The inhibitor of semicarbazide-sensitive amine oxidase, PXS-4728A, ameliorates key features of chronic obstructive pulmonary disease in a mouse model.

BACKGROUND AND PURPOSE: Chronic obstructive pulmonary disease (COPD) is a major cause of illness and death, often induced by cigarette smoking (CS). It is characterized by pulmonary inflammation and fibrosis that impairs lung function. Existing treatments aim to control symptoms but have low efficacy, and there are no broadly effective treatments. A new potential target is the ectoenzyme, semicarbazide-sensitive mono-amine oxidase (SSAO; also known as vascular adhesion protein-1). SSAO is elevated in smokers' serum and is a pro-inflammatory enzyme facilitating adhesion and transmigration of leukocytes from the vasculature to sites of inflammation. EXPERIMENTAL APPROACH: PXS-4728A was developed as a low MW inhibitor of SSAO. A model of COPD induced by CS in mice reproduces key aspects of human COPD, including chronic airway inflammation, fibrosis and impaired lung function. This model was used to assess suppression of SSAO activity and amelioration of inflammation and other characteristic features of COPD. KEY RESULTS: Treatment with PXS-4728A completely inhibited lung and systemic SSAO activity induced by acute and chronic CS-exposure. Daily oral treatment inhibited airway inflammation (immune cell influx and inflammatory factors) induced by acute CS-exposure. Therapeutic treatment during chronic CS-exposure, when the key features of experimental COPD develop and progress, substantially suppressed inflammatory cell influx and fibrosis in the airways and improved lung function. CONCLUSIONS AND IMPLICATIONS: Treatment with a low MW inhibitor of SSAO, PXS-4728A, suppressed airway inflammation and fibrosis and improved lung function in experimental COPD, demonstrating the therapeutic potential of PXS-4728A for this debilitating disease.

Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model.

BACKGROUND AND PURPOSE: P2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines. EXPERIMENTAL APPROACH: The activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund's adjuvant (CFA) in rat paws). KEY RESULTS: Compound A potently blocked P2X3 (pIC(50)= 7.39) and P2X2/3 (pIC(50)=6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC(50)< 4.7). Whole-cell voltage clamp electrophysiology confirmed these results. Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3mg·kg(-1) ). CONCLUSIONS AND IMPLICATIONS: The diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.

Design and application of a novel brain slice system that permits independent electrophysiological recordings from multiple slices.

We describe a novel brain slice system 'SliceMaster' that allows electrophysiological recordings from eight brain slices independently. The system consists of two autonomous units each supporting four modular brain slice chambers enabling high signal-to-noise ratio recordings, each chamber has one stimulation electrode, one recording electrode, a twin camera system and a solution application system. The positioning of both electrodes and cameras are controlled from a remote user console. The software both acquires and performs on-line analysis of the data. We have demonstrated utility of this system in obtaining recordings of spontaneous firing activity and evoked synaptic activity from mouse hippocampal slices, with reduced variability within and between experiments. Furthermore, we show recordings of population spikes from the perirhinal cortex, indicating applicability of this system for further brain regions. In addition, stable recordings could be maintained until recording was terminated after 3 h, permitting investigation of the induction and maintenance of synaptic plasticity. Recordings of spontaneous and synaptic activity, and effects of pharmacological and electrophysiological manipulation, were consistent with reports using conventional methods. However, the described system permits concurrent and independent recordings from eight brain slices, thus improving throughput, statistical design, and reducing animal use.

Neurological phenotype and synaptic function in mice lacking the CaV1.3 alpha subunit of neuronal L-type voltage-dependent Ca2+ channels.

Neuronal L-type calcium channels have been implicated in pain perception and neuronal synaptic plasticity. To investigate this we have examined the effect of disrupting the gene encoding the CaV1.3 (alpha 1D) alpha subunit of L-type Ca2+ channels on neurological function, acute nociceptive behavior, and hippocampal synaptic function in mice. CaV1.3 alpha 1 subunit knockout (CaV1.3 alpha 1(-/-)) mice had relatively normal neurological function with the exception of reduced auditory evoked behavioral responses and lower body weight. Baseline thermal and mechanical thresholds were unaltered in these animals. CaV1.3 alpha 1(-/-) mice were also examined for differences in N-methyl-D-aspartate (NMDA) receptor-dependent (100 Hz tetanization for 1 s) and NMDA receptor-independent (200 Hz in 100 microM DL-2-amino-5-phosphopentanoic acid) long-term potentiation within the CA1 region of the hippocampus. Both NMDA receptor-dependent and NMDA receptor-independent forms of long-term potentiation were expressed normally. Radioligand binding studies revealed that the density of (+)[3H]isradipine binding sites in brain homogenates was reduced by 20-25% in CaV1.3 alpha 1(-/-) mice, without any detectable change in CaV1.2 (alpha 1C) protein levels as detected using Western blot analysis. Taken together these data indicate that following loss of CaV1.3 alpha 1 subunit expression there is sufficient residual activity of other Ca2+ channel subtypes to support NMDA receptor-independent long-term potentiation and some forms of sensory behavior/function.

Molecular determinants of inactivation and dofetilide block in ether a-go-go (EAG) channels and EAG-related K(+) channels.

The major subunit of the cardiac delayed rectifier current I(Kr) is encoded by the human ether a-go-go related gene (HERG). HERG/I(Kr) channels are blocked selectively by class III antiarrhythmic methanesulfonanilide drugs such as dofetilide. The binding site for methanesulfonanilides is believed to be similar for nonantiarrhythmic drugs such as antihistamines, antibiotics, and antipsychotics. To gain further insight into the binding site, we examined the minimal structural changes necessary to transform low-affinity binding of dofetilide by the related bovine ether a-go-go channel bEAG to high-affinity binding of HERG. Previously, it was shown that high-affinity binding in HERG required intact C-type inactivation; the bovine ether a-go-go K(+) channel (bEAG), unlike HERG, is noninactivating. Therefore, we introduced C-type inactivation into noninactivating bEAG using site-directed mutagenesis. Two point mutations in the pore region, T432S and A443S, were sufficient to produce C-type inactivation. Low concentrations of dofetilide produced block of bEAG T432S/A443S; unlike HERG, block was almost irreversible. Substitution of an additional amino acid in transmembrane domain S6 made the block reversible. Dofetilide blocked the triply mutated bEAG T432S/A443S/A453S with an IC(50) value of 1.1 microM. The blocking potency was 30-fold greater than bEAG WT and about one third that of HERG WT. We conclude that high affinity methanesulfonanilide binding to HERG channels is strongly dependent on C-type inactivation.

Impaired inhibition of epileptiform activity by baclofen, but not by adenosine in the weaver hippocampus.

The weaver defect results in a loss of baclofen- and adenosine-gated K+ conductance in the hippocampus of adult homozygous (wv/wv) mice. In addition, suppression of hippocampal epileptiform activity by baclofen is impaired (Jarolimek, W., Bäurle, J., Misgeld, U., 1998. Pore mutation in a G protein-gated inwardly rectifying K+ channel subunit causes loss of K+ dependent inhibition in weaver hippocampus. Journal of Neuroscience 18, 4001-4007). We used wv/wv and wild-type (+/+) mice to determine whether K+ conductance increases are essential for the suppression of epileptiform activity by R-baclofen and adenosine in disinhibited hippocampal slices. In wv/wv mice R-baclofen was less potent by two orders of magnitude in reducing the frequency of spontaneous synchronous burst discharges than in +/+ mice. Endogenous adenosine and adenosine A1 receptor agonists differed only slightly in their efficacy to inhibit spontaneous synchronous burst discharges in wv/wv and +/+ mice. The findings on adenosine A1 receptors suggest that the varied efficacy of R-baclofen in wv/wv and +/+ mice may not be explained solely on the basis of a loss of ligand-gated K+ conductance. Therefore, we investigated the affinity of GABA(B) receptors for the antagonist CGP55845A in wv/wv and +/+ hippocampi. Schild plot analysis revealed a K(D) for the GABA(B) antagonist CGP55845A 10 fold higher in wv/wv than in +/+ mice. The data suggest that an alteration of GABA(B) receptors could contribute to the reduced efficacy of R-baclofen to suppress hippocampal epileptiform activity in weaver mice, while the suppression by adenosine remains largely unaffected.

A furosemide-sensitive K+-Cl- cotransporter counteracts intracellular Cl- accumulation and depletion in cultured rat midbrain neurons.

Efficacy of postsynaptic inhibition through GABAA receptors in the mammalian brain depends on the maintenance of a Cl- gradient for hyperpolarizing Cl- currents. We have taken advantage of the reduced complexity under which Cl- regulation can be investigated in cultured neurons as opposed to neurons in other in vitro preparations of the mammalian brain. Tightseal whole-cell recording of spontaneous GABAA receptor-mediated postsynaptic currents suggested that an outward Cl- transport reduced dendritic [Cl-]i if the somata of cells were loaded with Cl- via the patch pipette. We determined dendritic and somatic reversal potentials of Cl- currents induced by focally applied GABA to calculate [Cl-]i during variation of [K+]o and [Cl-] in the patch pipette. [Cl-]i and [K+]o were tightly coupled by a furosemide-sensitive K+-Cl- cotransport. Thermodynamic considerations excluded the significant contribution of a Na+-K+-Cl- cotransporter to the net Cl- transport. We conclude that under conditions of normal [K+]o the K+-Cl- cotransporter helps to maintain [Cl-]i at low levels, whereas under pathological conditions, under which [K+]o remains elevated because of neuronal hyperactivity, the cotransporter accumulates Cl- in neurons, thereby further enhancing neuronal excitability.

Pore mutation in a G-protein-gated inwardly rectifying K+ channel subunit causes loss of K+-dependent inhibition in weaver hippocampus.

Weaver (wv) mice carry a point mutation in the pore region of a G-protein-gated inwardly rectifying K+ channel subunit (Kir3.2). wvKir3.2 conducts inward currents that may cause the loss of neurons in the cerebellum and substantia nigra. Although Kir3.2 is widely expressed in the CNS, significant morphological or physiological changes have not been reported for other brain areas. We studied the role of wvKir3.2 in hippocampal slices of young [postnatal day (P) 4-18] and adult wv/wv (>/=P24) mice, because protein levels of Kir 3. 1 and Kir3.2 appear to be normal in the first 3 postnatal weeks and only decrease thereafter. In disinhibited slices, the GABAB receptor agonist R-baclofen reduced burst activity in wv/wv mice but was much more potent in wild-type mice. Mean resting membrane potential, slope input resistance, and membrane time constant of CA3 neurons of adult wv/wv and wild-type mice were indistinguishable. However, R-baclofen or chloroadenosine did not induce K+ currents or any other conductance change in wv/wv mice. Moreover, electrical or chemical stimulation of inhibitory neurons did not evoke slow IPSPs in adult wv/wv mice. Only in a few cells of young wv/wv mice did GABAB receptor activation by R-baclofen or presynaptic stimulation induce small inward currents, which were likely caused by a Na+ ion influx through wvKir3.2 channels. The data show that the pore mutation in wvKir3.2 channels results in a hippocampal phenotype resembling Kir3.2-deficient mutants, although it is not associated with the occurrence of seizures.

Molecular determinants of dofetilide block of HERG K+ channels.

The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac delayed rectifier K+ current (IKr). HERG/IKr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.

GABAB receptor-mediated inhibition of spontaneous inhibitory synaptic currents in rat midbrain culture.

1. Tight-seal, whole-cell recording was used to study GABAB receptor-mediated inhibition of spontaneous inhibitory synaptic currents in cultured rat midbrain neurones. 2. Spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were recorded in tetrodotoxin (TTX), Cd2+ and Ba2+. (R)-(-)-baclofen reduced the frequency of mIPSCs through a presynaptic mechanism. The EC50 for this effect was 7 microM. It was antagonized by the GABAB receptor antagonist CGP55845A (0.5 microM). 3. In pertussis toxin (PTX)-treated cultures, some GABAB receptor-mediated reduction of the frequency of mIPSCs persisted. In contrast, PTX treatment totally abolished inhibition of miniature excitatory postsynaptic currents (mEPSCs). 4. In PTX-treated cultures, a saturating concentration of (R)-(-)-baclofen inhibited action potential-generated IPSCs but no EPSCs. 5. PTX treatment abolished the (R)-(-)-baclofen-mediated inhibition of high voltage-activated somatic Ca2+ currents and of spontaneous IPSCs depending on presynaptic Ca2+ entry. 6. We conclude that cellular mechanisms underlying GABAB receptor-mediated inhibition of mIPSCs contribute to auto-inhibition of GABA release.

GABAB receptor-mediated inhibition of tetrodotoxin-resistant GABA release in rodent hippocampal CA1 pyramidal cells.

Tight-seal whole-cell recordings from CA1 pyramidal cells of rodent hippocampus were performed to study GABAB receptor-mediated inhibition of tetrodotoxin (TTX)-resistant IP-SCs. IPSCs were recorded in the presence of TTX and glutamate receptor antagonists. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs by a presynaptic action. The inhibition by (R)-(-)-baclofen was concentration-dependent, was not mimicked by the less effective enantiomer (S)-(+)-baclofen, and was blocked by the GABAB receptor antagonist CGP 55845A, suggesting a specific effect on GABAB receptors. The inhibition persisted in the presence of the Ca2+ channel blocker Cd2+. There was no requirement for an activation of K+ conductances by (R)-(-)-baclofen, because the inhibition of TTX-resistant IPSCs persisted in Ba2+ and Cd2+. Because the time courses of TTX-resistant IPSCs were not changed by (R)-(-)-baclofen, there was no evidence for a selective inhibition of quantal release from a subgroup of GABAergic terminals. (R)-(-)-baclofen reduced the frequency of TTX-resistant IPSCs in guinea pigs and Wistar rats, whereas the inhibition was much smaller in Sprague Dawley rats. In Cd2+ and Ba2+, beta-phorbol-12,13-dibutyrate and forskolin enhanced the frequency of TTX-resistant IPSCs. Only beta-phorbol-12, 13-dibutyrate reduced the inhibition by (R)-(-)-baclofen. We conclude that GABAB receptors inhibit TTX-resistant GABA release through a mechanism independent from the well known effects on Ca2+ or K+ channels. The inhibition of quantal GABA release can be reduced by an activator of protein kinase C.

(-)-Baclofen-induced and constitutively active inwardly rectifying potassium conductances in cultured rat midbrain neurons.

Biophysical and pharmacological properties, and development of the (-)-baclofen-induced potassium (KBac) conductance and the constitutively active inwardly rectifying potassium (KIR) conductance were characterised using the patch-clamp technique in cultured embryonic rat midbrain neurons. KBac conductance was induced by (-)-baclofen acting on gamma-aminobutyric acid B (GABAB) receptors, and displayed a high degree of selectivity for potassium ions, an approximate square-root dependence of conductance on extracellular potassium concentration and strongly voltage-dependent activation. Ba2+ blocked the conductance in a voltage-independent manner, whereas Cs+ produced a voltage-dependent block. In the same preparation, the KIR conductance displayed biophysical properties indistinguishable from those of the KBac conductance. Block of KIR currents by Ba2+ was voltage independent (KI, 4 microM), whereas Cs+ produced a voltage-dependent block (KI, 370 microM at -100 mV, equivalent valence, z', 1.67). The KBac and KIR conductances additionally displayed a strikingly similar pattern of development in culture; the specific conductance (nS/pF) of both conductances increased two- to three-fold between the first and second week in vitro, and remained constant thereafter.

Role of chloride-homeostasis in the inhibitory control of neuronal network oscillators.

1. Spontaneous synaptic activity in networks formed by dissociated neurons from embryonic rat midbrain was analyzed in tight seal whole cell recordings. 2. Application of furosemide (0.5 mM) to the cell and its surrounding area increased the frequency of spontaneous synaptic currents. Incubation of the culture with furosemide resulted in "rhythmic" burst activity. 3. Furosemide (0.1-0.5 mM) changed equilibrium potentials of inhibitory postsynaptic currents, gamma-aminobutyric acid-A (GABAA) or glycine receptor-mediated Cl- currents by a blockade of Cl(-)-outward transport. Furosemide did not alter the slope conductance of GABAA receptor-mediated currents. Membrane conductance and cell excitability were also unaffected. 4. We conclude that furosemide locked the activity of the network in "burst activity" mode through impairment of inhibition resulting from the disturbance of Cl- homeostasis.

Structure-activity relationship of quaternary ammonium ions at the external tetraethylammonium binding site of cloned potassium channels.

Changes in the chemical structure of the tetraethylammonium (TEA) ion reduce binding affinity at the external TEA receptor of outwardly rectifying potassium channels. To study the mechanism of selective binding, we applied a variety of hydrophilic quaternary ammonium (QA) ions to the noninactivating mutant of Shaker B T449Y, to Kv3.1, and to Kv3.1 mutants, expressed in Xenopus oocytes. In outside-out patches, QA ions in which ethyl groups of TEA were replaced by methyl groups had a lower affinity than TEA, whereas changes in binding affinity were minor when propyl groups were substituted for ethyl groups. All channels tested showed this pattern. Changes in free energy of binding correlated well with changes in the computed free energy of hydration of the TEA derivatives that we used. The affinity for TEA derivatives was reduced in Kv3.1Y407T, which is in support of the hypothesis that cation pi-electron interaction is involved. Binding affinities of QA ions were higher in Kv3.1 Y407F than in the wild-type, suggesting that the hydroxyl groups of the tyrosines reduce QA binding. The rank order of potency of the QA ions toward the different channels studied was the same. These results indicate that external QA ions bind strongly to hydrophobic pi-electron-rich functions. The selectivity, however, is determined by the physical properties of the QA ion.

The selectivity of different external binding sites for quaternary ammonium ions in cloned potassium channels.

Tetraethylammonium (TEA) is thought to be the most effective quaternary ammonium (QA) ion blocker at the external site of K+ channels, and small changes to the TEA ion reduce its potency. To examine the properties of the external QA receptor, we applied a variety of QA ions to excised patches from human embryonic kidney cells or Xenopus oocytes transfected with the delayed rectifying K+ channels Kv 2.1 and Kv 3.1. In outside-out patches of Kv 3.1, the relative potencies were TEA > tetrapropylammonium (TPA) > tetrabutylammonium (TBA). In contrast to Kv 3.1, the relative potencies in Kv 2.1 were TBA > TEA > TPA. In Kv 3.1 and Kv 2.1, external tetrapentylammonium (TPeA) blocked K+ currents in a fast, reversible and, in contrast to TEA, time-dependent manner. The external binding of TPeA appeared to be voltage independent, unlike the effects of TPeA applied to inside-out patches. External n-alkyl-triethylammonium compounds (C8, C10 chain length) had a lower affinity than TEA in Kv 3.1, but a higher affinity than TEA in Kv 2.1. In Kv 3.1, the decrease in QA affinity was large when one or two methyl groups were substituted for ethyl groups in TEA, but minor when propyl groups replaced ethyl groups. Changes in the free energy of binding could be correlated to changes in the free energy of hydration of TEA derivatives calculated by continuum methodology. These results reveal a substantial hydrophobic component of external QA ion binding to Kv 2.1, and to a lesser degree to Kv 3.1, in addition to the generally accepted electrostatic interactions. The chain length of hydrophobic TEA derivatives affects the affinity for the hydrophobic binding site, whereas the hydropathy of QA ions determines the electrostatic interaction energy.

A physiological role for GABAB receptors and the effects of baclofen in the mammalian central nervous system.

The inhibitory neurotransmitter GABA acts in the mammalian brain through two different receptor classes: GABAA and GABAB receptors. GABAB receptors differ fundamentally from GABAA receptors in that they require a G-protein. GABAB receptors are located pre- and/or post-synaptically, and are coupled to various K+ and Ca2+ channels presumably through both a membrane delimited pathway and a pathway involving second messengers. Baclofen, a selective GABAB receptor agonist, as well as GABA itself have pre- and post-synaptic effects. Pre-synaptic effects comprise the reduction of the release of excitatory and inhibitory transmitters. GABAergic receptors on GABAergic terminals may regulate GABA release, however, in most instances spontaneous inhibitory synaptic activity is not modulated by endogenous GABA. Post-synaptic GABAB receptor-mediated inhibition is likely to occur through a membrane delimited pathway activating K+ channels, while baclofen, in some neurons, may activate K+ channels through a second messenger pathway involving arachidonic acid. Some, but not all GABAB receptor-gated K+ channels have the typical properties of those G-protein-activated K+ channels which are also gated by other endogenous ligands of the brain. New, high affinity GABAB antagonists are now available, and some pharmacological evidence points to a receptor heterogeneity. The pharmacological distinction of receptor subtypes, however, has to await final support from a characterization of the molecular structure. The function importance of post-synaptic GABAB receptors is highlighted by a segregation of GABAA and GABAB synapses in the mammalian brain.

Differences in the Cs block of baclofen and 4-aminopyridine induced potassium currents of guinea pig CA3 neurons in vitro.

Single-electrode current- and voltage-clamp techniques were employed to study responses elicited by (-)baclofen or gamma-aminobutyric acid (GABA) and 4-aminopyridine (4-AP) induced inhibitory postsynaptic potentials in CA3 pyramidal neurons in guinea pig hippocampal slices. All drugs were applied by the bath to submerged slices in which fast synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM), bicuculline (50 microM), and picrotoxin (50 microM). (-)Baclofen (0.5 microM) and GABA (1 mM) induced equivalent-sized hyperpolarizations and input resistance decreases. The agonist induced hyperpolarization or current and 4-AP induced hyperpolarizations or currents (4-AP induced K-IPSPs or IPSCs) reversed in sign near the K-equilibrium potential (EK). The GABAB receptor antagonists, OH-saclofen (500 microM) and CGP 35348 (100 microM), reduced (-)baclofen responses, and 4-AP induced K-IPSPs, suggesting that they were mediated by GABAB receptors. Intracellular tetraethylammonium-, and extracellular barium-ions (1 mM) diminished the (-)baclofen induced current and 4-AP induced K-IPSCs. Intracellular Cs-ions blocked the (-)baclofen induced outward current at resting membrane potential but did not grossly affect the inward current recorded at membrane potentials negative to EK. 4-AP induced inwardly or outwardly directed K-IPSCs were not blocked by intracellular Cs-ions. Extracellular Cs-ions (5 mM) blocked the (-)baclofen induced inward K-current, but did not block 4-AP induced inwardly directed K-IPSCs. In conclusion, we found differences in the Cs block of activated by (-)baclofen or the endogenous transmitter GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of serotonin on hilar neurons and granule cell inhibition in the guinea pig hippocampal slice.

Intracellular recordings in guinea pig hippocampal slices were used to study the effects of serotonin (5-HT) on presumed inhibitory hilar neurons and on postsynaptic inhibition of granule cells. 5-HT applied by the bath hyperpolarized only 50% of the hilar neurons tested but all CA3 neurons and granule cells, presumably by activating a K-conductance. The bath application of 4-aminopyridine (4-AP, 50 microM) induced burst discharge activity in hilar neurons and giant inhibitory postsynaptic potentials (IPSPs) in granule cells consisting of a Cl- and K-component. 5-HT (5-10 microM) reversibly blocked the K-component of giant IPSPs in granule cells, but not their Cl-component. In the majority of hilar neurons 5-HT increased the frequency of 4-AP induced burst discharges even when hilar neurons were hyperpolarized. Only in a few hilar neurons 5-HT blocked 4-AP induced burst discharges. We conclude that the changes in burst discharge pattern of hilar neurons correspond with the differential effect of 5-HT on Cl- and K-mediated inhibition of granule cells.

4-Aminopyridine-induced synaptic GABAB currents in granule cells of the guinea-pig hippocampus.

Sharp-electrode and tight-seal perforated-patch and whole-cell recording techniques were used to evaluate K(+)-dependent inhibitory postsynaptic potentials (K-IPSPs) and currents (K-IPSCs) induced by the convulsant 4-aminopyridine (50 mumol l-1) in granule cells of guinea-pig hippocampal slices. The responses were recorded in the presence of blockers for glutamatergic and GABAA-receptor-mediated synaptic transmission, 6-cyano-7-nitroquinoxaline-2,3-dione, picrotoxin and bicuculline. The input resistance was much larger (approximately 300 M omega) in tight-seal recording than in sharp-electrode recording (approximately 100 M omega), but the amplitudes of K-IPSPs recorded at -65 mV holding potential were similar in all three recording configurations. The 4-aminopyridine-induced currents reversed near the K+ equilibrium potential, and the reversal potentials shifted with changes in [K+]out or [K+]in as expected for a K+ current. Slope conductance measurements indicated a conductance increase during the peak of the K-IPSP up to 5 nS (mean 2.4 nS). The peak conductance was underestimated in whole-cell recordings unless the pipette contained Cs+. Considering the high membrane resistance of granule cells, K-IPSCs induced by 4-aminopyridine hyperpolarize the cells considerably and thereby are likely to contribute to the failure of 4-aminopyridine to induce burst discharges in granule cells.

CGP 55845A blocks baclofen, gamma-aminobutyric acid and inhibitory postsynaptic potassium currents in guinea pig CA3 neurons.

Single electrode voltage-clamp recording from CA3 neurons in guinea pig hippocampal slices was applied to study effects of a new GABAB antagonist, CGP 55845A, on (-)baclofen (IBac)- or gamma-aminobutyric acid (IGABA)-induced potassium (K)-currents and on inhibitory postsynaptic K-currents (K-IPSCs) recorded in the presence of blockers for fast synaptic transmission. K-IPSCs were induced by bath application of 4-amino-pyridine (4-AP). CGP 55845A, in 10(-8) to 10(-7) M concentrations, blocked all these K-currents and was more potent than all GABAB antagonists known to date. However, onset of the CGP 55845A effect and recovery were slow. We conclude that a potent and selective GABAB antagonist is now available to study the physiological role of GABAB receptors in the mammalian brain.