current-voltage analysis revealed that IPSC reversal potentials (-70 to -75 mV) remained constant in the presence of halothane. Under control conditions, GABA(A slow) IPSC decay times increased linearly with membrane depolarization, and this IPSC decay time voltage dependence was not significantly altered by halothane. These results confirm the existence of separable GABA(A fast) and GABA(A slow) IPSCs in hippocampus, and further elucidate the effects of halothane on these currents.     

Dual actions of volatile anesthetics on GABA(A) IPSCs: dissociation of blocking and prolonging effects

BACKGROUND: Volatile agents alter inhibitory postsynaptic currents (IPSCs) at clinically relevant concentrations, an action that is thought to make an important contribution to their behavioral effects. The authors investigated the mechanisms underlying these effects by evaluating the concentration dependence of modulation by enflurane, isoflurane, and halothane of IPSCs in rat hippocampal slices. METHODS: Action potential-independent gamma-aminobutyric acid(A) IPSCs (miniature IPSCs [mIPSCs]) were recorded from CA1 pyramidal neurons. The effects on mIPSC amplitude were used to distinguish between presynaptic (altered release) and postsynaptic (altered receptor response) actions of volatile agents. The concentration dependence of blocking and prolonging actions was compared among the volatile agents to determine whether a single modulatory process could account for both effects. RESULTS: The application of volatile anesthetics prolonged the decay and reduced the amplitude of mIPSCs in a dose-dependent manner. The effects on decay time for isoflurane and enflurane could not be distinguished. However, the blocking effect of enflurane was significantly greater than that of isoflurane at all concentrations. Despite the blocking effect, the net action of these agents was enhanced inhibition, because charge transfer was always significantly greater than control. Isoflurane, and to a lesser extent enflurane and halothane, caused a picrotoxin-sensitive increase in baseline noise. Moderate increases in mIPSC frequency were also observed for all agents. CONCLUSIONS: These results show that enflurane, isoflurane, and halothane reduce IPSC amplitude through a direct postsynaptic action. Furthermore, the concentration dependence of the actions of the agents reveals a dissociation between the effects on the amplitude and the time course of IPSCs, suggesting that distinct mechanisms underlie the two actions.     

Spillover-mediated transmission at inhibitory synapses promoted by high affinity alpha6 subunit GABA(A) receptors and glomerular geometry

Divergence and convergence of synaptic connections make a crucial contribution to the information processing capacity of the brain. Until recently, it was thought that transmitter released at a synapse affected only a specific postsynaptic cell. We show here that spillover of inhibitory transmitter at the Golgi to granule cell synapse produces significant cross-talk to non-postsynaptic cells, which is promoted both by the anatomical specialization of this glomerular synapse and by the presence of the high affinity alpha6 subunit-containing GABA(A) receptor in granule cells. Cross-talk is manifested as a novel slow rising and decaying small amplitude inhibitory postsynaptic current (IPSC) that can also contribute a long-lasting component to more typical IPSCs, which is prolonged by inhibition of the neuronal GABA transporter GAT-1. Because of the long duration of IPSCs generated by spillover, the total charge carried is three times that of IPSCs generated by directly connected terminals. GABA spillover within the mossy fiber glomerulus may play an important role in regulating the number of granule cells active in the cerebellar cortex, a regulation that is suggested by theoretical models to optimize cerebellar information processing.     

Adrenoceptor-mediated elevation of ambient GABA levels activates presynaptic GABA(B) receptors in rat sensorimotor cortex

At inhibitory synapses in the mature neocortex and hippocampus in vitro, spontaneous action-potential-dependent and -independent release of gamma-aminobutyric acid (GABA) activates postsynaptic GABA(A) receptors but not pre- or postsynaptic GABA(B) receptors. Elevation of synaptic GABA levels with pharmacological agents or electrical stimulation can cause activation of GABA(B) receptors, but the physiological conditions under which such activation occurs need further elucidation. In rodent sensorimotor cortex, epinephrine produced a depression in the amplitude of evoked monosynaptic inhibitory postsynaptic currents (IPSCs) and a concomitant, adrenoceptor-mediated increase in the frequency of spontaneous IPSCs. Blockade of GABA(B) receptors prevented the depression of evoked IPSC amplitude by epinephrine but did not affect the increase in spontaneous IPSC frequency. These data show that adrenoceptor-mediated increases in spontaneous IPSCs can cause activation of presynaptic GABA(B) receptors and indirectly modulate impulse-related GABA release, presumably through elevation of synaptic GABA levels.     

Hippocampal CA1 lacunosum-moleculare interneurons: modulation of monosynaptic GABAergic IPSCs by presynaptic GABAB receptors

1. Whole cell patch-clamp recordings were employed to characterize monosynaptic inhibitory postsynaptic currents (IPSCs) in morphologically and electrophysiologically identified interneurons located in the stratum lacunosum moleculare, or near the border of the stratum radiatum (LM interneurons), in the CA1 region of hippocampal slices taken from 3- to 4-wk-old rats. Monosynaptic IPSCs, evoked in the presence of glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM) and D-2-amino-5-phosphopentanoate (APV; 50 microM) were biphasic. The gamma-aminobutyric acid-A (GABAA) receptor antagonist, bicuculline (20 microM), blocked the fast IPSC, and the slow IPSC was blocked by the GABAB receptor antagonist CGP35348 (500 microM). 2. Monosynaptic IPSCs were evoked by electrical stimulation in several distant regions including the stratum radiatum, the stratum oriens, the stratum lacunosum-moleculare, and the molecular layer of dentate gyrus, suggesting an extensive network of inhibitory interneurons in the hippocampus. In paired recordings of CA1 interneurons and pyramidal cells, IPSCs were evoked by electrical stimulation of most of these distal regions with the exception of the molecular layer of dentate gyrus, which evoked an IPSC only in LM interneurons. 3. Frequent (> 0.1 Hz) stimulation depressed the evoked IPSCs. With a paired-pulse protocol, the second IPSC was depressed and the maximal depression (40-50%) was observed with an interstimulus interval of 100-200 ms. 4. The GABAB receptor agonist baclofen (1 microM) reduced the amplitude of evoked IPSCs and the paired-pulse depression of the second IPSC. The GABAB receptor antagonist CGP35348 (0.5-1 mM) had no significant effect on the amplitude of isolated IPSCs. However, CGP35348 reduced but did not fully block paired-pulse depression, suggesting that this depression is partly due to the activation of presynaptic GABAB receptors. 5. The paired-pulse depression depended on the level of transmitter release. Potentiation of synaptic release of GABA, by increasing the extracellular Ca2+ concentration to 4 mM and reducing the extracellular Mg2+ concentration to 0.1 mM, enhanced the depression. Reduction of transmitter release by increasing extracellular Mg2+ concentration to 7 mM diminished the paired-pulse depression of IPSCs. After potentiation of transmitter release, CGP35348 was less efficient in reducing the paired-pulse depression, suggesting that enhancement of depression by high-calcium/low-magnesium medium was preferentially due to the potentiation of a GABAB-independent component. 6. In summary, monosynaptic IPSCs recorded in LM interneurons show similar features to those recorded in pyramidal cells. The strong correlation between the level of transmitter release and the degree of paired-pulse depression may have important physiological consequences, because in synapses with a high level of activity and a high level of GABA release, inhibition is powerful, but depression can develop more readily.     

Shaping of IPSCs by endogenous calcineurin activity

Synaptic inhibition, mediated by GABAA receptors, regulates neuronal firing, influences coincidence detection (K?nig et al., 1996), and can synchronize the output of neural circuits (Cobb et al., 1995). Although GABAA receptors can be modulated by phosphorylation, few studies have directly addressed the role of such modulation at synapses, where the nonequilibrium conditions of receptor activation are quite different from those often used to study GABAA receptors in vitro. Here we promoted endogenous phosphorylation by inhibiting specific phosphatases in rat hippocampal neurons and compared the effects on IPSCs with GABAA channel responses in outside-out patches. Brief and saturating GABA pulses (5 msec; 10 mM) activated patch currents resembling the IPSC. Inhibition of calcineurin (protein phosphatase 2B), but not phosphatases 1 or 2A, produced a similar shortening of IPSC and patch responses, as did nonspecific inhibition of dephosphorylation using ATPgammaS or high concentrations of intracellular phosphate. Calcineurin inhibition increased the microscopic ligand unbinding rate, which was measured using the competitive antagonist 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide, suggesting that the IPSC shortening was partly caused by destabilization of the ligand binding site. Calcineurin inhibition also increased the rate and extent of macroscopic receptor desensitization. These results show that endogenous regulation by kinases and calcineurin can produce substantial changes in the IPSC duration by altering the unbinding and gating kinetics of the GABAA receptor. Dynamic regulation of synaptic inhibition may thus allow for the tuning of circuit behavior at the level of individual inhibitory synapses.     

Regulation of the NMDA component of EPSPs by different components of postsynaptic GABAergic inhibition: computer simulation analysis in piriform cortex

Regulation of the NMDA component of EPSPs by different components of postsynaptic GABAergic inhibition: computer simulation analysis in piriform cortex. J. Neurophysiol. 78: 2546-2559, 1997. Physiological analysis in the companion paper demonstrated that gamma-aminobutyric acid-A (GABAA)-mediated inhibition in piriform cortex is generated by circuits that are largely independent in apical dendritic and somatic regions of pyramidal cells and that GABAA-mediated inhibitory postsynaptic currents (IPSCs) in distal dendrites have a slower time course than those in the somatic region. This study used modeling methods to explore these characteristics of GABAA-mediated inhibition with respect to regulation of the N-methyl--aspartate (NMDA) component of excitatory postsynaptic potentials. Such regulation is relevant to understanding NMDA-dependent long-term potentiation (LTP) and the integration of repetitive synaptic inputs that can activate the NMDA component as well as pathological processes that can be activated by overexpression of the NMDA component. A working hypothesis was that the independence and differing properties of IPSCs in apical dendritic and somatic regions provide a means whereby the NMDA component and other dendritic processes can be controlled by way of GABAergic tone without substantially altering system excitability. The analysis was performed on a branched compartmental model of a pyramidal cell in piriform cortex constructed with physiological and anatomic data derived by whole cell patch recording. Simulations with the model revealed that NMDA expression is more effectively blocked by the slow GABAA component than the fast. Because the slow component is present in greater proportion in apical dendritic than somatic regions, this characteristic would increase the capacity of dendritic IPSCs to regulate NMDA-mediated processes. The simulations further revealed that somatic-region GABAergic inhibition can regulate the generation of action potentials with little effect on the NMDA component generated by afferent fibers in apical dendrites. As a result, if expression of the NMDA component or other dendritic processes were enabled by selective block of dendritic inhibition, for example, by centrifugal fiber systems that may regulate learning and memory, the somatic-region IPSC could preserve system stability through feedback regulation of firing without counteracting the effect of the dendritic-region block. Simulations with paired inputs revealed that the dendritic GABAA-mediated IPSC can regulate the extent to which a strong excitatory input facilitates the NMDA component of a concurrent weak input, providing a possible mechanism for control of "associative LTP" that has been demonstrated in this system. Postsynaptic GABAB-mediated inhibition had less effect on the NMDA component than either the fast or slow GABAA components. Depolarization from a concomitant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) component also was found to have comparatively little effect on current through the NMDA channel because of its brief time course.     

Effect of zolpidem on miniature IPSCs and occupancy of postsynaptic GABAA receptors in central synapses

GABAA-mediated miniature IPSCs (mIPSCs) were recorded from layer V pyramidal neurons of the visual cortex using whole-cell patch-clamp recording in rat brain slices. At room temperature, the benzodiazepine site agonist zolpidem enhanced both the amplitude (to 138 +/- 26% of control value at 10 microM) and the duration (163 +/- 14%) of mIPSCs. The enhancement of mIPSC amplitude was not caused by an increase of the single-channel conductance of the postsynaptic receptors, as determined by peak-scaled non-stationary fluctuation analysis of mIPSCs. The effect of zolpidem on fast, synaptic-like (1 msec duration) applications of GABA to outside-out patches was also investigated. The EC50 for fast GABA applications was 310 microM. In patches, zolpidem enhanced the amplitude of currents elicited by subsaturating GABA applications (100-300 microM) but not by saturating applications (10 mM). The increase of mIPSC amplitude by zolpidem provides evidence that the GABAA receptors are not saturated during miniature synaptic transmission in the recorded cells. By comparing the facilitation induced by 1 microM zolpidem on outside-out patches and mIPSCs, we estimated the concentration of GABA seen by the postsynaptic GABAA receptors to be approximately 300 microM after single vesicle release. We have estimated a similar degree of receptor occupancy at room and physiological temperature. However, at 35 degreesC, zolpidem did not enhance the amplitude of mIPSCs or of subsaturating GABA applications on patches, implying that, in these neurons, zolpidem cannot be used to probe the degree of receptor occupancy at physiological temperature.     

Effects of small concentrations of volatile anesthetics on action potential firing of neocortical neurons in vitro

BACKGROUND: Volatile general anesthetics depress neuronal activity in the mammalian central nervous system and enhance inhibitory Cl- currents flowing across the gamma-aminobutyric acid A (GABA(A)) receptor-ion channel complex. The extent to which an increase in GABA(A)-mediated synaptic inhibition contributes to the decrease in neuronal firing must be determined, because many further effects of these agents have been reported on the molecular level. METHODS: The actions of halothane, isoflurane, and enflurane on the firing patterns of single neurons were investigated by extracellular recordings in organotypic slice cultures derived from the rat neocortex. RESULTS: Volatile anesthetics depressed spontaneous action potential firing of neocortical neurons in a concentration-dependent manner. The estimated median effective concentration (EC50) values were about one half the EC50 values for general anesthesia. In the presence of the GABA(A) antagonist bicuculline (20 microM), the effectiveness of halothane, isoflurane, and enflurane in reducing the discharge rates were diminished by 48-65%, indicating that these drugs act via the GABA(A) receptor. CONCLUSIONS: Together with recent investigations, our results provide evidence that halothane, isoflurane, and enflurane reduced spontaneous action potential firing of neocortical neurons in cultured brain slices mainly by increasing GABA(A)-mediated synaptic inhibition. At concentrations, approximately one half the EC50 for general anesthesia, volatile anesthetics increased overall GABA(A)-mediated synaptic inhibition about twofold, thus decreasing spontaneous action potential firing by half.     

Evidence for metabotropic glutamate receptor activation in the induction of depolarization-induced suppression of inhibition in hippocampal CA1

Depolarization-induced suppression of inhibition (DSI) is a transient reduction of GABAA receptor-mediated IPSCs that is mediated by a retrograde signal from principal cells to interneurons. Using whole-cell recordings, we tested the hypothesis that mGluRs are involved in the DSI process in hippocampal CA1, as has been proposed for cerebellar DSI. Group II mGluR agonists failed to affect either evoked monosynaptic IPSCs or DSI, and forskolin, which blocks cerebellar DSI, did not affect CA1 DSI. Group I and group III mGluR agonists reduced IPSCs, but only group I agonists occluded DSI. (S)-MCPG blocked (1S,3R)-ACPD-induced IPSC suppression and markedly reduced DSI, whereas group III antagonists had no effect on DSI. Many other similarities between DSI and the (1S,3R)-ACPD-induced suppression of IPSCs also were found. Our data suggest that a glutamate-like substance released from pyramidal cells could mediate CA1 DSI by reducing GABA release from interneurons via the activation of group I mGluRs.     

Increased probability of GABA release during withdrawal from morphine

Opioid receptors located on interneurons in the ventral tegmental area (VTA) inhibit GABA(A)-mediated synaptic transmission to dopamine projection neurons. The resulting disinhibition of dopamine cells in the VTA is thought to play a pivotal role in drug abuse; however, little is known about how this GABAA synapse is affected after chronic morphine treatment. The regulation of GABA release during acute withdrawal from morphine was studied in slices from animals treated for 6-7 d with morphine. Slices containing the VTA were prepared and maintained in morphine-free solutions, and GABAA IPSCs were recorded from dopamine cells. The amplitude of evoked IPSCs and the frequency of spontaneous miniature IPSCs measured in slices from morphine-treated guinea pigs were greater than placebo-treated controls. In addition, activation of adenylyl cyclase, with forskolin, and cAMP-dependent protein kinase, with Sp-cAMPS, caused a larger increase in IPSCs in slices from morphine-treated animals. Conversely, the kinase inhibitors staurosporine and Rp-CPT-cAMPS decreased GABA IPSCs to a greater extent after drug treatment. The results indicate that the probability of GABA release was increased during withdrawal from chronic morphine treatment and that this effect resulted from an upregulation of the cAMP-dependent cascade. Increased transmitter release from opioid-sensitive synapses during acute withdrawal may be one adaptive mechanism that results from prolonged morphine treatment.     

Isoflurane and halothane do not alter the enhanced afterload sensitivity of left ventricular relaxation in dogs with pacing-induced cardiomyopathy

BACKGROUND: The afterload dependence of left ventricular (LV) relaxation is accentuated in the failing heart. The authors tested the hypothesis that isoflurane and halothane alter the afterload sensitivity of LV relaxation in dogs with pacing-induced cardiomyopathy. METHODS: Dogs (n = 6) were chronically instrumented for measurement of LV and aortic pressures and subendocardial segment length. Hemodynamics were recorded, and LV relaxation was evaluated with a time constant of isovolumic relaxation (tau) under control conditions and during decreases and increases in LV load produced by abrupt inferior vena caval (IVC) occlusion and phenylephrine (intravenous infusion), respectively, in the conscious state and during isoflurane and halothane anesthesia (1.5 MAC) on separate days before and after the development of pacing-induced cardiomyopathy. The slope (R) of the tau versus LV end-systolic pressure (P[es]) relation was also used to determine the afterload sensitivity of LV relaxation. RESULTS: IVC occlusion and phenylephrine produced similar or less profound changes in P(es), regional end-systolic force (an index of LV afterload), and end-systolic segment length in cardiomyopathic compared with healthy dogs. However, IVC occlusion and phenylephrine caused more pronounced alterations in tau in conscious and isoflurane- and halothane-anesthetized dogs after the development of cardiomyopathy. R was also greater in cardiomyopathic compared with healthy dogs (e.g., 0.32 +/- 0.03 before pacing to 1.00 +/- 0.13 ms/mmHg in conscious dogs). No differences in the load dependence of LV relaxation were observed between the conscious and anesthetized states before and after production of LV dysfunction. CONCLUSIONS: The results indicate that isoflurane and halothane do not alter the afterload dependence of LV relaxation in the normal and cardiomyopathic heart. The lack of effect of the volatile anesthetics is probably related to anesthetic-induced reductions in the resistance to LV ejection concomitant with simultaneous negative inotropic effects.     

Enhanced bursts of IPSCs in dentate granule cells in mice with regionally inhibited long-term potentiation

Until recently, most studies on the synaptic-cellular basis of learning and memory concentrated on the activity-dependent changes occurring in principal cells such as hippocampal pyramidal cells and dentate granule cells. However, the ability of the inhibitory interneurons to regulate synaptic plasticity remains less understood. This study tested the hypothesis that the gamma-aminobutyric-acid (GABA)-mediated inhibitory neurotransmission is enhanced in mice that show no detectable long-term potentiation in the dentate gyrus in the absence of the GABAA receptor antagonist bicuculline. Patch clamp recordings were made from dentate granule cells in brain slices from wild-type and Thy-1 knockout (KO) mice. The frequency, amplitude and kinetics of miniature inhibitory postsynaptic currents (mIPSCs, generated by the action potential-independent release of GABA) was not different between animals. However, bursts of spontaneous IPSCs (sIPSCs, generated by both action potential-independent and -dependent GABA release) in KO mice were associated with larger synaptic charge transfers and increased durations. When pairs of IPSCs were evoked at varying intervals, the amplitude of the second response with respect to the first was significantly larger in KO animals. These results further support the concept that enhancement of interneuronal functions in cortical structures can have profound effects on the activity-dependent synaptic plasticity observed in principal cells.     

Glutamate metabotropic receptor agonists depress excitatory and inhibitory transmission on rat mesencephalic principal neurons

Intracellular and whole-cell patch-clamp recordings were used to evaluate the actions of different metabotropic glutamate receptor (mGluR) agonists on the synaptic inputs evoked on principal cells of the rat mesencephalon. Bath application of the group III mGluR agonists L-2-amino-4-phosphonobutyric acid (L-AP4) and L-serine-O-phosphonobutanoate (L-SOP) did not change the holding current of the cells held at resting potential (-60 mV) but produced a dose-dependent inhibition of the amplitude of the excitatory and inhibitory events. L-AP4 and L-SOP were more effective at inhibiting the excitatory postsynaptic currents (EPSCs) than the GABA(A) and GABA(B) inhibitory postsynaptic currents (IPSCs). The suppressing effects of L-AP4 and L-SOP were antagonized by (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP-4) but not by +/- -alpha-methyl-4-carboxyphenylglycine (MCPG). Moreover, the group II agonist (2S,1'S,2'S)-(carboxycyclopropyl)glycine (L-CCG1) and the group I agonist (RS)-3,5-dihydrophenylglycine (3,5-DHPG) depressed in a dose-related manner the EPSC, the GABA(A) IPSC and the GABA(B) IPSC. The suppressing effect of the two mGluRs agonists was partially antagonized by MCPG but not by MAP-4. In addition, both L-CCG1 and 3,5-DHPG caused an inward shift of the holding current. To characterize the site of action of the metabotropic receptor agonists, experiments were performed to examine the amplitude and ratio of EPSC and GABA(A) IPSC pairs. The increase of the s2/s1 ratio caused by the agonists suggests that the location of the inhibitory mGluRs was presynaptic. These results indicate that the activation of presynaptic mGluRs controls the release of excitatory and inhibitory transmitters on presumed dopaminergic cells within the ventral mesencephalon.     

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.     

Temporal patterns and depolarizing actions of spontaneous GABAA receptor activation in granule cells of the early postnatal dentate gyrus

Whole cell patch-clamp recordings were used to investigate the properties of the gamma-aminobutyric acid type A (GABAA) receptor-mediated spontaneous synaptic events in immature granule cells of the developing, early postnatal day (P0-P6) rat dentate gyrus. With Cs-gluconate-filled whole cell patch pipettes at 0 mV in control medium, spontaneous inhibitory postsynaptic currents (sIPSCs) occurred in prominent bursts (peak amplitude of the bursts 406.9 +/- 58.4 pA; intraburst IPSC frequency 71.0 +/- 12.4 Hz) at 0.05 +/- 0.02 Hz in every immature granule cell younger than P7. Between the bursts of IPSCs, lower frequency (1.7 +/- 0.7 Hz), interburst IPSCs could be observed. Bicuculline and picrotoxin as well as the intracellularly applied chloride-channel blockers CsF- and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) abolished the intraburst as well as the interburst IPSCs, indicating that the IPSCs were mediated by GABAA receptor channels. The bursts of IPSCs, but not the interburst IPSCs, were blocked by the simultaneous application of the glutamate receptor antagonists 2-amino-5-phosphovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione, indicating the importance of the glutamatergic excitatory drive onto the interneurons in the early postnatal dentate gyrus. The spontaneously occurring excitatory postsynaptic currents in immature granule cells, observable after the intracellular blockade of GABAA receptor channels with CsF- and DIDS, appeared exclusively as single events at low frequencies, i.e., they did not occur in prominent bursts. Gramicidin-based perforated patch-clamp recordings determined that the reversal potential for the burst of IPSCs (-46.6 +/- 3.1 mV) was more depolarized than the resting membrane potential (-54.2 +/- 4.2 mV) but more hyperpolarized than the action potential threshold (-41. 8 +/- 1.7 mV). The depolarizing action of the bursts of synaptic events most often evoked only a single action potential per burst. Simultaneous whole cell patch recordings, with KCl-filled patch pipettes at -60 mV in current clamp from pairs of immature granule cells of the developing dentate gyrus, determined that the bursts of IPSPs took place in a similar temporal pattern but with imperfect synchrony in neighboring granule cells (average lag between the onsets of the bursts between granule cell pairs 77.7 +/- 8.6 ms). These results show that the spontaneous activation of GABAA receptors in immature dentate granule cells displays unique properties that are distinct from the temporal patterns and biophysical features of spontaneous GABAA receptor activation taking place in the developing Ammon's horn and in the adult dentate gyrus.     

Enhancement of gamma-aminobutyric acidA receptor activity by alpha-chloralose

alpha-Chloralose is widely used as an anesthetic in the laboratory due to its minimal effects on autonomic and cardiovascular systems, yet little is known about its mechanism of action. We examined the effects of alpha-chloralose on gamma-aminobutyric acid type A (GABAA) receptor activity because recent studies have shown that several classes of general anesthetics modulate the function of this receptor. GABAA receptor activity was assayed by measuring the GABA-induced current in Xenopus oocytes expressed with human GABAA receptor alpha-1, beta-1 and gamma-2L subunits. alpha-Chloralose produced a concentration-dependent potentiation of the GABA-induced current with an EC50 value of 49 microM and a maximal effect of 239% of control. Membrane current was not affected by alpha-chloralose in the absence of GABA. alpha-Chloralose (100 microM) increased the affinity for GABA 5-fold and produced a small (17%) increase in the efficacy of GABA. Measurement of the reversal potentials for the alpha-chloralose response suggested that the effect is mediated through increased Cl- conductance. Studies of alpha-chloralose interactions with other allosteric modulators determined that alpha-chloralose binds to a site on the GABAA receptor complex distinct from the benzodiazepine, neurosteroid and barbiturate sites. Chloral hydrate, trichloroethanol and urethane also augmented GABA-induced currents. alpha-Chloralose had no effect on the hydroxytryptamine-induced currents in oocytes expressed with the 5-hydroxytryptamine3 receptor. These data extend the number of classes of anesthetics that allosterically modulate GABAA receptor activity and indicate that GABAA receptors may be a common site of action for diverse classes of general anesthetics.     

Mechanism of early anoxia-induced suppression of the GABAA-mediated inhibitory postsynaptic current

1. We investigated the mechanism of hypoxia-induced depression of gamma-aminobutyric acid-A (GABAA)-mediated inhibitory postsynaptic currents (IPSCs) in CA1 neurons of hippocampal slices from 21- to 28-day-old rats. Cells were examined by whole-cell patch-clamp recording and hypoxia was induced by switching perfusion of the slice from oxygenated artificial cerebral spinal fluid (ACSF) to ACSF saturated with 95% N2-5% CO2. 2. Synaptic responses evoked by stimulation of the Schaffer collateral-commissural projection at a fixed holding potential (VH = -60 mV) during anoxia revealed that the IPSC appeared more sensitive than the excitatory postsynaptic current to anoxia-induced depression. All subsequent studies examined the GABAA-mediated IPSC synaptic responses in isolation by direct stimulation of GABA interneurons in the stratum radiatum in the presence of extracellular 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) (20 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (50 microM) to block glutamatergic currents and intracellular QX-314 (lidocaine N-ethyl bromide, 1 mM) to block GABAB-mediated currents. When studied in this manner (VH = -60 mV) the GABAA-mediated IPSC appeared to change from an outward to inward current after exposure to anoxia. 3. The current-voltage relationship of GABAA-mediated IPSCs revealed that these changes resulted from a positive shift in the IPSC reversal potential without a significant change in the conductance. Thus under patch clamp apparent IPSC inhibition may result from a decrease in the extracellular concentration of chloride ions. Similar findings were observed with micropipettes that contained high intracellular chloride concentrations. 4. Miniature spontaneous IPSCs were examined in the presence of tetrodotoxin (1 microM) with micropipettes containing high intracellular chloride concentrations. The miniature IPSCs (mIPSCs) appeared as spontaneous transient inward currents. Consistent with an anoxia-induced decrease in extracellular chloride, the mean amplitude of the mIPSCs increased after the onset of anoxia. A significant decrease in rise and decay time was also noted during anoxia. The frequency of the mIPSCs also increased by approximately 300%. 5. The resting input resistance of the cells was examined by measuring the current resulting from a 20-mV hyperpolarizing pulse. A significant reduction in resistance was observed 2 min after the onset of anoxia. This still occurred, although to a lesser degree, in the presence of glutamatergic blockers (20 microM CPP plus 50 microM CNQX). In the presence of both GABAergic (picrotoxin, 100 microM) and glutamatergic blockers no significant reduction in resting input resistance was apparent after 2 min of anoxia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preservation of the ration of cerebral blood flow/metabolic rate for oxygen during prolonged anesthesia with isoflurane, sevoflurane, and halothane in humans

BACKGROUND: In several animal studies, an increase in cerebral blood flow (CBF) produced by volatile anesthetics has been reported to resolve over time during prolonged anesthesia. It is important to investigate whether this time-dependent change of CBF takes place in humans, especially in clinical situations where surgery is ongoing under anesthesia. In this study, to evaluate the effect of prolonged exposure to volatile anesthetics (isoflurane, sevoflurane, and halothane), the CBF equivalent (CBF divided by cerebral metabolic rate for oxygen (CMRO2) was determined every 20 min during anesthesia lasting more than 4h in patients. METHODS: Twenty-four surgical patients were assigned to three groups at random to receive isoflurane, sevoflurane, or halothane (8 patients each). End-tidal concentration of the selected volatile anesthetic was maintained at 0.5 and 1.0 MAC before surgery and then 1.5 MAC for the 3 h of surgical procedure. Normothermia and normocapnia were maintained. Mean arterial blood pressure was kept above 60 mmHg, using phenylephrine infusion, if necessary. CBF equivalent was calculated every 20 min as the reciprocal of arterial-jugular venous oxygen content difference. RESULTS: CBF equivalent at 0.5 MAC of isoflurane, halothane, and sevoflurane was 21 +/- 4, 20 +/- 3, and 21 +/- 5 ml blood/ml oxygen, respectively. All three examined volatile anesthetics significantly (P<0.01) increased CBF equivalent in a dose-dependent manner (0.5, 1.0, 1.5 MAC). AT 1.5 MAC, the increase of CBF equivalent with all anesthetics was maintained increased with minimal fluctuation for 3 h. The mean value of CBF equivalent at 1.5 MAC in the isoflurane group (45 +/- 8) was significantly (P<0.01) greater than those in the halothane (32 +/- 8) and sevoflurane (31 +/- 8) groups. Electroencephalogram was found to be relatively unchanged during observation periods at 1.5 MAC. CONCLUSIONS: These results demonstrate that CBF/CMRO2 ratio is markedly increased above normal and maintained during prolonged inhalation of volatile anesthetics in humans. It is impossible to determine whether these data indicate a stable CBF or whether CBF and CMRO2 are changing in parallel during the observation period. The unchanging electroencephalographic pattern suggests that the former possibility is more likely and that the increase of CBF produced by volatile anesthetics is maintained over time without decay, which has been reported in several animal studies. It also is suggested that isoflurane possesses greater capability to maintain global CBF relative to CMRO(2) than does halothane or sevoflurane. time.)     

Volatile anesthetics affect calcium mobilization in bovine endothelial cells

BACKGROUND: The site where volatile anesthetics inhibit endothelium-dependent, nitric oxide-mediated vasodilation is unclear. To determine whether anesthetics could limit endothelium-dependent nitric oxide production by inhibiting receptor-mediated increases in cytosolic Ca2+, experiments were performed to see if the inhalational anesthetics halothane, isoflurane, and enflurane affect intracellular Ca2+ ([Ca2+]i) transients induced by the agonists bradykinin and adenosine triphosphate in cultured bovine aortic endothelial cells. METHODS: Bovine aortic endothelial cells, which had been loaded with the fluorescent Ca2+ indicator Fura-2, were added to medium preequilibrated with volatile anesthetic (1.25% and 2.5% for isoflurane, 1.755 and 3.5% for enflurane, and 0.75% and 1.5% for halothane). In Ca(2+)-containing medium, intracellular Ca2+ transients were elicited in response to bradykinin (10 nM and 1 microM) or adenosine triphosphate (1 microM and 100 microM). RESULTS: Both bradykinin and adenosine triphosphate triggered a rapid rise to peak [Ca2+]i followed by a gradual decline to a plateau above the resting level. Although basal [Ca2+]i was unaltered by the anesthetics, both halothane and enflurane, in a dose-dependent manner, depressed the peak and plateau of the [Ca2+]i transient elicited by 10 nM bradykinin, whereas isoflurane had no effect. When [Ca2+]i transients were elicited by 1 microM bradykinin, halothane (1% and 5%) did not alter peak and plateau levels. Halothane and enflurane also decreased [Ca2+]i transients evoked by 1 microM and 100 microM adenosine triphosphate, whereas isoflurane also had no effect in this setting. CONCLUSIONS: Halothane and enflurane, but not isoflurane, inhibit bradykinin- and adenosine triphosphate-stimulated Ca2+ transients in endothelial cells. Limitations of Ca2+ availability to activate constitutive endothelial nitric oxide synthase could allow for part, but not all, of the inhibition of endothelium-dependent nitric oxide-mediated vasodilation by inhalational anesthetics.