Synaptically activated Cl- accumulation responsible for depolarizing GABAergic responses in mature hippocampal neurons.

It is known that GABA, a major inhibitory transmitter in the CNS, acts as an excitatory (or depolarizing) transmitter transiently after intense GABAA receptor activation in adult brains. The depolarizing effect is considered to be dependent on two GABAA receptor-permeable anions, chloride (Cl-) and bicarbonate (HCO3-). However, little is known about their spatial and temporal profiles during the GABAergic depolarization in postsynaptic neurons. In the present study, we show that the amplitude of synaptically induced depolarizing response was correlated with intracellular Cl- accumulation in the soma of mature hippocampal CA1 pyramidal cells, by using whole cell patch-clamp recording and Cl- imaging technique with a Cl- indicator 6-methoxy-N-ethylquinolinium iodide (MEQ). The synaptically activated Cl- accumulation was mediated dominantly through GABAA receptors. Basket cells, a subclass of fast-spiking interneurons innervating the somatic portion of the pyramidal cells, actually fired at high frequency during the Cl- accumulation accompanying the depolarizing responses. These results suggest synaptically activated GABAA-mediated Cl- accumulation may play a critical role in generation of an excitatory GABAergic response in the mature pyramidal cells receiving intense synaptic inputs. This may be the first demonstration of microscopic visualization of intracellular Cl- accumulation during synaptic activation.

Excitatory GABA input directly drives seizure-like rhythmic synchronization in mature hippocampal CA1 pyramidal cells.

GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABA(A) receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABA(A) receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average -38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABA(A) receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy.

Possible regulatory role of dendritic spikes in induction of long-term potentiation at hippocampal schaffer collateral-CA1 synapses.

The amplitude of backpropagating action potentials (BAPs) is attenuated, either activity- or neurotransmitter-dependently in the apical dendrite of hippocampal pyramidal neurons. To test the possibility that this BAP attenuation may contribute to regulating the inducibility of long-term potentiation (LTP), BAPs evoked by theta-burst stimulation (TBS), a standard protocol for LTP induction, to apical dendrite synapses were subjected to perturbation by conditioning stimuli to basal dendrite synapses. During this conditioned TBS (cTBS), the amplitude of BAPs was noticeably attenuated, but that of somatic action potentials was not. In the distal dendrite area, cTBS-induced LTP was much smaller than that induced by TBS. By contrast, no difference was observed between TBS- and cTBS-induced LTP in the proximal dendrite area. These findings suggest that the activity-dependent attenuation of BAPs, propagating along the apical dendrite, may serve to regulate hippocampal synaptic plasticity.

Overt congestive heart failure with mitral and aortic regurgitation due to antiphospholipid syndrome in a patient with systemic lupus erythematosus.

A 51-year-old woman with overt congestive heart failure with pleural and pericardial effusion was treated with furosemide and nifedipine, leading to improvement in her condition and a decrease in effusions. An echocardiography demonstrated mitral and aortic regurgitation with mitral valve prolapse, which caused the congestive heart failure. Since leukocytopenia and lymphocytopenia with arthralgia could be observed, serological investigations were performed. She was diagnosed as having systemic lupus erythematosus (SLE) with antiphospholipid syndrome, and started on a treatment of prednisolone and aspirin. Based on the treatment, the pleural and pericardial effusion went into complete remission, indicating that the serositis related to SLE had overlapped the heart failure. Since there was no evidence of any other diseases that could be responsible for the valvular lesions, we concluded that they were due to antiphospholipid syndrome. The administration of prednisolone had no significant effect on valvular morphology or function as demonstrated by echocardiography. When patients with valvular disease are seen, a valvulopathy related to antiphospholipid syndrome should be considered as part of the differential diagnosis.

Intracellular calcium releases facilitate induction of long-term depression.

In visual cortex layer II/III pyramidal neurons, long-term depression (LTD) of synaptic currents was induced by a combination of the intracellular increase of inositol-1,4,5-trisphosphate (IP(3)), achieved by photolyzing caged-IP(3), and the tetanization to nearby cortex, but not by either of these two procedures alone. A facilitatory role of an IP(3)-induced calcium release in LTD is suggested.

An IP3-assisted form of Ca2+-induced Ca2+ release in neocortical neurons.

Calcium increases induced by single action potentials in rat visual cortex layer II/III pyramidal neurons were shown to be augmented by muscarinic acetylcholine receptor (mAchR) stimulation. This augmentation was drastically reduced by intracellular injection of heparin but not ruthenium red, therefore involving inositol-1,4,5-trisphosphate (IP3)-sensitive rather than ryanodine-sensitive calcium stores. Only the calcium increase induced by the second or later spike of a spike train, but not that induced by the first spike, was augmented, indicating the requirement of both spike-induced calcium increase and mAchR activation. The calcium store depletor thapsigargin abolished this augmentation use-dependently. These findings suggest a neocortical occurrence of calcium-induced calcium release from IP3-sensitive calcium stores that have been sensitized beforehand by IP3 through mAchR-mediated mechanisms.

Occurrence of dentate granule cell LTP without proximal dendritic Ca2+ increase.

We investigated activity-dependent calcium increases in proximal dendrites of dentate granule cells in the rat hippocampus, and its relationship with induction of LTP at perforant path synapses (PP-synapses). LTP was induced at PP-synapses by high-frequency stimulation (HFS; 100 Hz for 0.4 s), and the same HFS evoked a dendritic calcium increase in the proximal dendrite. However, bath-application of the L-type voltage-dependent calcium channel (VDCC) blocker nimodipine noticeably reduced this calcium increase without abolishing induction of LTP. This calcium increase mediated by high-threshold VDCCs is likely to be evoked by action potentials. LTP induction at PP-synapses is hence suggested to be independent of action potential-induced calcium increases in the proximal dendrite.

Action potential-induced dendritic calcium dynamics correlated with synaptic plasticity in developing hippocampal pyramidal cells.

In hippocampal CA1 pyramidal cells, intracellular calcium increases are required for induction of long-term potentiation (LTP), an activity-dependent synaptic plasticity. LTP is known to develop in magnitude during the second and third postnatal weeks in the rats. Little is known, however, about development of intracellular calcium dynamics during the same postnatal weeks. We investigated postnatal development of intracellular calcium dynamics in the proximal apical dendrites of CA1 pyramidal cells by whole cell patch-clamp recordings and calcium imaging with the Ca(2+) indicator fura-2. Dendritic calcium increases induced by intrasomatically evoked action potentials were slight during the first postnatal week but gradually became robust 3 to 6-fold during the second and third postnatal weeks. These calcium increases were blocked by application of 250 microM CdCl(2), a nonspecific blocker for high-threshold voltage-dependent calcium channels (VDCCs). Under the voltage-clamp condition, both calcium currents and dendritic calcium accumulations induced by depolarization were larger at the late developmental stage (P15-18) than the early stage (P4-7), indicating developmental enhancement of calcium influx mediated by high-threshold VDCCs. Moreover, theta-burst stimulation (TBS), a protocol for LTP induction, induced large intracellular calcium increases at the late developmental stage, in synchrony with maturation of TBS-induced LTP. These results suggest that developmental enhancement of intracellular calcium increases induced by action potentials may underlie maturation of calcium-dependent functions such as synaptic plasticity in hippocampal neurons.

Developmental regulation of action potential-induced Ca2+ entry in neocortical neurons.

Intracellular calcium plays a critical role in the regulation of membrane excitability, synaptic integration and synaptic plasticity. Using whole-cell recording and calcium imaging, we investigated intracellular calcium dynamics in apical dendrites of layer V pyramidal cells in the immature rat neocortex. Dendritic calcium increases induced by action potentials were only small on postnatal days 4-6 (P4-6), then underwent a gradual enhancement during the second post-natal week and were 2- to 3-fold larger on P16-18 than on P4-6. At P15-18 there were no significant differences in the calcium increases among the three subclasses of layer V pyramidal neurons: adapting regular spiking (RS), non-adapting RS and intrinsically bursting (IB) neurons. Developmental regulation of dendritic calcium dynamics may be crucial for functional maturation of the neocortex.

Distinct temporal profiles of activity-dependent calcium increase in pyramidal neurons of the rat visual cortex.

1. Using fluo-3-based fluorometry, we studied variation in depolarization-induced calcium increases in the proximal dendrites or soma of pyramidal neurons in layer II/III of the rat visual cortex. 2. Depolarization for all durations tested (0.1-2 s; 0.5 nA) evoked a train of action potentials and a small increase in calcium signal (mean 26 %) which peaked within 1 s of the onset of depolarization. With depolarization for longer than 1 s, this small increase was often followed by a larger increase (73 %). This later phase of calcium increase occurred without sudden changes in action potential firing. 3. Application of ryanodine, which suppresses intracellular calcium release, abolished the second phase without affecting the early phase in a use-dependent manner. Meanwhile, no major changes were observed in the pattern of action potential firing. 4. In calcium-free medium, both the early and late phases were almost undetectable, although action potential firing was still evoked by injection of depolarizing currents. Since the late phase depended on intracellular calcium release, this effect of calcium-free medium on the late phase is likely to be indirect through an influence on the early phase. 5. This two-phase profile was observed with somatic depolarization or with antidromic action potentials induced by tetanization. Neocortical pyramidal neurons can thus recruit calcium from different sources, even without chemical sensitization, generating temporally diverse profiles of intracellular calcium signal in response to action potential firing. 6. Such variety in the mechanisms of calcium increase may be relevant to the role of calcium as a versatile second messenger for various types of synaptic plasticity.

Postnatal development of action potential-induced dendritic calcium entry in neocortical layer II/III pyramidal cells.

By whole-cell patch-clamp recording and calcium imaging with fura-2, we investigated postnatal development of intracellular calcium dynamics in apical dendrites of layer II/III pyramidal cells in the rat visual cortex. Dendritic calcium increases, induced by single action potentials, occurred only slightly on postnatal days 8-10 (P8-10), and then underwent a gradual enhancement during the second postnatal week to become 2-3-fold larger on P16-18 than on P8-10. The results suggest that the developmental growth of calcium dynamics may play a critical role for functional development of neocortical neurons.

Enhancement of activity-dependent calcium increase by neurotrophin-4 in visual cortex pyramidal neurons.

In pyramidal neurons from rat visual cortex slices, bath-application of NT-4 (20 ng/ml) did not much affected the baseline calcium signal, but did enhance calcium signals elicited by injections of depolarizing currents (+0.5 nA, 1 s). This enhancing effect of NT-4 was abolished by co-applying K252a. With ryanodine injected intracellularly, the effect of NT-4 was significantly reduced, suggesting an involvement of intracellular calcium release in this NT-4-induced enhancement of calcium transient.

Peptide based interleukin-1 beta converting enzyme (ICE) inhibitors: synthesis, structure activity relationships and crystallographic study of the ICE-inhibitor complex.

Based on the X-ray structure of the complex of Ac-Tyr-Val-Ala-Asp-H (L-709049) and interleukin-1 beta converting enzyme (ICE), we synthesized compounds which were derived from 2-NapCO-Val-Pro-Asp-CH2OPh (1) to obtain a potent inhibitor in the cell assay. Among these compounds, (3S)-N-methanesulfonyl-3-[[1-[N-(2-naphthoyl)-L-valyl]-L-prolyl]amino]- 4-oxobutanamide (27c) showed high potency not only in the enzyme assay but also cell assay with IC50 values of 38 nM and 0.23 microM, respectively. Compound 27c, with a c log P value of 1.76, had a more hydrophilic character compared with 1. Compound 27c also dose dependently inhibited LPS-primed ATP-induced IL-1 beta release in mice. The crystal structure of the complex of compound 27c and ICE revealed that compound 27c had further interactions with ICE in the naphthoyl group at the P4 position and in the methyl group of the methanesulfonamidecarbonyl group at the P1 position, compared with L-709049. To our knowledge, compound 27c is the first example that shows a strong inhibitory activity without the carboxyl group at the P1 position.

Studies on novel bone resorption inhibitors. II. Synthesis and pharmacological activities of fused aza-heteroarylbisphosphonate derivatives.

Two new series of fused aza-heteroarylbisphosphonates (5, 8), which are structurally quite different from incadronate (YM175), and related compounds were synthesized and evaluated for antiresorptive activity using a parathyroid hormone(PTH)-induced hypercalcemia model in rats (PIH model). Among these compounds, several exhibited more potent antiresorptive activity than pamidronate. In particular, [1-hydroxy-2-(imidazo[1,2-a]pyridin-3-yl)ethylidene]bisphosphonic acid (5b, minodronate) was 100-fold more potent than pamidronate in not only the PIH model, but also in an immobilization bone atrophy model in rats (DA model), and was selected for clinical development. The structure-activity relationships in these new series of bisphosphonates are discussed.

Spiro-substituted piperidines as neurokinin receptor antagonists. III. Synthesis of (+/-)-N-[2-(3,4-dichlorophenyl)-4-(spiro-substituted piperidin-1'-yl)butyl]-N-methylbenzamides and evaluation of NK1-NK2 dual antagonistic activities.

To discover a novel NK1-NK2 dual antagonist, we have synthesized a series of spiro-substituted piperidines utilizing YM-35375 as a lead compound, and evaluated affinities for NK1 and NK2 receptors. In the N-methylbenzamide moiety, introduction of methoxy groups increased affinity for the NK1 receptor without a significant loss of affinity for the NK2 receptor. We also found that a conformation in which the phenyl groups of the N-methylbenzamide and 3,4-dichlorophenyl moieties are close to each other through a cis-amide bond, may be favorable for showing high affinity for the NK1 receptor and that a hydrogen bond-accepting group in the spiro-substituted piperidine moiety may be crucial for exhibiting high affinity for the NK2 receptor. Among the compounds prepared, YM-44778 (31) showed high and well-balanced affinity for NK1 and NK2 receptors (IC50 values of 18 and 16 nM, respectively). This compound also exhibited potent antagonistic activities against both NK1 and NK2 receptors (IC50 values of 82 and 62 nM, respectively) in isolated tissues.

Selective muscarinic antagonists. I. Synthesis and antimuscarinic properties of 4-piperidyl benzhydrylcarbamate derivatives.

A series of 1-substituted-4-piperidyl benzhydrylcarbamate derivatives were synthesized and evaluated for binding affinity to M1, M2 and M3 receptors, and for antimuscarinic activities. Receptor binding assays indicated that 1-benzyl-4-piperidyl benzhydrylcarbamate derivatives showed higher affinities for M1 and M3 receptors, and good selectivities for M3 over M2 receptor, than the corresponding ester analog. These results indicate that the urethane bond is a novel linker for muscarinic antagonists, and serves to lock the molecular conformation and allows the hydrophobic portion and cationic site of the molecule to bind to M1 and M3 muscarinic receptors. Among the prepared compounds, 1-(4-methylaminobenzyl)-4-piperidyl benzhydrylcarbamate monohydrochloride (18b, YM-58790) exhibited potent inhibitory activity on bladder pressure in reflexly-evoked rhythmic contraction, comparable to oxybutynin and was approximately ten times less inhibitory on oxotremorine-induced salivary secretion than oxybutynin in rats. Further evaluation of antimuscarinic effects on bradycardia and pressor in pithed rats, and on tremor in mice, demonstrated that YM-58790 can be useful for treatment of urinary urge incontinence as a bladder-selective M3 antagonist with fewer side effects.

Selective muscarinic antagonists. II. Synthesis and antimuscarinic properties of biphenylylcarbamate derivatives.

A novel series of biphenylylcarbamate derivatives were synthesized and evaluated for binding to M1, M2 and M3 receptors and for antimuscarinic activities. Receptor binding assays indicated that biphenyl-2-ylcarbamate derivatives had high affinities for M1 and M3 receptors and good selectivities for M3 receptor over M2 receptor, indicating that the biphenyl-2-yl group is a novel hydrophobic replacement for the benzhydryl group in the muscarinic antagonist field. In this series, quinuclidin-4-yl biphenyl-2-ylcarbamate monohydrochloride (8l, YM-46303) exhibited the highest affinities for M1 and M3 receptors, and selectivity for M3 over M2 receptor. Compared to oxybutynin, YM-46303 showed approximately ten times higher inhibitory activity on bladder pressure in reflexly-evoked rhythmic contraction, and about 5-fold greater selectivity for urinary bladder contraction against salivary secretion in rats. Moreover, selective antagonistic activity was also observed in vitro. Further evaluation of antimuscarinic effects on bradycardia and pressor in pithed rats, and on tremor in mice, showed that YM-46303 can be useful for the treatment of urinary urge incontinence as a bladder-selective M3 antagonist with potent activities and fewer side effects.

Spiro-substituted piperidines as neurokinin receptor antagonists. II. Syntheses and NK2 receptor-antagonistic activities of N-[2-aryl-4-(spiro-substituted piperidin-1'-yl)butyl]carboxamides.

In the course of our research on spiro-compounds as neurokinin receptor antagonists, N-[2-aryl-4-(spiro-substituted piperidin-1'-yl)butyl]carboxamides were designed, based on YM-35375 (3) as a lead compound, and evaluated for NK2 receptor-antagonistic activities. Some derivatives inhibited the binding of radio-labeled neurokinin A to the NK2 receptor with IC50 values at the level of 10(-9) M. Among these compounds, (+/-)-1'-[4-(N-benzoyl-N-methylamino)-3- (3,4-dichlorophenyl)butyl]spiro[benzo[c]thiophene-1(3H), 4'-piperidine] 2-oxide (58, YM-38336) showed 10 times more potent NK2 receptor binding affinity than compound 3 (IC50 values of 8.9 and 84 nM, respectively). It showed more potent inhibitory activity (ID50 20 micrograms/kg (i.v.)) against [beta-Ala8]-NKA(4-10)-induced bronchoconstriction in guinea pigs than compound 3 (ID50 41 micrograms/kg (i.v.)). This compound was also effective intraduodenally in the same model, exhibiting an ID50 value of 0.41 microgram/kg.

Spiro-substituted piperidines as neurokinin receptor antagonists. I. Design and synthesis of (+/-)-N-[2-(3,4-dichlorophenyl)-4-(spiro [isobenzofuran-1(3H),4'piperidin]-1'-yl)butyl]-N-methylbenzamide, YM-35375, as a new lead compound for novel neurokinin receptor antagonists.

Analysis of the structural requirements of compound 1 (SR48968), a potent NK2 receptor antagonist, revealed that the 4-phenyl group of the piperidine is essential for binding with the NK2 receptor and occupies an equatorial position. Energy calculation of a variety of substituted 4-phenyl piperidines revealed that spiro[isobenzofuran-1(3H),4'-piperidine] possesses a conformationally restricted equatorial phenyl group. Our compound 12 (YM-35375) possessing this spiro-substituted piperidine bound to the NK2 receptor with an IC50 value of 84 nM and to the NK1 receptor with an IC50 value of 710 nM. It showed more potent inhibitory activity (ID50 41 micrograms/kg (i.v.)) against [beta-Ala8]-NKA(4-10)-induced bronchoconstriction in guinea pigs than (+/-)-SR48968 (ID50 68 micrograms/kg (i.v.)). YM-35375 may be a new lead compound for novel NK2 receptor antagonists or NK1-NK2 dual antagonists.

Synthesis and NK1 receptor antagonistic activity of (+/-)-1-acyl-3-(3,4- dichlorophenyl)-3-[2-(spiro-substituted piperidin-1'-yl)ethyl]piperidines.

(+/-)-1-Acyl-3-(3,4-dichlorophenyl)-3-[2-(spiro-substituted piperidin-1'-yl)ethyl]piperidines and their quaternary ammonium salts were prepared and evaluated for their NK1 receptor antagonistic activity. Some of these inhibited SP-induced contraction in guinea pig ileum with IC50 values at a level of 10(-9) M and showed potent inhibitory activity against selective NK1 receptor agonist-induced bronchoconstriction in guinea pigs.