Sparse Labeling and Neural Tracing in Brain Circuits by STARS Strategy: Revealing Morphological Development of Type II Spiral Ganglion Neurons.

Elucidating axonal and dendritic projection patterns of individual neurons is a key for understanding the cytoarchitecture of neural circuits in the brain. This requires genetic approaches to achieve Golgi-like sparse labeling of desired types of neurons. Here, we explored a novel strategy of stochastic gene activation with regulated sparseness (STARS), in which the stochastic choice between 2 competing Cre-lox recombination events is controlled by varying the lox efficiency and cassette length. In a created STARS transgenic mouse crossed with various Cre driver lines, sparse neuronal labeling with a relatively uniform level of sparseness was achieved across different brain regions and cell types in both central and peripheral nervous systems. Tracing of individual type II peripheral auditory fibers revealed for the first time that they undergo experience-dependent developmental refinement, which is impaired by attenuating external sound input. Our results suggest that STARS strategy can be applied for circuit mapping and sparse gene manipulation.

Slit/Robo signaling mediates spatial positioning of spiral ganglion neurons during development of cochlear innervation.

During the development of periphery auditory circuits, spiral ganglion neurons (SGNs) extend their neurites to innervate cochlear hair cells (HCs) with their soma aggregated into a cluster spatially segregated from the cochlear sensory epithelium. The molecular mechanisms underlying this spatial patterning remain unclear. In this study, in situ hybridization in the mouse cochlea suggests that Slit2 and its receptor, Robo1/2, exhibit apparently complementary expression patterns in the spiral ganglion and its nearby region, the spiral limbus. In Slit2 and Robo1/2 mutants, the spatial restriction of SGNs was disrupted. Mispositioned SGNs were found to scatter in the space between the cochlear epithelium and the main body of spiral ganglion, and the neurites of mispositioned SGNs were misrouted and failed to innervate HCs. Furthermore, in Robo1/2 mutants, SGNs were displaced toward the cochlear epithelium as an entirety. Examination of different embryonic stages in the mutants revealed that the mispositioning of SGNs was due to a progressive displacement to ectopic locations after their initial normal settlement at an earlier stage. Our results suggest that Slit/Robo signaling imposes a restriction force on SGNs to ensure their precise positioning for correct SGN-HC innervations.

Broadening of inhibitory tuning underlies contrast-dependent sharpening of orientation selectivity in mouse visual cortex.

Orientation selectivity (OS) in the visual cortex has been found to be invariant to increases in stimulus contrast, a finding that cannot be accounted for by the original, purely excitatory Hubel and Wiesel model. This property of OS may be important for preserving the quality of perceived stimulus across a range of stimulus intensity. The synaptic mechanisms that can prevent a broadening of OS caused by contrast-dependent strengthening of excitatory inputs to cortical neurons remain unknown. Using in vivo loose-patch recordings, we found in excitatory neurons in layer 4 of mouse primary visual cortex (V1) that the spike response to the preferred orientation was elevated as contrast increased while that to the orthogonal orientation remained unchanged, resulting in an overall sharpening rather than a weakening of OS. Whole-cell voltage-clamp recordings further revealed that contrast increases resulted in a scaling up of excitatory conductance at all stimulus orientations. Inhibitory conductance was enhanced at a similar level as excitation for the preferred orientation, but at a significantly higher level for the orthogonal orientation. Modeling revealed that the resulting broadening of inhibitory tuning is critical for maintaining and sharpening OS at high contrast. Finally, two-photon imaging guided recordings from parvalbumin-positive (PV) inhibitory neurons revealed that the broadening of inhibition can be attributed to a contrast-dependent broadening of spike-response tuning of PV neurons. Together our results suggest that modulation of synaptic inhibition in the mouse V1 cortical circuit preserves the sharpness of response selectivity during changes of stimulus strength.

Functional elimination of excitatory feedforward inputs underlies developmental refinement of visual receptive fields in zebrafish.

In many sensory systems, receptive fields (RFs) measured by spike responses undergo progressive refinement during development. It has been proposed that elimination of excitatory synaptic inputs underlies such functional refinement. However, despite many extracellular recording and anatomical studies, direct in vivo intracellular evidence has remained limited. In this study, by cell-attached recordings in the developing optic tectum of zebrafish, we found that during a short period after the initial formation of retinotectal synapses, spike visual RFs of tectal neurons underwent a two-stage developmental modulation: from an initial expansion to a later refinement. Whole-cell voltage-clamp recordings revealed that the underlying excitatory synaptic RF exhibited a similar developmental progression, with its spatial extent first increased and then reduced, and its spatial tuning profile gradually sharpened. The inhibitory RF was initially larger than the excitatory RF but became matched with the excitatory RF at later stages. Simulation with the integrate-and-fire neuron model suggested that the developmental changes of excitatory RFs primarily accounted for the initial enlargement and later refinement of spike RFs, whereas inhibitory inputs generally reduced the size of the spike RF without affecting its developmental progression. In addition, spike RF of individual retinal ganglion cells did not significantly change in size during the same period, and the spatial extent and tuning profile of the tectal excitatory RF barely changed after intratectal excitatory connections were silenced. Together, our results demonstrate that the functional refinement of tectal visual RFs results primarily from a selective elimination of feedforward retinotectal inputs.

[Thermo-chemotherapy enhances the immunogenicity of oral squamous cell carcinoma cells by inducing the expression of damage-associated molecular patterns].

OBJECTIVE: To investigate whether hyperthermia, chemotherapy and thermo-chemotherapy could trigger the expression of damage-associated molecular patterns (DAMPs). METHODS: The optimal working concentration of pingyangmycin (PYM) was detected by CCK-8 assay, and temperatures of 39, 42, and 45 ℃ were applied to the oral squamous cell carcinoma CAL27, SCC-15, and Tca8113 cell lines. The effects of different treatments on the apoptosis, calreticulin (CRT) membrane expression and high-mobility group box 1 (HMGB1) secretion of the cells were detected by using Annexin V/propidium iodide (PI), flow cytometry and enzyme-linked immunosorbent assay (ELISA) assay. SPSS 20.0 software was used for statistical analysis. RESULTS: Both hyperthermia and chemotherapy could increase the membrane expression of CRT and the secretion of HMGB1, and furthermore, thermo-chemotherapy group showed significantly increased in apoptosis, CRT membrane expression rate and HMGB1 secretion compared with chemotherapy group, and the difference was statistically significant (P<0.05). CONCLUSIONS: Hyperthermia, chemotherapy and thermo-chemotherapy could induce oral squamous cell carcinoma cells succumb to death, and at the same time, they can effectively induce the membrane expression of CRT, and promote the secretion of HMGB1. Moreover, thermo-chemotherapy is significantly better than that of chemotherapy alone in the induction of cell apoptosis and DAMPs expression.

[Differential Diagnostic Value of 18F-FDG PET/CT Imaging in Multiple Myeloma and Bone Metastases].

OBJECTIVE: To investigate the imaging characteristics of 18F-FDG positron emission computed tomography (18F-FDG PET/CT) in multiple myeloma (MM) patients and to analyze its application value in MM and bone metastases. METHODS: A retrospective analysis was made on MM patients (n=72) and bone metastases patients (n=50) admitted to Hainan Western Central Hospital from January 2017 to March 2019. All patients underwent 18F-FDG PET/CT examination. The distribution of lesions, bone destruction, maximum standardized uptake (SUVmax) and metabolic homogeneity were analyzed in both groups. RESULTS: More than 80% of MM and bone metastases involved thoracic bone, spine and pelvis, followed by limbs. MM was more common in the lesions of thoracic bone and skull than those in bone metastases, the difference was statistically significant (P＜0.05). The majority of MM patients presented osteolytic bone destruction (97.2%), mostly showing "insect-like phagocytic pattern", so the bone showed dilated changes, and osteogenic changes were rarely seen (2.8%). Osteolytic bone destruction accounted for 74.0% in patients with bone metastatic tumor, presenting "focal" appearance more often, and osteogenic changes accounted for 26.0%. Osteolytic bone destruction in patients with MM was significantly higher than that in patients with bone metastases（χ2=14.757，P＜0.05）. The SUVmax of MM (4.25±2.16)was significantly lower than that of bone metastases (7.84±3.25) (t=6.830, P＜0.05). Diffuse mild uptake of 18F-FDG was more common in patients with MM, and heterogeneous high uptake of 18F-FDG was more common in patients with bone metastasis, the difference was statistically significant (P＜0.05). CONCLUSION: 18F-FDG PET/CT examination is helpful to acquire the imaging features of bone structure and metabolic changes, and shows an important clinical value in the differential diagnosis of MM and bone metastases.

Disruption of glial function regulates the effects of electro-acupuncture at Tsusanli on gastric activity in rats.

According to recent evidence, acupuncture at Tsusanli (ST 36) can regulate gastric activity. And this regulation mainly depends upon neural basis or structure and may probably relate to the central neurons in the dorsal vagal complex. However, whether the glias of the dorsal vagal complex participate in the regulation of gastric activity, when electro-acupuncture (EA) at Tsusanli, still remains to be interpreted. In this study, we observed the effect of EA at Tsusanli (ST 36) on regulation of gastric activity. Propentofylline (PPF), a glial metabolic inhibitor, was used to inhibit the function of glial cells. EA at Tsusanli showed that the expressions of glial fibrillary acidic protein (GFAP) and OX42 increased significantly compared to that of the control group, and gastric electric change was obvious, with significantly higher frequency and wave amplitude compared to the control group. The expressions of GFAP and OX42 were decreased markedly when pretreated with PPF group than without PPF pretreatment group. Compared to the Tsusanli group and the control group, the changes of electro gastric graph (EGG) were significantly decreased in PPF pretreatment group. On the other hand, we observed the changes of spontaneous electro-activity of the DVC (dorsal vagal complex) in our previous experiment. The results indicated that EA at Tsusanli could activate glial cells in the dorsal vagal complex and regulate gastric activity. PPF blocked the function of glia, thus the effect of EA at Tsusanli on gastric activity was weakened. Our study suggested that this electro-acupuncture regulation of gastric activity was possibly related with glia of the dorsal vagal complex.

Clonal relationships of CSF B cells in treatment-naive multiple sclerosis patients.

A role of B cells in multiple sclerosis (MS) is well established, but there is limited understanding of their involvement during active disease. Here, we examined cerebrospinal fluid (CSF) and peripheral blood (PB) B cells in treatment-naive patients with MS or high-risk clinically isolated syndrome. Using flow cytometry, we found increased CSF lymphocytes with a disproportionate increase of B cells compared with T cells in patients with gadolinium-enhancing (Gd+) lesions on brain MRI. Ig gene heavy chain variable region (Ig-VH) repertoire sequencing of CSF and PB B cells revealed clonal relationships between intrathecal and peripheral B cell populations, which could be consistent with migration of B cells to and activation in the CNS in active MS. In addition, we found evidence for bystander immigration of B cells from the periphery, which could be supported by a CXCL13 gradient between CSF and blood. Understanding what triggers B cells to migrate and home to the CNS may ultimately aid in the rational selection of therapeutic strategies to limit progression in MS.

B cell repertoire expansion occurs in meningeal ectopic lymphoid tissue.

Ectopic lymphoid tissues (ELT) can be found in multiple sclerosis (MS) and other organ-specific inflammatory conditions. Whether ELT in the meninges of central nervous system (CNS) autoimmune disease exhibit local germinal center (GC) activity remains unknown. In an experimental autoimmune encephalomyelitis model of CNS autoimmunity, we found activation-induced cytidine deaminase, a GC-defining enzyme, in meningeal ELT (mELT) densely populated by B and T cells. To determine GC activity in mELT, we excised meningeal lymphoid aggregates using laser capture microscopy and evaluated B cell repertoires in mELT and secondary lymphoid organs by next-generation immune repertoire sequencing. We found immunoglobulin heavy chain variable region sequences that were unique to mELT and had accumulated functionally relevant somatic mutations, together indicating localized antigen-driven affinity maturation. Our results suggest that B cells in mELT actively participate in CNS autoimmunity, which may be relevant to mELT in MS and ELT in other chronic inflammatory conditions.

Dcc Mediates Functional Assembly of Peripheral Auditory Circuits.

Proper structural organization of spiral ganglion (SG) innervation is crucial for normal hearing function. However, molecular mechanisms underlying the developmental formation of this precise organization remain not well understood. Here, we report in the developing mouse cochlea that deleted in colorectal cancer (Dcc) contributes to the proper organization of spiral ganglion neurons (SGNs) within the Rosenthal's canal and of SGN projections toward both the peripheral and central auditory targets. In Dcc mutant embryos, mispositioning of SGNs occurred along the peripheral auditory pathway with misrouted afferent fibers and reduced synaptic contacts with hair cells. The central auditory pathway simultaneously exhibited similar defective phenotypes as in the periphery with abnormal exit of SGNs from the Rosenthal's canal towards central nuclei. Furthermore, the axons of SGNs ascending into the cochlear nucleus had disrupted bifurcation patterns. Thus, Dcc is necessary for establishing the proper spatial organization of SGNs and their fibers in both peripheral and central auditory pathways, through controlling axon targeting and cell migration. Our results suggest that Dcc plays an important role in the developmental formation of peripheral and central auditory circuits, and its mutation may contribute to sensorineural hearing loss.

EphA7 regulates spiral ganglion innervation of cochlear hair cells.

During the development of periphery auditory circuitry, spiral ganglion neurons (SGNs) form a spatially precise pattern of innervation of cochlear hair cells (HCs), which is an essential structural foundation for central auditory processing. However, molecular mechanisms underlying the developmental formation of this precise innervation pattern remain not well understood. Here, we specifically examined the involvement of Eph family members in cochlear development. By performing RNA-sequencing for different types of cochlear cell, in situ hybridization, and immunohistochemistry, we found that EphA7 was strongly expressed in a large subset of SGNs. In EphA7 deletion mice, there was a reduction in the number of inner radial bundles originating from SGNs and projecting to HCs as well as in the number of ribbon synapses on inner hair cells (IHCs), as compared with wild-type or heterozygous mutant mice, attributable to fewer type I afferent fibers. The overall activity of the auditory nerve in EphA7 deletion mice was also reduced, although there was no significant change in the hearing intensity threshold. In vitro analysis further suggested that the reduced innervation of HCs by SGNs could be attributed to a role of EphA7 in regulating outgrowth of SGN neurites as knocking down EphA7 in SGNs resulted in diminished SGN fibers. In addition, suppressing the activity of ERK1/2, a potential downstream target of EphA7 signaling, either with specific inhibitors in cultured explants or by knocking out Prkg1, also resulted in reduced SGN fibers. Together, our results suggest that EphA7 plays an important role in the developmental formation of cochlear innervation pattern through controlling SGN fiber ontogeny. Such regulation may contribute to the salience level of auditory signals presented to the central auditory system.

[Effects of thermochemotherapy on immunologic function of patients with lip cancer].

OBJECTIVE: To evaluate the effects of thermochemotherapy on the immunologic function of lip cancer patients and to provide a theoretical basis for the clinical application of thermochemotherapy. METHODS: Twenty patients were heated by microwave at (42.5 +/- 0.2) degree C for 40 minutes after venoclysis of Pingyangmycin (8 mg) and Methotrexate (20 mg). Each of the patients received the therapy twice a week for 5 weeks. Venous blood was obtained before the first thermochemotherapy and after the tenth thermochemotherapy. Lymphocyte transformation index was examined by 3H-TdR method, and CD4+ and CD8+ T cells were assayed by flow cytometry. RESULTS: The tumors of the 20 patients were completely extinctive. The lymphocyte transformation index after treatment was significantly higher than that before treatment (P < 0.01). The CD4+ T cells and CD4+/CD8+ after treatment were significantly higher than those before treatment (P < 0.05); the CD8+ T cells after treatment was lower than that before treatment, but there was no statistically significant difference (P > 0.05). CONCLUSION: Thermochemotherapy can enhance the lip cancer patient's T lymphocyte immunologic function, which possibly plays an important role in the treatment of lip cancer.

[Change of T lymphocyte subsets, interleukin-2 and tumor necrosis factor-alpha in tumor-bearing mice and patients with oral cancer receiving thermo-chemotherapy].

OBJECTIVE: To investigate the change of T lymphocyte subsets, interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha in the tumor- bearing mice and patients with oral cancer receiving thermo-chemotherapy, and investigate the correlation among them. METHODS: After treatments, the expression of lymphocyte transformation index (LTI), IL-2 and TNF-alpha in the tumor-bearing mice were detected with methyl thiazolyl tetrazolium (MTT), the expression of IL-2 and TNF-alpha in the patients with oral cancer were detected with MTT, the expression of LTI, CD4+ and CD8+ were detected with 3H-TdR incorporation. RESULTS: LTI, IL-2 and TNF-alpha of thermo-chemotherapy group (HP group) had no significant difference comparing with those of normal mice group (N group) (P>0.05), but which were significantly higher than those of chemotherapy group (P group) and no treatment group (NT group) (P<0.01). In clinical trials, the expression of LTI, CD4+, CD4+/CD8+, IL-2 and TNF-alpha on oral cancer patients after thermo-chemotherapy were significantly higher than those before thermo-chemotherapy (P<0.01). CONCLUSION: After thermo-chemotherapy, the expression of LTI, IL-2 and TNF-alpha of tumor-bearing hosts are significantly improved, there is a significant correlation between IL-2 and T cell.

Polymorphisms of tumor-related genes IL-10, PSCA, MTRR and NOC3L are associated with the risk of gastric cancer in the Chinese Han population.

BACKGROUND: Gastric cancer is the fourth most common cancer in the world. Environmental and genetic factors both play critical roles in the etiology of gastric cancer. Hundreds of SNPs have been identified to have association with the risk of gastric cancer in many races. In this study, 25 SNPs in genes for IL-10, IL-1B, MTRR, TNF-а, PSCA, PLCE1 and NOC3L were analyzed to further evaluate their associations with gastric cancer susceptibility in the Chinese Han population. METHODS: Two hundred and seventy nine gastric cancer patients and 296 healthy controls were recruited in this study. SNP genotyping was conducted using Sequenom MassARRAY RS1000. Data management and statistical analyses were conducted by Sequenom Typer 4.0 Software and Pearson's χ(2) test. RESULTS: One protective allele and three risk alleles for gastric cancer patients were found in this study. The allele "G" of rs1801394 in MTRR showed an association with a decreased risk of gastric cancer: odds ratio (OR) = 0.74, 95% confidence interval (95% CI) = 0.57-0.97, P = 0.030 in the additive model; OR = 0.495, 95% CI = 0.26-0.95, P = 0.034 in the recessive model. The other three SNPs, the allele "C" of rs1800871 in IL10 (OR = 1.33, 95% CI = 1.04-1.90; P = 0.026 in the additive model; OR = 1.46, 95% CI = 1.04-2.06; P = 0.030 in the recessive model), the allele "A" of rs2976391 in PSCA (OR = 1.30, 95% CI = 1.01-1.66; P = 0.041 in the additive model and OR = 1.48, 95% CI = 1.04-2.11, P = 0.028 in the recessive model), and the allele "G" of rs17109928 in NOC3L gene (OR = 1.34, 95% CI = 1.01-1.78; P = 0.042 by additive model analysis; OR = 1.47, 95% CI = 1.04-2.07, P = 0.028 by dominant model analysis), showed an association with an increased risk of gastric cancer. CONCLUSIONS: These results indicate the importance of four gastric cancer susceptibility polymorphisms of IL-10, NOC3L, PSCA and MTRR in the Chinese Han population, which could be used in the determination of gastric cancer risk in clinical practice.

History matters: illuminating metaplasticity in the developing brain.

Metaplasticity refers to an activity-dependent regulation of the plastic state of neurons. In this issue of Neuron, Dunfield and Haas demonstrate that in intact developing brain circuits, specific patterns of visual stimulation drive functional plasticity of individual neurons with variable outcomes, predisposed by time-averaged postsynaptic activity recent to visual training.

A genetic strategy for stochastic gene activation with regulated sparseness (STARS).

It remains a challenge to establish a straightforward genetic approach for controlling the probability of gene activation or knockout at a desired level. Here, we developed a method termed STARS: stochastic gene activation with genetically regulated sparseness. The stochastic expression was achieved by two cross-linked, mutually-exclusive Cre-mediated recombinations. The stochastic level was further controlled by regulating Cre/lox reaction kinetics through varying the intrachromosomal distance between the lox sites mediating one of the recombinations. In mammalian cell lines stably transfected with a single copy of different STARS transgenes, the activation/knockout of reporter genes was specifically controlled to occur in from 5% to 50% of the cell population. STARS can potentially provide a convenient way for genetic labeling as well as gene expression/knockout in a population of cells with a desired sparseness level.

[Influence of thermochemotherapy on the activity of cytotoxic T lymphocyte in oral maxillofacial cancer patients].

OBJECTIVE: To study the influence of thermochemotherapy on the activity of cytotoxic T lymphocyte (CTL) in peripheral blood of patients with oral maxillofacial cancer. METHODS: Twenty-one subjects with oral maxillofacial cancer were treated by thermochemotherapy, and the activity of CTL in peripheral blood was analyzed. RESULTS: Thermochemotherapy can obviously enhance the activity of CTL (P<0.01). CONCLUSION: Thermochemotherapy can enhance the activity of CTL, thus enhance the patient's immune function. Therefore, it can enhance the antitumor response in whole body.

[Effect of hypoxia on the expression of urokinase-type plasminogen activator in human tongue squamous cell carcinoma cells (Tca8113)].

OBJECTIVE: To observe the expression of urokinase-type plasminogen activator (uPA) in human tongue squamous carcinoma cells (Tca8113) under hypoxia, in order to explore the relation between hypoxia and invasion and metastasis of oral cancer. METHODS: Under different hypoxic times(0, 3, 6, 12, 24 h), the expression of uPA protein was examined quantitatively using immunohistochemical technique (IH) and flow cytometry (FCM). RESULTS: Hypoxia promoted the expression of uPA. The longer hypoxic time was, the higher expression of uPA was. CONCLUSION: Hypoxia can promote invasion and metastasis of oral squamous carcinoma through stimulating the expression of uPA.

[Influence of the expression of heat shock protein 70 in maxillofacial squamous cell carcinoma by thermochemotherapy].

OBJECTIVE: To study the influence of thermochemotheraphy on the expression of HSP70 in maxillofacial squamous cell carcinoma. METHODS: 12 patients were treated with thermochemotheraphy twice a week, altogether 10 times. After 8 mg of Pingyangmycin infused, the patients were treated with microwave hyperthermia at 43 degrees C for 40 min. The part of carcinoma tissue was removed with surgical operations at before treatment and aftre five times of treatment. The expression of HSP70 in tumor cells was determined by SP immunohistochemcial method. RESULTS: The expression of HSP70 in tumor cells was enhanced obviously by thermochemotherapy. CONCLUSION: Special high expression of HSP70 in the tumor cells was induced by thermochemotherapy. With the antigen presenting action and other action, HSP70 have special antitumor effect.