[Clinical and pathological characteristics of gastric adenocarcinoma of fundic gland type].

To analyze the clinical, endoscopic and pathological feature of gastric adenocarcinoma of fundic gland type (GA-FG) (18 cases) collected from Sir Run Run Shaw Hospital, affiliated with Zhejiang University School of Medicine and Taizhou Hospital of Zhejiang Province from January 2019 to July 2022. There were 18 cases of patient of GA-FG, including male 12 cases, female 6 cases, aged from 38 to 78 years old, with average age of 60.5 years old. Gastroscopy showed that bulging or flat lesions of gastric fundus were 0.2-5.5 cm in size, and the mucosal surface was smooth, redness or rough. Histologic examination showed that tumor cells were dominated by chief cells and scattered with a few oxyntic cells, formed a complex gland that anastomoses each other, and infiltrated to the submucosa. The results of immunohistochemistry showed that tumor cells were positive for the expression of mucin-6 (MUC6) and Pepsinogen 1, and partial expression of synaptophysin (Syn). GA-FG is a rare type of gastric adenocarcinoma with good differentiation, and currently only a few cases have been reported, and often easily been misdiagnosed or missed. Therefore, to master the characteristics of clinic and pathology is helpful to improve the ability of clinical pathologists in differential diagnosis.

[Effects of femtosecond laser treatment on surface characteristics and flexural strength of zirconia].

OBJECTIVE: To evaluate the effects of femtosecond laser treated microgrooved surface on microscopic topography, phase transformation, and three-points flexural strength of zirconia, and to provide reference for surface microstructure optimization of zirconia implant. METHODS: According to different surface treatment methods, 57 computer aided design/computer aided manufacture (CAD/CAM) zirconia bars (20.0 mm×4.0 mm×1.4 mm) were evenly divided into three groups: sintered group, no treatment after sintering, taken as control; sandblasted group, sandblasted with 110 µm aluminium oxide (Al2O3) after sintering; microgrooved group, femtosecond laser fabricated microgrooves with 50 µm width, 30 µm depth, and 100 µm pitch. Surface microscopic topography was observed with scanning electron microscope (SEM) and 3D laser microscope. Further, surface roughness in each group and microgroove size were measured. Crystal phase was analyzed with X-ray diffraction. Specimens were subjected to three- points flexural strength test, and Weibull distribution was used to analyze their strength characteristics. RESULTS: SEM showed that sintered surface was flat with clear grain structure; sandblasted surface exihibited bumps and holes with sharp margins and irregular shape; microgrooves were regularly aligned without evident defect, and nano-scale particles were observed on the surface inside of the microgrooves. Ra value of microgrooved group [(9.42±0.28)] µm was significantly higher than that of sandblasted group [(1.04±0.03) µm] and sintered group [(0.60±0.04) µm], and there was statistical difference between sandblasted group and sintered group (P < 0.001). The microgroove size was precise with (49.75±1.24) µm width, (30.85±1.02) µm depth, and (100.58±1.94) µm pitch. Crystal phase analysis showed that monoclinic volume fraction of sandblasted group (18.17%) was much higher than that of sintered group (1.55%), while microgrooved group (2.21%) was similar with sintered group. The flexural strength of sandblasted group (986.22±163.25) MPa had no statistical difference with that of sintered group (946.46±134.15) MPa (P=0.847), but the strength in microgrooved group (547.92±30.89) MPa dropped significantly compared with the other two groups (P < 0.001). Weibull modulus of sintered, sandblasted, microgrooved groups were 7.89, 6.98, and 23.46, respectively. CONCLUSION: Femtosecond laser was able to form micro/nanostructured microgrooves on zirconia surface, which deleteriously affected the flexural strength of zirconia.

Accuracy of detecting vertical root fractures in non-root filled teeth using cone beam computed tomography: effect of voxel size and fracture width.

AIM: To investigate simultaneously the effect of voxel size and fracture width on the accuracy of detecting vertical root fractures (VRFs) in non-root filled teeth when using cone beam computed tomography. METHODOLOGY: Fifty-one of 161 extracted human permanent teeth (16 anterior teeth, 132 premolars and 13 mandibular molars) were selected randomly for VRF induction with two fracture widths. All teeth were scanned with four CBCT units at different voxel sizes provided by the units. Three observers classified the presence or absence of VRF using a 5-point scale. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the ROC curve (AUC) were calculated. AUCs amongst voxel sizes and between the fracture widths were compared using the Z test. Intra- and inter-observer agreement was assessed using weighted Cohen kappa. RESULTS: For the NewTom VGi and ProMax 3D Mid CBCT unit, no significant differences were found amongst voxel sizes for the AUCs, irrespective of the fracture width (P > 0.05). There were significant differences between images scanned with voxel size 250 and 160 µm (P = 0.02), and images scanned with voxel size 250 and 80 µm for AUCs in the narrow VRF group for the 3D Accuitomo 170 unit (P = 0.03). For i-CAT FLX, significant differences were found between the voxel protocols of 300 µm and of the other three voxel sizes for AUC, sensitivity and NPV (P < 0.05). Significant differences between the wide and the narrow VRF groups for AUCs were found for 3D Accuitomo 170 (P = 0.01) and ProMax 3D Mid (P < 0.01). CONCLUSIONS: Cone beam computed tomography was accurate for detecting VRF in non-root filled teeth. Fracture width had an effect on the detection of VRF. The effect of the voxel size on the detection of VRF depended on the CBCT unit used.

Genome-wide identification, phylogenetic relationships, and expression analysis of the carotenoid cleavage oxygenase gene family in pepper.

Carotenoid cleavage oxygenases (CCOs) are a family of dioxygenases, which specifically catalyze the cleavage of conjugated double bonds in carotenoids and apocarotenoids in plants. In this study, genome-wide analysis of CCO genes in pepper plants was performed using bioinformatic methods. At least 11 members of the CCO gene family were identified in the pepper genome. Phylogenetic analysis showed that pepper and tomato CCO genes could be divided into two groups (CCDs and NCEDs). The CCD group included five sub-groups (CCD1, CCD4, CCD7, CCD8, and CCD-like). These results indicate that there is a close genetic relationship between the two species. Sequence analysis using the online tool, Multiple Expectation Maximization for Motif Elicitation (MEME), showed that the CCO proteins comprise multiple conserved motifs, with 20 to 41 amino acids. In addition, multiple cis-acting elements in the promoter of CCO genes were identified using the online tool PlantCARE, and were found to be involved in light responsiveness, plant hormone regulation, and biotic and abiotic stresses, suggesting potential roles of these proteins under different conditions. RNA-seq analysis revealed that the CCO genes exhibit distinct patterns of expression in the roots, stems, leaves, and fruit. These findings suggest that the CCO genes have important roles in the vegetative and reproductive development of pepper plants.

Effect of continuous infusion of midazolam on immune function in pediatric patients after surgery.

The current study was performed to investigate the effects of midazolam on immune function in pediatric patients after surgery and possible mechanism involved. Patients who needed sedation for more than 2 consecutive days after undergoing surgery in the Pediatric Surgery Department of our hospital were enrolled for the study. Fifty-six patients (5-14 years old) were randomly divided into midazolam and propofol treatment groups (N = 28 each in each group). Pediatric patients received midazolam or profolol via continuous intravenous administration, and their plasma cytokine levels were compared after 48 h. Cultured rat C6 brain glioma cells were pretreated with a range of concentrations of midazolam or propofol for 60 minutes prior to incubation with 10 ng/mL IL-1ß in serum-free medium or vehicle for 36 h. IL-6 concentration was subsequently measured using ELISA. In comparison with levels measured before the infusion of midazolam for 48 h, concentrations of all cytokines decreased, with the differences in IL-1ß, IL-8, and TNF-α concentrations reaching significance (all P < 0.05). Midazolam significantly suppressed the IL-1ß-induced release of IL-6 in rat C6 glioma cells. This inhibition was concentration-dependent between 0.3 and 3 µM, with 3 µM concentration of midazolam decreasing the IL-1ß-induced release of IL-6 by 43.58%. Midazolam can significantly inhibit the release of cytokines in pediatric patients after surgery. One of the mechanisms may be the inhibition of IL-1ß- induced release of IL-6 in the central nervous system.

STAT3 gene polymorphism in chronic obstructive pulmonary disease.

OBJECTIVE: The aim of this study was to explore the correlation between rs8069115, rs41289087, and rs11079042 polymorphisms of the signal transducer and activator of transcription 3 (STAT3) gene and chronic obstructive pulmonary disease (COPD). PATIENTS AND METHODS: A total of 200 patients diagnosed with COPD were enrolled in the disease group. Meanwhile, 200 normal subjects were selected as the control group. Peripheral blood was collected from subjects in the disease group and control group. Subsequently, nucleated cells were isolated for determination of STAT3 gene polymorphisms. Quantitative Polymerase Chain Reaction (qPCR) was utilized to detect the expression level of STAT3. Samples from 12 patients with differences in STAT3 haplotypes and 12 cases with no difference were collected and treated with transcriptome sequencing to analyze pathways enriched with differentially expressed genes. RESULTS: There were statistically significant differences in allele distributions at rs8069115 between the disease group and control group (p=0.000), and the allele frequency of G was higher in disease group. Genotype distributions of rs8069115 (p=0.000) and rs41289087 (p=0.000) of the STAT3 gene in disease group were significantly different in comparison with the control group. The frequency of rs8069115 GG genotype was remarkably higher, while the frequency of rs41289087 TG genotype was lower in the disease group (p<0.05). In addition, compared with the control group, the distributions of the dominant model (p=0.002) and recessive model (p=0.004) of rs8069115 of the STAT3 gene were markedly different in the disease group. A significantly higher frequency of dominant model GG+GA and lower frequency of recessive model GA+AA were observed at rs8069115 in the disease group (p<0.05). Moreover, the haplotype distributions of AGC (p=0.002), ATC (p=0.001), GTA (p=0.010), and GTC (p=0.035) at rs8069115, rs41289087, and rs11079042 were different between the disease group and control group. Besides, rs8069115 locus and rs11079042 locus were linked to each other (D'=0.523). There was a remarkable association between rs11079042 polymorphism of the STAT3 gene and gene expression (p<0.05). STAT3 was highly expressed in patients with genotype CC (p<0.05). Furthermore, changes in transcriptome levels among different haplotype populations (haplotype with different distributions vs. haplotype with no difference in distribution) were analyzed. The results demonstrated that multiple pathways, such as ECM-receptor interactions, cell cycle checkpoints, and protein processing were notably enriched (p<0.05). CONCLUSIONS: According to our results, we confirmed that the polymorphisms (rs8069115, rs41289087, and rs11079042) of STAT3 gene are noticeably correlated with the occurrence and progression of COPD.

Long-range signaling in growing neurons after local elevation of cyclic AMP-dependent activity.

Cyclic AMP-dependent activity at the growth cone or the soma of cultured Xenopus spinal neurons was elevated by local extracellular perfusion of the neuron with culture medium containing 8-bromoadenosine 3',5'-cyclic monophosphate (8-br-cAMP) or forskolin. During local perfusion of one of the growth cones of multipolar neurons with these drugs, the perfused growth cone showed further extension, while the distant, unperfused growth cones were inhibited in their growth. Local perfusion of the growth cone with culture medium or local perfusion with 8-br-cAMP at a cell-free region 100 microns away from the growth cone did not produce any effect on the extension of the growth cone. Reduced extension of all growth cones was observed when the perfusion with 8-br-cAMP was restricted to the soma. The distant inhibitory effect does not depend on the growth of the perfused growth cone since local coperfusion of the growth cone with 8-br-cAMP and colchicine inhibited growth on both perfused and unperfused growth cones, while local perfusion with colchicine alone inhibited only the perfused growth cone. The distant inhibitory effect was abolished when the perfusion of 8-br-cAMP was carried out together with kinase inhibitor H-8, suggesting the involvement of cAMP-dependent protein kinase and/or its downstream factors in the long-range inhibitory signaling. Uniform exposure of the entire neuron to bath-applied 8-br-cAMP, however, led to enhanced growth activity at all growth cones. Thus, local elevation of cAMP-dependent activity produces long-range and opposite effects on distant parts of the neuron, and a cytosolic gradient of second messengers may produce effects distinctly different from those following uniform global elevation of the messenger, leading to differential growth regulation at different regions of the same neuron.

Neutrophil elastase inhibitor suppresses oxidative stress in obese asthmatic rats by activating Keap1/Nrf2 signaling pathway.

OBJECTIVE: The aim of this study was to detect the oxidative stress response in the rat model of obesity, asthma and obese asthma. Meanwhile, we aimed to investigate the inhibitory effect of neutrophil elastase inhibitor (NEI) on cellular oxidative stress in the body and whether it exerted an effect on the oxidative stress response in obese asthma through the Kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (Keap1/Nrf2) pathway. MATERIALS AND METHODS: The obesity and asthma models were established using a total of 70 Sprague-Dawley (SD) rats. All rats were randomly divided into 7 groups. The rats with normal weight were divided into the control (CTR) group (n=10), asthma (ATM) group (n=10) and ATM+NEI group (n=10). Meanwhile, the obese rats were divided into the obesity (OBS) group (n=10), the OBS+NEI group (n=10), the OBS+ATM group (n=10) and the OBS+ATM+NEI group (n=10). After modeling, rats in NEI intervention groups were injected with Sivelestat (5 mg/kg) via the caudal vein twice a day for 1 week. The tests of cough sensitivity to capsaicin and bronchial responsiveness were performed 24 h after the last administration. Lung tissues of rats were collected for hematoxylin-eosin (HE) staining. Meanwhile, the levels of reactive oxygen species (ROS) in heart, lung and kidney tissues were detected via 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The activities of reduced glutathione (GSH), glutathione peroxidase (GSH-Px), H2O2 and total superoxide dismutase (T-SOD) in the heart, lung and kidney tissues were detected using the colorimetric method. The mRNA and protein expressions of Keap1 and Nrf2 messenger ribonucleic acid expressions in the heart, lung and kidney tissues were measured via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting, respectively. RESULTS: NEI significantly improved the symptoms and lung pathology in rats with asthma. The level of ROS in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly increased. However, NEI markedly inhibited the level of ROS in rats with asthma. The activities of antioxidant stress-related enzymes (reduced GSH, GSH-Px, H2O2 and SOD) in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group were significantly decreased. However, NEI markedly promoted the activities of the related antioxidant enzymes in oxidative stress response in asthma rats. Besides, the Keap1/Nrf2 signaling pathway in the heart, lung and kidney tissues of the OBS group, ATM group and OBS+ATM group was significantly inhibited, while NEI activated the Keap1/Nrf2 signaling pathway in rats with asthma. CONCLUSIONS: NEI promotes the release of a variety of antioxidant factors, enhances the activity of antioxidant enzymes and improves the symptoms of rats with obese asthma. The possible underlying mechanism may be the activation of the Keap1/Nrf2 signaling pathway.

[Preliminary study on the automatic preparation of dental implant socket controlled by micro-robot].

Objective: To analyze the quantitative relationship between the number of layers of laser pulses and the amount of step in ultra-short pulse laser cutting of cortical bone, optimize the robot's vertical single stepping parameters, and to explore the feasibility of automatic preparation of dental implant cavity using robot controlling ultra-short pulse laser, in order to lay the foundation for automated dental implant surgery. Methods: Eight pig ribs were segmented into to make 16 specimens. Using the robotic surgical system and path planning software independently developed by our group, circular holes with a diameter of 4 mm were cut two-dimensionally in the rib segments to obtain the quantification relationship of the number of laser pulse layers (n) and the depth of two-dimensional (2D) cutting (d). When conducting the three-dimensional (3D) cutting procedure, the number of pulse layers were set to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 layers, the vertical single step amount was an integer value corresponding to the results of 2D cutting depth, and the number of pulses (n') corresponding to the minimum difference between the theoretical depth of cut and the actual depth of cut was obtained. The n' was taken as the most suitable single step pulse layer, the rib segment was cut, and the depth of single cut was measured while the integer value was taken as the most appropriate vertical single step amount (d'). The vertical parameters of laser single stepping were set as n' layer pulse and d' µm step size. The 3D cutting produces a cylindrical cavity with a diameter of 4 mm and a height of 2 mm to evaluate the 3D cutting accuracy (the difference between the measured value and the theoretical value of cutting diameter or depth). Ten 4 mm×3 mm implant holes were automatically prepared on the bilateral femurs of 5 Japanese big white rabbits, and ten 4 mm×3 mm implants made by 3D printer were artificially implanted, and the preparation effect of the implant cavities was evaluated. Results: The quantitative relationship curve between the number of laser pulses (n) and 2D depth of cut (d) showed a linear upward trend. The linear fitting obtained the quantitative relation function formula d=9.278 4 n±26.763 0, R(2)=0.988 9. The optimum number of single step pulse layers was 5 layers, and the vertical single step amount was 50 µm, so as to set the vertical parameters of a single step of a 3D cutting, and the 3D cutting diameter accuracy was (3.98±2.87) µm, with a depth accuracy of (15.42±5.44) µm. Automated preparation of 10 implant cavities on the femur of the rabbit were completed. When the implants were placed into the implant cavities, there was resistance, but they were fully seated and primary stability has been achieved after seating implant placement. Conclusions: The method of non-contact automatic preparation of dental implant cavities using robot controlling ultra-short pulse laser is feasible. By optimizing the single cutting process parameters, precise control of laser cutting cortical bone can be realized.

A functional role for intra-axonal protein synthesis during axonal regeneration from adult sensory neurons.

Although intradendritic protein synthesis has been documented in adult neurons, the question of whether axons actively synthesize proteins remains controversial. Adult sensory neurons that are conditioned by axonal crush can rapidly extend processes in vitro by regulating the translation of existing mRNAs (Twiss et al., 2000). These regenerating processes contain axonal but not dendritic proteins. Here we show that these axonal processes of adult sensory neurons cultured after conditioning injury contain ribosomal proteins, translational initiation factors, and rRNA. Pure preparations of regenerating axons separated from the DRG cell bodies can actively synthesize proteins in vitro and contain ribosome-bound beta-actin and neurofilament mRNAs. Blocking protein synthesis in these regenerating sensory axons causes a rapid retraction of their growth cones when communication with the cell body is blocked by axotomy or colchicine treatment. These findings indicate that axons of adult mammalian neurons can synthesize proteins and suggest that, under some circumstances, intra-axonal translation contributes to structural integrity of the growth cone in regenerating axons. By immunofluorescence, translation factors, ribosomal proteins, and rRNA were also detected in motor axons of ventral spinal roots analyzed after 7 d in vivo after a peripheral axonal crush injury. Thus, adult motor neurons are also likely capable of intra-axonal protein synthesis in vivo after axonal injury.

Insights into the stimulatory mechanism of 2-aminoethoxydiphenyl borate on TREK-2 potassium channel.

2-Aminoethoxydiphenyl borate (2-APB) has been recently identified as a common agonist of TWIK-related K(+) channel (TREK)/TRAAK channels, a subfamily of two-pore domain K(+) (K2P) channels. TREK-2 displays much higher sensitivity to 2-APB compared with TREK-1, despite that these two channels share the highest homology among K2P members. However, the structural basis for their difference in response to 2-APB still remains unknown. Here we identified that the cytosolic C-terminus (Ct) domain plays a dominant role in controlling the stimulatory effects of 2-APB on TREK-2 channel. The distal Ct region negatively regulates the effect of 2-APB, while the proximal Ct is sufficient to evoke the full 2-APB activation of the channel. Further mapping within the proximal Ct revealed that His368 is required for 2-APB activation, and the cooperation of the other non-conserved residues is also necessary. We also identified a secondary active site for 2-APB, which is located at the bottom of the transmembrane segment M2. Finally, we demonstrated that key residues or domains required for 2-APB activation are not involved in the gating mechanism of the selectivity filter. In summary, we reveal a unique modulatory model of TREK-2-Ct that distinguishes it from TREK-1 in high sensitivity to 2-APB. The cooperation of the non-conserved residues within the proximal Ct of TREK-2 plays a dominant role in the 2-APB-induced channel opening, whereas the distal Ct negatively regulates the process.

Direct synaptic projections to esophageal motoneurons in the nucleus ambiguus from the nucleus of the solitary tract of the rat.

Neurons of the nucleus of the solitary tract (NTS) serve as interneurons in swallowing. We investigated the synaptology of the terminals of these neurons and whether they project directly to the esophageal motoneurons in the compact formation of the nucleus ambiguus (AmC). Following wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) injection into the NTS, many anterogradely labeled axodendritic terminals were found in the neuropil of the AmC. The majority of labeled axodendritic terminals (89%) contained round vesicles and made asymmetric synaptic contacts (Gray's type I), but a few (11%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II). More than half of the labeled terminals contacted intermediate dendrites (1-2 microm diameter). There were no retrogradely labeled medium-sized motoneurons, but there were many retrogradely labeled small neurons having anterogradely labeled axosomatic terminals. A combined retrograde and anterograde transport technique was developed to verify the direct projection from the NTS to the esophageal motoneurons. After the esophageal motoneurons were retrogradely labeled by cholera toxin subunit B conjugated HRP, the injection of WGA-HRP into the NTS permitted ultrastructural recognition of anterogradely labeled axosomatic terminals contacting directly labeled esophageal motoneurons. Serial sections showed that less than 20% of the axosomatic terminals were labeled in the esophageal motoneurons. They were mostly Gray's type I, but a few were Gray's type II. In the small neurons, more than 30% of axosomatic terminals were labeled, which were exclusively Gray's type I. These results indicate that NTS neurons project directly not only to the esophageal motoneurons, but also to the small neurons which have bidirectional connections with the NTS.

Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat.

During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium-sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) injections into the pharyngeal muscles of male Sprague-Dawley rats, many nerve terminals anterogradely labeled with WGA-HRP were found to contact PH motoneurons retrogradely labeled with CT-HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Gray's type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Gray's type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Gray's type II. Observations of serial ultrathin sections revealed that both the small and the medium-sized neurons received only a few labeled axosomatic terminals that were exclusively Gray's type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS.

Direct parabrachial nuclear projections to the pharyngeal motoneurons in the rat: an anterograde and retrograde double-labeling study.

The parabrachial nucleus consists of several subnuclei which contains autonomic, gustatory, visceral sensory, nociceptive, and respiratory related neurons. We have investigated the direct projections from the rat parabrachial region, including the K olliker-Fuse nucleus, to the pharyngeal motoneurons with an anterograde and retrograde double-tracing technique. The cholera toxin subunit-B was injected into the lower pharynx or the esophagus after injection of biotinylated dextran amine into the ventrolateral parabrachial nuclear region, including the external medial, the external lateral, and the crescent area of the central lateral parabrachial nuclei and into the Kölliker-Fuse nucleus. The anterogradely dextran amine-labeled fibers from these nuclei projected to the semicompact, loose and external formations besides the compact formation of the nucleus ambiguus. Many anterogradely labeled fibers and terminals were found to contact retrogradely cholera toxin-labeled pharyngeal neuronal soma and dendrites in the semicompact formation of the nucleus ambiguus. The medial half of the parabrachial nucleus, including the medial and the medial part of the central lateral parabrachial nuclei, sent a few fibers to the reticular formation just dorsal to the esophageal motoneurons but no fibers to either the pharyngeal or to the esophageal motoneurons. These results suggested that the visceral sensory, gustatory, nociceptive or respiratory related neurons in the parabrachial nucleus project directly to the pharyngeal motoneurons, but there are no parabrachial projections to the esophageal motoneurons in the nucleus ambiguus.

Neostigmine competitively inhibited nicotinic acetylcholine receptors in sympathetic neurons.

The present experiment investigates the effect of neostigmine on nicotinic acetylcholine receptors (nAChRs) in the cultured neurons from neonatal rat superior cervical ganglia (SCG). Using whole-cell patch clamp techniques, we found that the amplitudes of the currents induced by 50 microM dimethylphenylpiperazinium (DMPP) were 21.5+/-10.7%, 52.9+/-9.2% and 86.9+/-4.9% depressed at the increased concentrations of neostigmine 100, 200 and 400 microM, respectively. The inhibition of neostigmine decreased gradually with the increased concentration of nicotine from 10 to 160 microM. Lineweaver-Burk's double-reversible plot illustrated that neostigmine blocked neuronal nAChRs in a competitive manner. Hyperpolarization of membrane potential from -40 mV to -100 mV did not significantly influence the blockade of neostigmine. Neostigmine could not accelerate the decay of the DMPP-induced currents, neither evoke any detectable currents in SCG neurons. The results indicate that neostigmine depress neuronal nAChRs in a competitive, concentration-dependent and voltage-independent manner, and can not facilitate desensitization of the receptors. The present data suggest that neostigmine blocks neuronal nAChRs by interacting with the ACh binding sites of the receptors.

Fine structure of the semicompact formation of the nucleus ambiguus of the rat.

We investigated the fine structure of the semicompact formation of the nucleus ambiguus (AmS), which was identified by retrogradely labeled pharyngeal (PH) motoneurons. When cholera toxin subunit B-conjugated horseradish peroxidase was injected into the lower pharyngeal muscle, many retrogradely labeled PH neurons were found throughout the AmS. Besides the PH neurons, two types of neurons were recognized in the AmS: unlabeled medium-sized neurons and unlabeled small neurons. The PH neuron was large (27.6 x 44.1 microm) and polygonal, and contained many Nissl bodies and well-developed cell organelles with a prominent spherical nucleus. The medium-sized neuron was dark and oval (19.3 x 33.2 microm), and contained many free ribosomes and much swollen rough endoplasmic reticulum with a distorted oval nucleus. The small neuron was spindle-shaped (12.3 x 20.2 microm), and had poorly developed cell organelles with an irregularly shaped nucleus. The average number of axosomatic terminals in a sectional plane was largest in the PH neurons (32.8), smaller in the medium-sized neurons (23.1), and smallest in the small neurons (6.3). The number of axo-somatic terminals containing round vesicles (Gray's type I) was almost equal to that of terminals containing pleomorphic vesicles (Gray's type II) in the PH neuron, and slightly smaller in the small and the medium-sized neurons. About 60% of the axodendritic terminals were Gray's type I, and 40% were type II. These results indicate that there are two different types of interneurons besides the PH motoneurons in the AmS.

Synaptology and ultrastructural characteristics of laryngeal cricothyroid and posterior cricoarytenoid motoneurons in the nucleus ambiguus of the rat.

The laryngeal motoneurons innervating the cricothyroid muscle (CT) are located in the semicompact formation just ventral to the rostral part of the compact formation of the nucleus ambiguus. The motoneurons innervating the posterior cricoarytenoid muscle (PCA) are located in the loose formation. We retrogradely labeled the CT and the PCA motoneurons using cholera toxin subunit B-conjugated horseradish peroxidase, and determined the ultrastructure and synaptic organization of these neurons. The CT and the PCA motoneurons had the appearance of alpha-motoneurons, i.e., large, oval or polygonal cells containing well-developed organelles and a prominent spherical nucleus. Two kinds of neurons were recognized among the PCA motoneurons. The one (PCA-A) was significantly smaller than the other (PCA-B). The average number of axosomatic terminals in a section was significantly largest in the PCA-B (56.6), smaller in the PCA-A (36.0), and smallest in the CT (32.3) neurons. Most of the axosomatic terminals (64.7%) contained pleomorphic vesicles and made symmetric synaptic contacts (Gray's type II) with the PCA-A neurons, while more than 60% contained round vesicles with asymmetric synaptic contacts (Gray's type I) in the CT (69.5%) and the PCA-B (60.6%) neurons. A few terminals associated with subsurface cisterns were present on all laryngeal motoneurons. These results indicated that the CT motoneurons may receive mostly excitatory terminals, whereas the PCA muscle may be regulated by neurons having many inhibitory terminals, and neurons having many excitatory terminals.

Descending projections from the paraventricular hypothalamic nucleus to the spinal cord: anterograde tracing study in the rat.

We investigated the direct projections from the paraventricular hypothalamic nucleus (PVH) to the spinal cord. When Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into the PVH, labeled descending fibers were observed running bilaterally through three pathways. The first pathway ran into the dorsal longitudinal fasciculus and projected to the central gray matter, Edinger-Westphal nucleus, pedunculopontine tegmental nucleus, nucleus of the locus ceruleus and parabrachial nucleus. The second and third pathways coursed through the medial forebrain bundle, ventral tegmental area, and ventral part of the medulla oblongata. At the medulla oblongata, the second pathway curved dorsolaterally and joined Rexed's lamina V of C1 after giving many projections to the nucleus ambiguus, nucleus of the solitary tract, dorsal motor nucleus of the vagus, and a few to the area postrema. The fibers descended through lamina V until C5, and coursed through lamina I from C6 to the upper coccygeal segments. They gave collateral projections to lamina I from the cervical through the upper coccygeal segments. The third pathway coursed laterally and descended through the lateral funiculus after giving projections to the lateral reticular nucleus and the marginal layer of the spinal trigeminal nucleus. These fibers gave off many projections to the intermediolateral cell column of the thoracic cord and the sacral parasympathetic nucleus. Lamina X received many projections from the fibers of the lateral funiculus at C5 through the b-segment of sacral spinal cord. These results indicate that the PVH may integrate directly with the medullary and spinal autonomic regulatory nuclei, including the vagus complex, sympathetic intermediolateral cell column, laminae I and X, and sacral parasympathetic nucleus.

[Cortical projections from the reuniens nucleus of the thalamus in the rat].

Direct projections from the reuniens nucleus to the cerebral cortex were examined using the anterograde tracing method of Phaseolus vulgaris leucoagglutinin (PHA-L) in the rat. The reuniens nucleus was found to project to widespread cortical regions. The labeled fibers from the reuniens nucleus terminated in layers I, III and IV of the frontal cortex including the medial and ventrolateral orbital cortices, the cingulate cortex, and the infralimbic cortex. A few fibers from the nucleus reuniens also terminated in layer I of the agranular insular cortex, the temporal cortex and the parietal cortex. The labeled terminals were also present in the retrosplenial granular and agranular cortical regions of the layer I. The heavy PHA-L labeled fibers terminated in the stratum lacunosum and molecular of field CA1 of Ammon's horn of the hippocampus, and moderately in the subiculum, the presubiculum and the parasubiculum, mainly in the molecular layer. There were also may labeled fibers and terminals in the perirhinal cortex of layer I, the entorhinal cortex of layers I, III-IV and V, and the occipital cortex of layer I. These results indicate that the reuniens nucleus projects to much more widespread regions than previously known.