GABAergic synaptic transmission onto striatal cholinergic interneurons in dopamine D2 receptor knock-out mice.

Whole-cell or cell-attached analysis was carried out in dopamine (DA) D2 receptor (D2R) knock-out (KO) mice to elucidate the function of this receptor in the regulation of GABAergic synaptic transmission onto striatal cholinergic interneurons as well as their spontaneous firing. In slice preparation obtained from wild-type mice, evoked GABAergic inhibitory postsynaptic currents (IPSCs) showed frequency-dependent suppression, and this suppression significantly decreased in the presence of a D2-like receptor antagonist or in D2R KO mice. Contribution of N-type calcium channel was also significantly reduced in the striatal cholinergic interneurons of the D2R KO mice compared with that in the wild-type mice. Spontaneous firing of striatal cholinergic interneurons was inhibited by 5- or 10-Hz stimulation, and the suppression was decreased in the presence of a D2-like receptor antagonist or in D2R KO mice. These findings substantiate the physiological role of D2R in the regulation of GABAergic synaptic transmission onto striatal cholinergic interneurons as well as their excitability, confirming the tight coupling between D2R and N-type calcium channels in the regulation of GABA release.

Charon's size and an upper limit on its atmosphere from a stellar occultation.

Pluto and its satellite, Charon (discovered in 1978; ref. 1), appear to form a double planet, rather than a hierarchical planet/satellite couple. Charon is about half Pluto's size and about one-eighth its mass. The precise radii of Pluto and Charon have remained uncertain, leading to large uncertainties on their densities. Although stellar occultations by Charon are in principle a powerful way of measuring its size, they are rare, as the satellite subtends less than 0.3 microradians (0.06 arcsec) on the sky. One occultation (in 1980) yielded a lower limit of 600 km for the satellite's radius, which was later refined to 601.5 km (ref. 4). Here we report observations from a multi-station stellar occultation by Charon, which we use to derive a radius, R(C) = 603.6 +/- 1.4 km (1sigma), and a density of rho = 1.71 +/- 0.08 g cm(-3). This occultation also provides upper limits of 110 and 15 (3sigma) nanobar for an atmosphere around Charon, assuming respectively a pure nitrogen or pure methane atmosphere.

Dopamine D(2)-like receptors selectively block N-type Ca(2+) channels to reduce GABA release onto rat striatal cholinergic interneurones.

1. The modulatory roles of dopamine (DA) in inhibitory transmission onto striatal large cholinergic interneurones were investigated in rat brain slices using patch-clamp recording. 2. Pharmacologically isolated GABA(A) receptor-mediated IPSCs were recorded by focal stimulation within the striatum. Bath application of DA reversibly suppressed the amplitude of evoked IPSCs in a concentration-dependent manner (IC(50), 10.0 microM). 3. A D(2)-like receptor agonist, quinpirole (3-30 microM), also suppressed the IPSCs, whereas a D(1)-like receptor agonist, SKF 81297, did not affect IPSCs. Sulpiride, a D(2)-like receptor antagonist, blocked the DA-induced suppression of IPSCs (apparent dissociation constant (K(B)), 0.36 microM), while a D(1)-like receptor antagonist, SCH 23390 (10 microM), had no effect. 4. DA (30 microM) reduced the frequency of spontaneous miniature IPSCs (mIPSCs) without changing their amplitude distribution, suggesting that GABA release was inhibited, whereas the sensitivity of postsynaptic GABA(A) receptors was not affected. The effect of DA on the frequency of mIPSCs was diminished when extracellular Ca(2+) was replaced by Mg(2+) (5 mM), indicating that DA affected the Ca(2+) entry into the presynaptic terminal. 5. An N-type Ca(2+) channel selective blocker, omega-conotoxin GVIA (omega-CgTX, 3 microM), suppressed IPSCs by 65.4 %, whereas a P/Q-type Ca(2+) channel selective blocker, omega-agatoxin IVA (omega-Aga-IVA, 200 nM), suppressed IPSCs by 78.4 %. Simultaneous application of both blockers suppressed IPSCs by 95.9 %. Assuming a 3rd power relationship between Ca(2+) concentration and transmitter release, the contribution of N-, P/Q- and other types of Ca(2+) channels to presynaptic Ca(2+) entry is estimated to be, respectively, 29.8, 40.0 and 34.5 % at this synapse. After the application of omega-CgTX, DA (30 microM) no longer affected IPSCs. In contrast, omega-Aga-IVA did not alter the level of suppression by DA, suggesting that the action of DA was selective for N-type Ca(2+) channels. 6. A G protein alkylating agent, N-ethylmaleimide (NEM), significantly reduced the DA-induced suppression of IPSCs. 7. These results suggest that presynaptic D(2)-like receptors are present on the terminals of GABAergic afferents to striatal cholinergic interneurones, and down-regulate GABA release by selectively blocking N-type Ca(2+) channels through NEM-sensitive G proteins.

Presynaptic dopamine D2-like receptors inhibit excitatory transmission onto rat ventral tegmental dopaminergic neurones.

1. The effects of dopamine (DA) on non-NMDA glutamatergic transmission onto dopaminergic neurones in the ventral tegmental area (VTA) were examined in rat midbrain slices using the whole-cell patch-clamp technique. EPSCs in dopaminergic neurones evoked by focal stimulation within the VTA were reversibly blocked by 5 microM CNQX in the presence of bicuculline (20 microM), strychnine (0.5 microM) and D-amino-5-phosphonopentanoic acid (D-AP5, 25 microM). 2. Bath application of DA reduced the amplitude of EPSCs up to 65.1 +/- 9. 52% in a concentration-dependent manner between 0.3-1000 microM (IC50, 16.0 microM) without affecting the holding current at -60 mV measured using a Cs+-filled electrode. 3. The effect of DA on evoked EPSCs was mimicked by the D2-like receptor agonist quinpirole but not by the D1-like receptor agonist SKF 81297, and was antagonized by the D2-like receptor antagonist sulpiride (KB, 0.96 microM), but not by the D1-like receptor antagonist SCH 23390 (KB, 228.6 microM). 4. Dopamine (30 microM) reduced the mean frequency of spontaneous miniature EPSCs (mEPSCs) without affecting their mean amplitude, and the DA-induced effect on the mEPSCs was dependent on the external Ca2+ concentration. 5. These results suggest that afferent glutamatergic fibres which terminate on VTA dopaminergic neurones possess presynaptic D2-like receptors, activation of which inhibits glutamate release by reducing Ca2+ influx.

Laparoscopic-assisted surgery for Crohn's disease: reduced surgical stress following ileocolectomy.

Recent progress in laparoscopic techniques has enabled operations for various intestinal disorders to be performed under laparoscopic assistance. This study was conducted to assess the benefits of performing laparoscopic-assisted surgery (LAS) in patients with Crohn's disease. LAS was performed in 24 selected patients with Crohn's disease, most of whom underwent ileocolic resection for ileitis and/or colitis with stenosis. To determine the benefits of LAS, the postoperative inflammatory parameters of these patients were examined and compared with those of 17 patients who underwent conventional open surgery. Despite giving all patients total parenteral nutrition (TPN) for more than 2 weeks preoperatively, two patients with large inflammatory masses involving enteroenteric fistulas required conversion to laparotomy. No laparoscopic procedure was converted for adhesions after previous resection or intraoperative complications. The maximum C-reactive protein values and body temperatures were significantly lower, and the time taken to normalize body temperature and leukocyte counts was significantly shorter in the LAS group than in the laparotomy group. LAS should be performed for patients with Crohn's disease in the inactive phase after appropriate nutritional support. Patients with terminal ileitis without a fistula are considered to have the highest indication for this procedure.

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells.

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.

Inhibition by gamma-aminobutyric acid system activation of epileptic seizures in spontaneously epileptic rats.

The effects of muscimol, a gamma-aminobutyric acid (GABA)A-receptor agonist, and aminooxy-acetic acid (AOAA), an inhibitor of GABA-converting enzyme, on tonic and absence-like seizures in spontaneously epileptic rats (SER: zi/zi, tm/tm) were investigated to elucidate whether GABAergic function operates normally in these animals. Muscimol at doses of 1 and 3 mg/kg (i.p.) induced high-voltage slow waves in the cortical and hippocampal EEG of SER, although the behavioral observation suggested inhibition of absence-like seizures. Similar high-voltage slow waves were also observed in the cortical and hippocampal EEG of normal rats with muscimol (1 and 3 mg/kg). Tonic convulsions in SER were dose-dependently inhibited by muscimol. AOAA (3 and 10 mg/kg, i.v.) inhibited both tonic and absence-like seizures in SER, although there were no obvious changes in EEG pattern. The inhibitory effects of AOAA on tonic convulsions appeared more slowly and lasted longer than those on absence-like seizures. Cerebral, hippocampal and cerebellar GABA levels were significantly higher in SER than the normal Kyo:Wistar and zitter rat (zi/zi), which were both the parent strains. These findings suggest that GABA receptors and GABAergic neurons are functional in SER and that the GABA system is involved in the inhibition of both seizures.

Functional involvement of serum response factor in the transcriptional regulation of caldesmon gene.

A 22-bp fragment including the CArG element (CArG1) is essential for the transcription of the caldesmon gene. In this study, we investigated the effects of serum response factor (SRF) on the functional regulation of caldesmon promoter in smooth muscle cells. Gel supershift assay revealed that SRF was one component of the CArG1-protein complex. Dominant-negative mutants of SRF suppressed the promoter activity of caldesmon, whereas wild-type SRF overcame this suppression. These results suggest that SRF functions as a core activating factor of the caldesmon promoter. Furthermore, fractionation of smooth muscle cells' nuclear extracts using DNA affinity paramagnetic particles suggests that SRF transactivates the caldesmon promoter in concert with additional factors in the flow-through fraction recruited to the CArG element.

Smooth muscle cell phenotype-dependent transcriptional regulation of the alpha1 integrin gene.

The expressional regulation of chicken alpha1 integrin in smooth muscle cells was studied. The alpha1 integrin mRNA was expressed developmentally and was distributed dominantly in vascular and visceral smooth muscles in chick embryos. In a primary culture of smooth muscle cells, alpha1 integrin expression was dramatically down-regulated during serum-induced dedifferentiation. Promoter analyses revealed that the 5'-upstream region (-516 to +281) was sufficient for transcriptional activation in differentiated smooth muscle cells but not in dedifferentiated smooth muscle cells or chick embryo fibroblasts. Like other alpha integrin promoters, the promoter region of the alpha1 integrin gene lacks TATA and CCAAT boxes and contains binding sites for AP1 and AP2. The essential difference from other alpha integrin promoters is the presence of a CArG box-like motif. Deletion and site-directed mutation analyses revealed that the CArG box-like motif was an essential cis-element for transcriptional activation in differentiated smooth muscle cells, whereas the binding sites for AP1 and AP2 were not. Using specific antibodies, a nuclear protein factor specifically bound to the CArG box-like motif was identified as serum response factor. These results indicate that alpha1 integrin expression in smooth muscle cells is regulated transcriptionally in a phenotype-dependent manner and that serum response factor binding plays a crucial role in this regulation.

Laparoscopic cholecystectomy in patients with cardiac disease: hemodynamic advantage of the abdominal wall retraction method.

We examined the use of an abdominal wall retraction method instead of pneumoperitoneum in laparoscopic cholecystectomy for patients with cardiac disease to prevent the hemodynamic deterioration associated with pneumoperitoneum. Eight patients with cardiac diseases, mainly valvular or coronary artery diseases, underwent laparoscopic cholecystectomy under hemodynamic monitoring. Five patients without cardiac disease served as controls. As hemodynamic parameters, heart rate, mean systemic arterial pressure (mAP), mean pulmonary arterial pressure (mPAP), central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP), and cardiac index (CI) were measured. The patients with cardiac disease showed significantly elevated mPAP and PCWP compared with the control group under pneumoperitoneum, and one patient showed critically decreased CI due to increased tricuspid regurgitation under pneumoperitoneum. These changes, however, were resolved on the abdominal wall retraction. There was no major perioperative complication. This abdominal wall retraction method is, therefore, favorable for patients with underlying cardiac disease to minimize the hemodynamic deterioration during laparoscopic cholecystectomy.

D2 receptor activation in distinct striatal neurons in comparison with D3 receptors.

The role of D2/D3 receptors in striatum was electrophysiologically examined in vitro in chloralose-anesthetized rats. In addition, in vitro patch clamp method with rat brain slices was followed. Stimulations of the substantia nigra pars compacta (SN) in vivo elicited spike generation which was inhibited by microiontophoretically applied domperidone, a D2 antagonist. These domperidone-sensitive neurons were activated by microiontophoretic application of D2 agonists such as talipexole, quinpirole and bromocriptine as well as the D2 agonist, 7-OH-DPAT. They were also excited by either intravenous injection of bromocriptine or talipexole in a dose-dependent manner. Furthermore, the SN-induced increases in neuronal firing were blocked during microiontophoretic application of domperidone. In patch clamp whole-cell recording large-sized cells, identified visually under Ramanosky microscope, were depolarized with repetitive firing on bath application of talipexole and 7-OH-DPAT at a current clamp mode. Talipexole-induced depolarization in the large-sized cell was similarly observed in the presence of TTX and high Mg2+ in Ca(2+)-free physiological solution. In contrast, the medium-sized cells were hyperpolarized on bath application of talipexole without being affected by 7-OH-DPAT. These findings suggest that the large-sized cells, which were presumably cholinergic interneurons, are activated by dopamine derived from the SN via D2 and/or D3 receptors, while the medium-sized cells are inhibited by dopamine via D2 receptors.

Laparoscopic intragastric surgery for gastric leiomyoma.

Laparoscopic intragastric surgery (LIGS) was performed on a 63-year-old man with a gastric leiomyoma adjacent to the cardia. Because the tumor was about 5 cm in maximum diameter and showed ulceration, the possibility that the tumor was a leiomyosarcoma could not be ruled out preoperatively. Conventionally, major surgery has been performed on patients with a tumor located near the cardia, although it was not always malignant. Enucleation by LIGS enabled us to avoid excessive invasiveness and provided a favorable result. LIGS may be an appropriate new, minimally-invasive operation for gastric myogenic tumors and should be considered for such cases.

Modulation of inhibitory transmission by dopamine in rat basal forebrain nuclei: activation of presynaptic D1-like dopaminergic receptors.

The effects of dopamine (DA) on inhibitory transmission onto identified magnocellular neurons were examined in rat basal forebrain slices using whole-cell recording. IPSCs evoked by focal stimulation within basal forebrain nuclei were reversibly blocked by 10 microM bicuculline and had a decay time constant of 20.1 +/- 0.77 msec in the presence of 6-cyano-7-nitroquinoxalline-2,3-dione (5 mM). Bath application of DA reduced the amplitude of IPSCs up to 71.1 +/- 1.49% in a concentration-dependent manner between 0.003 and 1 mM (the IC50 value being 6.6 microM), without any effect on the holding current at -70 mV. DA (10 microM) reduced the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), without affecting their mean amplitude, rise time, and decay time constant. Furthermore, the DA-induced effect on mIPSCs remained unaffected by 100 microM cadmium, suggesting a presynaptic mechanism independent of calcium influx. SKF 81297, a D1-like agonist, mimicked DA-induced effect on evoked IPSCs (IC50, 10.9 microM), whereas R(-)-TNPA or (-)-quinpirole, D2-like agonists (30 microM), had little or no effect on the amplitude of evoked IPSCs. R(+)-SCH 23390, a D1-like antagonist, antagonized the DA-induced effect on IPSCs (K(B) 0.82 microM), whereas S(-)-eticlopride, a D2-like antagonist, showed slight antagonism (K(B) 7.8 microM). Forskolin (10 microM) reduced the amplitude of evoked IPSCs to approximately 58% of the control and occluded the inhibitory effect of DA. These findings indicate that DA reduces inhibitory transmission onto magnocellular basal forebrain neurons by activating presynaptic D1-like receptors.

Injury to a duplicated cystic duct during laparoscopic cholecystectomy.

Duplication of the cystic duct is a rare variation of the biliary system. It is important to pay attention to this variation to avoid intraoperative biliary system injury, especially during laparoscopic cholecystectomy. In a 66-year-old woman with gallstones, endoscopic retrograde cholangiopancreatography (ERCP) revealed no anomaly of the biliary system. At laparoscopic cholecystectomy, although intraoperative cholangiography demonstrated the second cystic duct, we misdiagnosed the duplication of cystic duct because preoperative ERCP had demonstrated normal anatomy. It was thought that the second cystic duct was dissected during the operation and the patient suffered from postoperative bile leakage. After reoperation, the patient recovered well. The diagnostic and therapeutic problems of such an anomaly are discussed below with the review of the literature.

Dopamine D1-like receptor-mediated presynaptic inhibition of excitatory transmission onto rat magnocellular basal forebrain neurones.

1. Excitatory postsynaptic currents (EPSCs) following focal afferent stimulation were recorded from patch-clamped magnocellular neurones in a thin-slice preparation of the rat basal forebrain. Evoked EPSCs had a mean decay time constant of 3.81 +/- 0.09 ms and were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM). 2. Bath-applied dopamine (DA) reduced evoked EPSC amplitude by up to 54.2 +/- 2.3% with an IC50 of 19.9 microM in normal Krebs solution (2.5 mM Ca2+, 1.2 mM Mg2+) without effect on postsynaptic holding current. 3. DA (30 microM) reduced the mean frequency of spontaneous miniature EPSCs recorded in 0.5 microM tetrodotoxin without affecting their mean amplitude, rise time or decay time constant. This effect was diminished by 100 microM Cd2+. 4. The effect of DA on evoked EPSCs was mimicked by the D1-like receptor agonist, SKF 81297 (IC50 25.6 microM), but not by the D2-like receptor agonist R(-)-TNPA (30 microM) or (-)-quinpirole (30 microM), and was antagonized by the D1-like receptor antagonist R(+)-SCH 23390 (estimated dissociation constant KB = 1.7 microM) but not by the D2-like receptor antagonist S(-)-eticlopride (10 microM). 5. Forskolin (10 microM) reduced evoked EPSCs to approximately 60% of the control amplitude, and occluded the effect of subsequent application of DA. 6. These results suggest that glutamatergic afferents to magnocellular basal forebrain neurones possess presynaptic D1-like DA receptors, and that activation of these receptors reduces excitatory glutamatergic transmission, probably via an adenylyl cyclase-dependent pathway.

Inhibition by a putative antipsychotic quinolinone derivative (OPC-14597) of dopaminergic neurons in the ventral tegmental area.

The effects of the newly synthesized quinolinone derivative, OPC-14597 (7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy}-3, 4-dihydro-2(1 H)-quinolinone), on dopaminergic neuronal activity in the ventral tegmental area were examined using both in vivo microiontophoretic methods in chloral hydrate-anesthetized rats and the tight-seal whole-cell patch-clamp technique in thin-slice preparations of the rat brain. Neurons in the ventral tegmental area were classified as type I or type II according to their responses to antidromic stimulation of the nucleus accumbens, probably corresponding to dopaminergic and non-dopaminergic neurons, respectively. Antidromic spikes elicited by nucleus accumbens stimulation were inhibited by microiontophoretic application of dopamine and OPC-14597 in type I, but not in type II neurons. Although the OPC-14597-induced inhibition was antagonized by simultaneous application of domperidone (5-chloro-1-[1-[3-(2,3-dihydro-2-oxo-1 H-benzimidazo-1-yl)-propy]-4-piperidinyl]-1,3-dihydro-2H- benzimidazol-2-one; dopamine D2 receptor antagonist), SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1 H-3-benzazepine hydrochloride; dopamine D1 receptor antagonist) had no such effect. Spontaneous firing of type I neurons was also inhibited by iontophoretically applied OPC-14597 and dopamine, whereas that of type II neurons was unaffected. The inhibitory effect of OPC-14597 on the spontaneous firing of type I neurons was antagonized by domperidone, but not by SCH 23390. In a whole-cell patch-clamp study using a thin-slice preparation of the rat brain, bath application of OPC-14597 induced hyperpolarization accompanied by inhibition of spontaneously occurring action potentials in the large neurons (> 20 microns in diameter) in a concentration-dependent manner. These results suggest that OPC-14597 acts on dopaminergic neurons in the ventral tegmental area as a dopamine D2 receptor agonist to inhibit neuronal activities, probably by increasing membrane potassium conductance.

Long-term antiepileptic effects of chronic intake of CNK-602A, a thyrotropin-releasing hormone analogue, on spontaneously epileptic rats.

Spontaneously epileptic rats (SER), which represent a double mutation (zi/zi, tm/tm), spontaneously exhibit both tonic and absence-like seizures. We examine the long-term effects of a thyrotropin-releasing hormone (TRH) analogue, CNK-602A, acute administration of which was effective inhibiting both types of seizures in SER, to determine if this agent could be used to treat epilepsy for long periods. Food pellets containing 0.001% CNK-602A were given ad libitum to SER from age 7 weeks. CNK-602A significantly inhibited tonic convulsions and prolonged survival. There were no alterations in body weight or plasma levels of triiodotHyronine (T3) and thyroxine (T4). These findings indicate that chronic intake of CNK-602A in a dose that does not affect plasma levels of T3 and T4 inhibits tonic convulsions in SER and suggest that this drug may be an effective treatment for convulsive seizures in patients with epilepsy.

Membrane depolarization by activation of prostaglandin E receptor EP3 subtype of putative serotonergic neurons in the dorsal raphe nucleus of the rat.

A whole-cell current-clamp study using a thin slice preparation of the rat brain was carried out to elucidate the function of prostaglandin E (PGE) receptor EP3 subtype in the dorsal raphe nucleus (DR), where mRNA of this subtype is highly expressed. Bath application of PGE2 or M&B 28767, an EP3 agonist, depolarized the membrane of the large DR neurons in a concentration-dependent manner between 10(-9) and 10(-6) M. These neurons showed hyperpolarization of membrane potential to 10 or 50 microM serotonin. Neither an EP2 receptor agonist, butaprost, an EP2/EP4 receptor agonist, 11-deoxy-PGE1, nor an EP1 receptor agonist, 17-phenyl-PGE2, had any effect on large DR neurons between 10(-9) and 10(-6) M. The M&B 28767-induced depolarization was observed in a Ca(2+)-free, high Mg2+ (5 mM) solution containing 0.3 microM tetrodotoxin, and occurred equally well when intracellular Cl- was replaced by gluconate. These results suggest that direct agonist-activation of EP3 receptor depolarizes the membrane by a cationic conductance, leading to excitation of DR neurons, and indicate a physiological implication that EP3 receptor may modulate the serotonergic inhibition of neuronal activities.