Cooling positronium to ultralow velocities with a chirped laser pulse train.

When laser radiation is skilfully applied, atoms and molecules can be cooled1-3, allowing the precise measurements and control of quantum systems. This is essential for the fundamental studies of physics as well as practical applications such as precision spectroscopy4-7, ultracold gases with quantum statistical properties8-10 and quantum computing. In laser cooling, atoms are slowed to otherwise unattainable velocities through repeated cycles of laser photon absorption and spontaneous emission in random directions. Simple systems can serve as rigorous testing grounds for fundamental physics-one such case is the purely leptonic positronium11,12, an exotic atom comprising an electron and its antiparticle, the positron. Laser cooling of positronium, however, has hitherto remained unrealized. Here we demonstrate the one-dimensional laser cooling of positronium. An innovative laser system emitting a train of broadband pulses with successively increasing central frequencies was used to overcome major challenges posed by the short positronium lifetime and the effects of Doppler broadening and recoil. One-dimensional chirp cooling was used to cool a portion of the dilute positronium gas to a velocity distribution of approximately 1 K in 100 ns. A major advancement in the field of low-temperature fundamental physics of antimatter, this study on a purely leptonic system complements work on antihydrogen13, a hadron-containing exotic atom. The successful application of laser cooling to positronium affords unique opportunities to rigorously test bound-state quantum electrodynamics and to potentially realize Bose-Einstein condensation14-18 in this matter-antimatter system.

Injection and confinement of positron bunches in a magnetic dipole trap.

We demonstrate the efficient injection of a pulsed positron beam into a magnetic dipole trap and investigate the ensuing particle dynamics in the inhomogeneous electric and magnetic fields. Bunches of ∼10^{5}e^{+} were transferred from a buffer-gas trap into the field of a permanent magnet using a lossless E×B drift technique. The Δt≈0.2µs pulses were short compared to the toroidal rotation period, τ_{d}≈16µs, and e^{+} confinement time, τ_{c}≈0.6 s. The redistribution dynamics were studied by measuring the delayed γ-ray emission as the trap was emptied. This work extends the record for the number of low-energy positrons held in a dipole trap by two orders of magnitude and represents a significant advance toward the confinement of an electron-positron pair plasma.

Possible presence of hydrophilic SO3H nanoclusters on the surface of dry ultrathin Nafion® films: a positron annihilation study.

Solutions of Nafion® with an ion exchange capacity (IEC) of 0.91 meq g(-1), which are on the verge of the formation of SO(3)H nanoclusters, were spin coated on silicon (Si), glassy carbon (GC) and platinum/silicon (Pt/Si) substrates to form films of up to 256 nm thickness. Nanostructure of the films was studied using Doppler broadening of annihilation radiation (DBAR), positron annihilation lifetime (PAL), X-ray photoelectron spectroscopy (XPS), an atomic force microscope (AFM) and contact angle measurements. Contact angles as low as 10 degrees indicate that the surface of dry ultrathin Nafion® films on Si is highly hydrophilic. XPS data of 10 nm thick, ultrathin film on Si show that oxygen concentration is enhanced and the SO(3)H group concentration, in other words, IEC on the surface is much higher than other films. The S parameter measured by DBAR of an ultrathin Nafion® film on Si is much higher than that of the films on the other substrates. We consider that a large number of hydrophilic, reversed micelle like SO(3)H groups are on the surface of the ultrathin Nafion® film on Si but not on the surface of other films. Positrons implanted into the film are trapped by the SO(3)H clusters, annihilating with the electrons of oxygen and exhibit the high S parameter. The SO(3)H concentration on the surface of thin Nafion® films on GC and Pt/Si substrates may not be so high as the threshold for the formation of a large number of SO(3)H clusters. Positrons implanted into the films annihilate mostly with fluorine atoms, resulting in a low S parameter. The film-substrate interaction plays an essential role in nanostructuring of Nafion® thin films, which may also be the case for Nafion® on the catalysts of polymer electrolyte fuel cells.

A20 is an early responding negative regulator of Toll-like receptor 5 signalling in intestinal epithelial cells during inflammation.

Several negative regulatory mechanisms control Toll-like receptor (TLR)-mediated inflammatory responses and restore immune system balance, including the zinc-finger protein A20, a negative regulator of TLR signalling that inhibits nuclear factor kappa B (NF-kappaB) activity. In the present study, we investigated TLR-5-mediated A20 expression and its role in intestinal epithelial cells (IECs) during inflammation. HCT-15 and HT-29 cells were stimulated with flagellin, then the expressions of A20, interleukin-1 receptor-associated kinase (IRAK-M) and Tollip were evaluated using RNase protection assay. Furthermore, experimental colitis was induced in tlr4-deficient CH3/HeJ mice by administration of dextran sodium sulphate (DSS), then flagellin was injected anally, and the colonic expression of A20 was examined by real-time polymerase chain reaction (PCR) and immunohistochemistry. To confirm flagellin-induced expression of A20, we employed an organ culture system. The role of A20 in flagellin-induced tolerance induction was evaluated in vitro, using a gene knock-down method targeting A20. A20 expression increased rapidly and peaked at 1 h after flagellin stimulation in cultured IECs, then declined gradually to the basal level. In vivo, anal injection of flagellin induced epithelial expression of A20 in injured colonic tissue, whereas flagellin did not cause a significant increase in A20 expression in non-injured normal tissue, which was also confirmed in vitro using the organ culture system. Gene knock-down using A20 siRNA did not influence tolerance induced by restimulation with flagellin. A20 is an early response negative regulator of TLR-5 signalling in IECs that functions during intestinal inflammation. Our results provide new insights into the negative feedback regulation of TLR-5 signalling that maintains the innate immune system in the gut.

Down-regulation of single immunoglobulin interleukin-1R-related molecule (SIGIRR)/TIR8 expression in intestinal epithelial cells during inflammation.

Single immunoglobulin (Ig) interleukin-1R-related molecule (SIGIRR) is an Ig-like membrane protein critical for negative regulation of Toll-like receptor (TLR)-4-mediated signalling. We investigated SIGIRR expression and its regulation mechanism in intestinal epithelial cells (IECs) during inflammation. Endoscopic biopsy specimens were obtained from active and inactive colonic mucosa of ulcerative colitis (UC) patients, then SIGIRR expression was examined using real-time polymerase chain reaction (PCR) and immunohistochemistry (IH). Mice experimental colitis models were established by administrations of sulphonic acid (TNBS) and dextran sodium sulphate (DSS), and epithelial expression of SIGIRR was examined using real-time PCR, IH and flow cytometry. The effects of lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α on SIGIRR expression were evaluated in vitro using cultured IECs. To elucidate SIGIRR expression regulation in IECs, binding ability of the transcription factor SP1 at the responsive element of the SIGIRR promoter was examined using gel-shift and chromatin immunoprecipitation (ChIP) assays. In human colonic samples, SIGIRR was expressed mainly in IECs at levels significantly higher in inactive compared to active mucosa. In the mice, SIGIRR colonic expression decreased rapidly after colitis development and returned gradually to basal levels. Experimental colitis-mediated down-regulation of SIGIRR in IECs was also confirmed by IH and flow cytometry results. Further, inflammatory conditions induced by TLR ligands and TNF-α caused significant down-regulation of SIGIRR expression in IECs, which was dependent upon decreased SP1 binding at the responsive element of the SIGIRR promoter. We found that SIGIRR is expressed in IECs and serves as a negative regulator to maintain gut innate immunity, which is down-regulated during inflammation by inhibition of an SP1-mediated pathway.

[Papillary fibroelastoma of the tricuspid valve with mitral regurgitation].

We report a 64-year-old male patient with papillary fibroelastoma of the tricuspid valve associated with moderate mitral regurgitation. Echocardiography had revealed a 10 x 10 mm tumor attaching to the anterior tricuspid leaflet 3 years before. Because the tumor was enlarged to 19 x 15 mm, we excised it with a part of the anterior tricuspid leaflet, and performed tricuspid and mitral valvoplasty. The histological findings suggested papillary fibroelastoma. The postoperative course was uneventful.

[Combined pulmonary resection and cardiac operation; report of a case].

A 59-year-old woman was admitted because of an abnormal shadow on the chest X-ray film. Transbronchial lung biopsy revealed adenocarcinoma of the right lung, and chest computed tomography showed left atrial tumor. First, we performed a resection of left atrial tumor (myxoma) under cardiopulmonary bypass (CPB), followed by a right upper lobectomy with lymph node dissection. The postoperative course was uneventful, and she was discharged on the 14th postoperative day. It is safe and efficient that pulmonary resection and cardiac operation under CPB are surgically treated in a one-stage operation.

A high-quality and energy-tunable positronium beam system employing a trap-based positron beam.

We constructed a new apparatus, built upon a trap-based slow positron beam, for the production of a collimated, energy-tunable positronium beam under ultra-high vacuum conditions employing the photodetachment of positronium negative ions. A slow positron generator consisting of a 22Na radioisotope (20 mCi) combined with a buffer-gas positron trap is employed to generate high-quality, nano-second positron bursts with a repetition rate of 1 Hz-1 kHz. The positron bursts are focused onto an efficient positron-to-positronium negative ion converter, a Na-coated W thin film in a transmission geometry, using a magnetic lens system. The ions emitted from the opposite surface of the film are electrostatically accelerated to a given energy and photodetached by a pulsed infrared laser to form a mono-energetic positronium beam with kinetic energies of 0.2 keV-3.3 keV. The achieved detection rate of Ps atoms is 23 cps at the energy of 3.3 keV with a signal-to-background ratio as high as 300. The energy spread of the beam was evaluated by comparing the result of the time-of-flight measurements and particle-tracking simulations. With the use of a collimator of 1 mm diameter, a coherent beam with an angular divergence of less than 0.3° is obtained. The obtained Ps beam, having a much higher quality than those reported hitherto, will open up a new field of experimental investigations, such as Ps interacting with a variety of materials and fundamental studies on Ps spectroscopy.

iBTA-induced bovine Biosheet for repair of abdominal wall defects in a beagle model: proof of concept.

INTRODUCTION: We evaluated the usefulness of xeno-Biosheets, an in-body tissue architecture-induced bovine collagenous sheet, as repair materials for abdominal wall defects in a beagle model. MATERIALS AND METHODS: Biosheets were prepared by embedding cylindrical molds into subcutaneous pouches of three Holstein cows for 2-3 months and stored in 70% ethanol. The Biosheets were 0.5 mm thick, cut into 2 cm × 2 cm, and implanted to replace defects of the same size in the abdominal wall of nine beagles. The abdominal wall and Biosheets were harvested and subjected to histological evaluation at 1, 3, and 5 months after implantation (n = 3 each). RESULTS: The Biosheet and bovine pericardiac patch (control) were not stressed during the suture operation and did not split, and patches were easily implanted on defective wounds. After implantation, the patch did not fall off and was not perforated, and healing was observed nacroscopically in all cases. During the first month of implantation, accumulation of inflammatory cells was observed along with decomposition around the Biosheet. Decomposition was almost complete after 3 months, and the Biosheet was replaced by autologous collagenous connective tissue without rejection. After 5 months, the abdominal wall muscle elongated from the periphery of the newly formed collagen layer and the peritoneum was formed on the peritoneal cavity surface. Regeneration of almost all layers of the abdominal wall was observed. However, almost all pericardium patches were remained even at 5 months with inflammation. CONCLUSION: Bovine Biosheets requiring no special post-treatment can be useful as off-the-shelf materials for abdominal wall repair.

[Tricuspid valve replacement for elderly patients with isolated tricuspid valve regurgitation; report of 2 cases].

Isolated tricuspid valve regurgitation (TR) is a rare clinical entity. We report 2 patients, 80 and 74-year-old with isolated TR. They underwent valve replacement with the Carpentier-Edwards bioprosthesis because of resistance to medical treatment. The causes of insufficiency were suspected as congenital in case 1 and infective endocarditis in case 2, respectively. Postoperative course was free from major complications in both patients. Valvuloplasty and/or annuloplasty are recommended for TR, however, replacement of the tricuspid valve is sometimes necessary in isolated TR patients. The higher occurrence of thrombosis of mechanical prosthesis in the tricuspid position has been reported. The bioprosthesis in tricuspid position may reduce the rate of thromboembolism, thrombosis and structural dysfunction, therefore it may be an option for radical therapy in isolated TR especially in aged patients.

[Emergent coronary artery bypass grafting for a patient with cardiopulmonary arrest].

A 59-year-old man was admitted to our hospital due to sudden onset of unconsciousness caused by myocardial infarction with ventricular fibrillation. Emergent coronary angiography under intraaortic balloon pumping revealed 90% stenosis of the left main trunk and left anterior descending artery (LAD), and complete obstruction of the left circumflex artery (Cx) and right coronary artery (RCA). Emergent coronary artery bypass grafting (CABG) to LAD, Cx, and RCA was performed. During the postoperative course, the patient developed ventricular tachycardia/fibrillation. After implantation of an implantable cardioverter defibrillator (ICD), he was discharged on the postoperative day 36. The patient has now resumed normal daily life.

Effects of famotidine, mosapride and tandospirone for treatment of functional dyspepsia.

BACKGROUND: An effective therapeutic strategy for functional dyspepsia (FD) has not been well-established. AIM: We investigated and compared the therapeutic effects of famotidine, mosapride and tandospirone for the control of dyspeptic symptoms. METHODS: Fully examined FD patients of outpatient clinics at seven different medical centres were enrolled in the study. They were randomly assigned to three groups based on the type of drug administered: famotidine, mosapride and tandospirone. The effects of treatment over 4 weeks were assessed by visual analogue scales. RESULTS: All of the drugs showed beneficial effects, although famotidine was the most effective for symptom relief, which was significantly greater than tandospirone, while the effect of mosapride was similar to that of famotidine. No subtype of FD showed a better response to a particular type of drug. CONCLUSIONS: For the treatment of FD, famotidine demonstrated the best therapeutic effect, followed by mosapride, while that of tandospirone was significantly lower.

[Reoperative off-pump coronary artery bypass via left thoracotomy with porcelain aorta; left axillary artery to circumflex artery bypass; report of a case].

A 62-year-old man who underwent coronary artery bypass grafting (CABG) [left internal thoracic artery (LITA)-left anterior descending (LAD), saphenous vein graft (SVG) right coronary artery (RCA)] 13 years previously developed angina pectoris and congestive heart failure because of occlusion of SVG and native vessels. Coronary angiography (CAG) revealed that inflow to the coronary artery remained only from LITA. Repeat off-pump CABG (OPCAB) with SVG to the circumflex artery via left thoracotomy was performed. The proximal end of SVG was anastomosed to the left axillary artery because of the porcelain aorta and the patent LITA graft. The patient developed no complications and was discharged from hospital on postoperative day 21. OPCAB for circumflex artery by left thoracotomy is an effective and safe approach in redo CABG, particularly in instances of patent LITA.

Uric acid, indoxyl sulfate, and methylguanidine activate bulbospinal neurons in the RVLM via their specific transporters and by producing oxidative stress.

Patients with chronic renal failure often have hypertension, but the cause of hypertension, other than an excess of body fluid, is not well known. We hypothesized that the bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are stimulated by uremic toxins in patients with chronic renal failure. To investigate whether RVLM neurons are sensitive to uremic toxins, such as uric acid, indoxyl sulfate, or methylguanidine, we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons of Wister rats using the whole-cell patch-clamp technique during superfusion with these toxins. A brainstem-spinal cord preparation that preserved the sympathetic nervous system was used for the experiments. During uric acid, indoxyl sulfate, or methylguanidine superfusion, almost all the RVLM neurons were depolarized. To examine the transporters for these toxins on RVLM neurons, histological examinations were performed. The uric acid-, indoxyl sulfate-, and methylguanidine-depolarized RVLM neurons showed the presence of urate transporter 1 (URAT 1), organic anion transporter (OAT)1 or OAT3, and organic cation transporter (OCT)3, respectively. Furthermore, the toxin-induced activities of the RVLM neurons were suppressed by the addition of an anti-oxidation drug (VAS2870, an NAD(P)H oxidase inhibitor), and a histological examination revealed the presence of NAD(P)H oxidase (nox)2 and nox4 in these RVLM neurons. The present results show that uric acid, indoxyl sulfate, and methylguanidine directly stimulate bulbospinal RVLM neurons via specific transporters on these neurons and by producing oxidative stress. These uremic toxins may cause hypertension by activating RVLM neurons.

Alzheimer beta amyloid deposition enhanced by apoE epsilon4 gene precedes neurofibrillary pathology in the frontal association cortex of nondemented senior subjects.

To clarify how Alzheimer disease pathology develops in the brains of nondemented subjects, we examined the interrelations among the amounts and morphology of Abeta deposition, neurofibrillary pathology, and apolipoprotein E (ApoE) genotype in the frontal association cortex of 101 autopsy brains from patients aged between 40 to 83. Senile plaque density correlated well with the logarithmic data of insoluble Abeta measured by enzyme immunoassay (EIA). The amounts of Abeta42-ETA increased dramatically in the late preclinical stage, whereas the AP42+ plaque density increased in the early preclinical stage. Neurofibrillary pathology appeared only in the areas with severe Abeta deposition and in subjects aged over 70. The ApoE epsilon4 allele enhanced the Abeta3 deposition in presenile subjects. Plaque-associated glial Abeta was prominent in subjects with mild to moderate Abeta deposition. The morphology of cerebral Abeta deposition changed from diffuse plaques with small amounts of Abeta in each plaque in the early preclinical stage to primitive/neuritic plaques with larger amounts of Abeta in each plaque in the late preclinical stage. Our findings suggest that the prevention of Abeta deposition in the late preclinical stage can be a rational therapeutic target, especially in elderly people with ApoE epsilon4 allele.

Improvement in baroreflex function by an oral angiotensin receptor antagonist in rats with myocardial infarction.

Impaired baroreflex function is a factor responsible for poor prognosis in myocardial infarction patients. Using logistic function curves, we calculated the maximal gain of the baroreflex control of renal sympathetic nerve activity (RSNA) and heart rate in conscious Wistar-Kyoto and spontaneously hypertensive rats whose left anterior descending artery had been ligated 4 weeks earlier. We further investigated whether 3-week oral treatment with the angiotensin II type 1 receptor antagonist TCV-116 would improve the baroreflex in rats with myocardial infarction. The maximal gain of the mean arterial pressure-RSNA relation in spontaneously hypertensive rats with myocardial infarction and treated with vehicle (1.7 +/- 0.1% control per mm Hg) was smaller than the gain in sham-operated hypertensive rats (2.3 +/- 0.1% control per mm Hg). After 3-week oral treatment with TCV-116, the maximal gain of the arterial pressure-RSNA relation in hypertensive rats with myocardial infarction was 2.3 +/- 0.1% control per mm Hg and significantly greater than the gain in infarcted and vehicle-treated hypertensive rats. In hypertensive rats, the maximal gain of the arterial pressure-heart rate relation of infarcted and TCV-116-treated rats was larger than in infarcted and vehicle-treated rats but significantly smaller than in sham-operated rats. These results demonstrate that oral treatment with an angiotensin receptor antagonist is effective in restoring the impaired baroreflex caused by myocardial infarction and that endogenous angiotensin II is one of the critical factors involved in the impaired baroreflex in myocardial infarction.

Use of a series of ompF-ompC chimeric proteins for locating antigenic determinants recognized by monoclonal antibodies against the ompC and ompF proteins of the Escherichia coli outer membrane.

A method is presented for the efficient location of antigenic determinants using a series of chimeric proteins. By means of in vivo homologous recombination between the ompC and ompF genes coding for OmpC and OmpF, homologous proteins of the Escherichia coli outer membrane, a series of ompF-ompC chimeric genes was constructed (Nogami, T., Mizuno, T., & Mizushima, S. (1985) J. Bacteriol. 164, 797-801, and this work). The OmpF-OmpC chimeric proteins expressed by these genes were successfully used to locate antigenic determinants recognized by monoclonal antibodies, which specifically react with either the OmpC or OmpF protein. Interaction between monoclonal antibodies and the chimeric proteins was examined by means of either enzyme-linked immunosorbent assay or immunoblot analysis. The antigenic determinants recognized by three anti-OmpC antibodies and one anti-OmpF antibody were thus located. Finally, the polypeptides covering these regions were chemically synthesized for two of them and then tested as to their reactivity with the antibodies. The peptides reacted with the corresponding antibodies when the former were chemically coupled with bovine serum albumin. Most of the monoclonal antibodies isolated in this work were highly specific to the unfolded monomer of the protein against which the antibody was raised. But they did not react with the trimer, the native form. These results are discussed in relation to the structures and functions of the OmpC and OmpF proteins. The use of a series of monoclonal antibodies for studying the mechanism of protein translocation across the cytoplasmic membrane is also discussed.

Interaction between endothelin and nitric oxide in sympathetic nerve modulation in hypertensive rats.

To test the hypothesis that endothelin (ET) and nitric oxide (NO) interact in modulating the sympathetic nervous system in conscious rats, as they do in the endothelium, mean arterial pressure (MAP), heart rate, and renal sympathetic nerve activity (RSNA) were recorded in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) given losartan and compared before and during intravenous infusion of an NO synthase inhibitor, L-NMMA (0.25 mg/kg/min). The slope of the relation between RSNA and MAP to blood pressure reduction was increased in the presence of L-NMMA (from 0.6 +/- 0.1 to 2.8 +/- 0.2), suggesting that endogenous NO suppresses the reflex increase in RSNA. Since NO inhibits ET production in the endothelium, we speculated that the increase in MAP-RSNA slope was due partly to an unmasking of ET, and thus recorded MAP, heart rate, and RSNA during intravenous infusions of both L-NMMA and the ET-type-A-receptor antagonist BQ-485 (0.10 mg/kg/min). The slope decreased significantly in SHR when BQ-485 was added (1.5 +/- 0.2), but not in WKY, implying that unmasked ET enhanced the sympathetic increase via ETA receptors in hypertensive rats. An ETB-receptor antagonist potentiated the sympathetic response only in WKY rats. These results suggest that NO suppressed the reflex increase in RSNA to blood pressure reduction, while ET uncovered by L-NMMA enhanced the sympatho-activation, indicating an interaction between ET and NO in modulating the sympathetic nervous system in conscious hypertensive animals in vivo. In contrast, the interaction was not observed in normotensive rats.

Further improvements to the photoelectric method for measuring motile responses of chromatophores.

With the intention of simplifying construction and operation, improvements have now been made to a photoelectric system for measuring the motile responses of chromatophores. Introduction of chop-per-stabilized operational amplifiers with a complimentary metal-oxide semiconductor (C-MOS) input has brought about a much improved stability of the electronics. Such a feature has been found to be especially suitable for measurements requiring higher amplification and longer periods of time, e.g., the detection of the effects of various factors on bright-colored chromatophores. The use of appropriate color filters that limit the spectral range of light used for measurement has also proven to be important. By installing a small filter close to the photosensor, we can now record the responses of particular types of chromatophores more selectively, while visually monitoring the states of all kinds of chromatophores in natural color. To minimize the influence of motile activities of xanthophores and/or erythrophores, the use of an orange-to-red long-pass filter is appropriate to optimize recording the melanophore responses. By contrast, the responses of xanthophores or erythrophores can be recorded more easily by employing a violet-to-blue band-pass filter, because that increases the contrast of images of these cells against the background. Using an orange-red variety of the medaka Oryzias, we have also recorded photometrically the responses of leucophores, whose organelles are light-scattering. A long-pass filter was efficient in excluding the influences of co-existing xanthophores.

Three types of putative presympathetic neurons in the rostral ventrolateral medulla studied with rat brainstem-spinal cord preparation.

To study the electrophysiological properties of presympathetic neurons in the rostral ventrolateral medulla (RVLM), intracellular recordings were performed by the whole-cell patch-clamp technique. We utilized the neonatal rat brainstem-spinal cord preparation, in which the sympathetic neuronal network is thought to be preserved, unlike in slice preparation. In response to stimulation in the ipsilateral Th2 spinal segment including intermediolateral cell column (IML), 33 of 151 non-respiratory RVLM neurons showed antidromic action potentials with a constant latency of 45 ms, and can be considered as presympathetic neurons. We classified and characterized the RVLM presympathetic neurons into three types: 'regularly firing neurons (n=7)', which showed ramp depolarization and frequent action potentials (4.2+/-0.9 spikes/s) with rare excitatory postsynaptic potentials (EPSPs); 'irregularly firing neurons (n=21)', which exhibited many EPSPs that modulated the firing rate; and 'silent-type neurons (n=5)', which discharged action potentials only during current-induced depolarization. Lucifer-Yellow staining showed that the irregularly firing neurons were significantly larger and had more dendrites than the regularly firing neurons. All regularly firing neurons retained their discharges during low-Ca2+ -high-Mg2+ superfusion that blocks synaptic input, whereas the discharges in 11 of 16 irregularly firing neurons were abolished, suggesting that the regularly firing neurons discharged independently of synaptic input. Seven of 31 RVLM neurons were hyperpolarized by stimulation of vagal afferent nerves. In summary, three types of RVLM presympathetic neurons were characterized by the patch-clamp technique in the brainstem-spinal cord preparation, in which the connection was preserved from vagal afferent to the Th2 spinal segment through the RVLM. Since antidromic action potentials were demonstrated by stimulation in the Th2 spinal segment in 33 neurons of all three types, all types of RVLM neurons constitute a part of the sympathetic neuronal network.