[The association between baseline TPOAb and/or TgAb positivity and thyroid immune-related adverse events in patients with malignancies following treatment with immune checkpoint inhibitors].

Objective: To investigate the association between positive anti-thyroid peroxidase antibody (TPOAb) and/or anti-thyroglobulin antibody (TgAb) and the occurrence of thyroid immune-related adverse events (irAEs) in patients with malignant tumors who treated with immune checkpoint inhibitors (ICIs). Methods: A case-control study. A total of 116 patients with malignant tumor who received ICIs treatment and underwent thyroid function evaluation at Peking Union Medical College Hospital from January 2017 to April 2023 were enrolled retrospectively, including 77 males and 39 females, with a median age of (M(Q1, Q3)) 63.0 (55.0, 70.0) years. The patients were divided into the euthyroid group (n=58) and the thyroid irAEs group (n=58) according to whether thyroid irAEs occurred after ICIs treatment. The clinical characteristics and baseline anti-thyroid antibodies associated with the occurrence of thyroid irAEs after ICIs treatment in patients with malignant tumors were evaluated. Variables with statistical significance in univariate analysis were included in multivariate logistic regression model to analyze the risk factors for thyroid irAEs in patients with malignant tumors who received ICIs treatment. Results: In irAEs group, therewore 4 (3.4%) cases of clinical thyrotoxicosis, 23(19.8%) cases of subclinical thyrotoxicosis, 23 (19.8%) cases of clinical hypothyroidism, and 8(6.9%) cases of subclinical hypothyroidism. The positive rate of anti-thyroid antibodies at baseline in the thyrioid irAEs group was higher than that in the euthyroid group［16/58（27.6%）vs 3/58（5.2%），P=0.001］. After at least one course of ICIs treatment, the incidence of thyroid irAEs in patients with positive anti-thyroid antibodies at baseline was 84.2% (16/19), whereas it was 43.3% (42/97) in patients with negative anti-thyroid antibodies(P=0.001). Univariate logistic regression analysis showed that gender (OR=2.812, 95%CI:1.257-6.293), baseline thyroid autoantibodies were positive (OR=6.984, 95%CI: 1.909-25.547), baseline TgAb positivity (OR=8.909, 95%CI: 1.923-41.280), and baseline TPOAb positivity (OR=7.304, 95%CI: 1.555-34.308) were associated with thyroid irAEs (all P<0.05). Multivariate logistic regression analysis indicated that baseline TgAb positivity (OR=7.637, 95%CI: 1.617-36.072) was a risk factor for thyroid irAEs (P=0.01). Conclusions: The incidence of thyroid irAEs is higher in patients who are positive for baseline TPOAb and/or TgAb compared to those who are negative for TPOAb and TgAb. Patients with positive TgAb at baseline are at high risk of developing thyroid irAEs.

Cell-free DNA from bile outperformed plasma as a potential alternative to tissue biopsy in biliary tract cancer.

BACKGROUND: Biliary tract cancers (BTCs) are rare and highly heterogenous malignant neoplasms. Because obtaining BTC tissues is challenging, the purpose of this study was to explore the potential roles of bile as a liquid biopsy medium in patients with BTC. PATIENTS AND METHODS: Sixty-nine consecutive patients with suspected BTC were prospectively enrolled in this study. Capture-based targeted sequencing was performed on tumor tissues, whole blood cells, plasma, and bile samples using a large panel consisting of 520 cancer-related genes. RESULTS: Of the 28 patients enrolled in this cohort, tumor tissues were available in eight patients, and plasma and bile were available in 28 patients. Somatic mutations were detected in 100% (8/8), 71.4% (20/28), and 53.6% (15/28) of samples comprising tumor tissue DNA, bile cell-free DNA (cfDNA), and plasma cfDNA, respectively. Bile cfDNA showed a significantly higher maximum allele frequency than plasma cfDNA (P = 0.0032). There were 56.2% of somatic single-nucleotide variant (SNVs)/insertions and deletions (indels) shared between bile and plasma cfDNA. When considering the genetic profiles of tumor tissues as the gold standard, the by-variant sensitivity and positive predictive value for SNVs/indels in bile cfDNA positive for somatic mutations were both 95.5%. The overall concordance for SNVs/indels in bile was significantly higher than that in plasma (99.1% versus 78.3%, P < 0.0001). Moreover, the sensitivity of CA 19-9 combined with bile cfDNA achieved 96.4% in BTC diagnosis. CONCLUSION: We demonstrated that bile cfDNA was superior to plasma cfDNA in the detection of tumor-related genomic alterations. Bile cfDNA as a minimally invasive liquid biopsy medium might be a supplemental approach to confirm BTC diagnosis.

Association between maximum mouth opening and area of bony fusion in simulated temporomandibular joint bony ankylosis.

The aim of this study was to investigate the quantitative association between active/passive maximum mouth opening (AMMO/PMMO) and the severity of simulated temporomandibular joint (TMJ) bony ankylosis. Twenty-eight male sheep were divided randomly and equally into surgical and control groups. Surgical group animals underwent bilateral TMJ osteotomy during which left lateral pterygoid muscle function was blocked. Control animals did not undergo surgery. Body weight, AMMO/PMMO, and TMJ morphological features were evaluated preoperatively and at 12 and 24 weeks post-surgery. In the surgical group, only the right TMJ complexes with maintained lateral pterygoid muscle function developed TMJ bony ankylosis. The AMMO/PMMO and end-feel distance in the surgical group were significantly lower than those in the control group (P < 0.001, both) at 12 and 24 weeks post-surgery. Moreover, AMMO (r = -0.940 and -0.952, P < 0.001, both) and PMMO (r = -0.944 and -0.953, P < 0.001, both) were negatively correlated with the area (mm2) of bony fusion post-surgery. These findings may be useful for the clinical treatment of early mandibular condyle fracture, with the use of occlusal pads/open-mouth plates to relax the lateral pterygoid muscle and block its function. When bony ankylosis developed in the TMJ, the greater the area of bony fusion, the more limited were AMMO/PMMO.

[Influence of environmental factors on the two-species biofilm formed by Streptococcus oligofermentans and Streptococcus mutans].

Objective: To study the influence of environmental factors on the two-species biofilm formed by the combinations of Streptococcus oligofermentans (So) with Streptococcus mutans (Sm) and Streptococcus sanguinis (Ss) with Sm so as to evaluate the role of So in maintaining the microecological balance of the oral cavity. Methods: Single-and two-species biofilms were grown on saliva-coated surfaces (glass tube and 96-well plate). Colony-counting method and safranin staining method were used to measure the biofilms formed under various oxygen conditions (aerobic and anaerobic), sucrose conditions (0%, 1% and 5% sucrose concentrations) and pH conditions (5.5, 6.0, 6.5, 7.0, 7.5 and 8.0). Results: Comparing the numbers of Sm in two co-cultures under various conditions, Sm counts in So+Sm group [(7.70±2.46)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(9.00±1.13)×10(8) CFU/ml] in aerobic environment (P<0.05). Sm counts in So+Sm group [(2.80±0.52)×10(8) CFU/ml] were also significantly lower than those in the Ss+Sm group [(4.00±1.25)×10(8) CFU/ml] in anaerobic environment (P<0.05). The Sm counts in So+Sm group [(8.90±0.82)×10(8) CFU/ml] were significantly higher than those in Ss+Sm group [(7.50±1.73)×10(8) CFU/ml] in 0% sucrose environment (P<0.05). The Sm counts in So+Sm group [(5.70±2.94)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(10.30±3.21) ×10(8) CFU/ml] in 1% sucrose environment (P<0.05). The Sm counts in So+Sm group [(6.10±1.71)×10(8) CFU/ml] were also significantly lower than those in Ss+Sm group [(7.40±1.20)×10(8) CFU/ml] in 5% sucrose environment (P<0.05). The Sm counts in So+Sm group [(3.50±1.50)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(10.70±2.80)×10(8) CFU/ml] in pH7.0 environment (P<0.05). Comparing the formation of biofilm after 24 h cultivation, the Sm counts in So+Sm group were significantly lower than those in Ss+Sm group both in aerobic and anaerobic environments (P<0.05). The Sm counts in So+Sm group were significantly higher than those in Ss+Sm group in 0% sucrose environment (P<0.05). The Sm counts in So+Sm group were significantly lower than those in Ss+Sm group in 1% and 5% sucrose and pH 7.0 environments (P<0.05). Both So and Ss had no inhibitory effect on Sm in pH5.5 and pH8.0 environments. Conclusions: In the in vitro two-species co-culture systems, So showed stronger inhibitory effects than Ss on Sm and its inhibitory ability might influenced by various environmental factors.

MiR-124 regulates osteoblast differentiation through GSK-3ß in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a spastic and spinal joint disease with the characteristic of pathological ossification. Bioinformatics analysis demonstrated that there is a complementary binding site between microRNA-124 (miR-124) and the 3'-UTR of glycogen synthase kinase-3ß (GSK-3ß) mRNA. We aimed to investigate the role of miR-124 in regulating GSK-3ß expression, Wnt/ß-catenin pathway activity, and osteoblast differentiation of spinal ligament fibroblasts. PATIENTS AND METHODS: The ligament tissues of AS and the femoral neck fracture patients were collected. MiR-124 and GSK-3ß mRNA expressions were detected by using quantitative Real-time PCR (qRT-PCR). GSK-3ß and ß-catenin protein expressions were detected by using Western blot. Ligament fibroblasts were isolated and induced to differentiate into osteoblasts. Alizarin red S staining (ARS) was used to identify osteoblast differentiation. Expressions of miR-124, GSK-3ß, ß-catenin, Osterix, and runt-related transcription factor 2 (RUNX2) were detected during differentiation. The cells were divided into two groups as agomiR-normal control (NC) transfection group and agomir miR-124 transfection group. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were detected. RESULTS: MiR-124 and ß-catenin expressions in the ligament of AS patients increased, while GSK-3ß level reduced compared with control. MiR-124, ß-catenin, Osterix, and RUNX2 expressions gradually elevated, whereas GSK-3ß level gradually declined following increased osteoblasts differentiation. Antagomir miR-124 transfection significantly up-regulated the expression of GSK-3ß in osteoblast differentiation, significantly decreased the expression of ß-catenin, Osterix, and RUNX2, and significantly inhibited osteoblast differentiation. CONCLUSIONS: MiR-124 decreased and GSK-3ß elevated in AS ligament tissue. Down-regulation of miR-124 expression enhanced GSK-3ß expression, weakened Wnt/ß-catenin pathway activity, and inhibited the differentiation of ligament fibroblasts into osteoblasts.

[Antibacterial and residual antimicrobial activities of five final irrigants in infected root canal: an in vitro comparative study].

OBJECTIVE: To evaluate antibacterial and residual antimicrobial activities of five root canal irrigants including Qmix, MTAD(mixture of a tetracycline isomer, an acid, and a detergent), 0.2% cetrimide(CTR), 2% chlorhexidine(CHX) and 17% ethylene diaminetetraacetic acid(EDTA) and to find the most optimal final irrigants for using in root canal therapy. METHODS: The standard enterococcus infection models were built up in 100 single rooted incisors with single canal. Totally 30 teeth were selected by using random number tablefor detecting the quality of the bacteria model. Crown-down technique with rotary ProTaper system was used to prepare the root canals. Then the teeth were randomly divided into seven groups of which five groups were irrigated with five different irrigants respectively, one group was irrigated with distilled water(distilled water group) and one group was no-irrigation group. Each tooth was sectioned into three parts: apical 1/3, middle 1/3 and coronal 1/3. After irrigation, specimenswere cultivated from day 0 to day 14. All statistical analyses were performed by means of SPSS 17.0 software. Chi-squared test was used to evaluate antibacterial activities. Generalized estimating equations was used to evaluate residual antimicrobial activities. RESULTS: All samples rinsed with Qmix, MTAD, CTR, CHX were bacteria-free in 0 day. The samples rinsed with EDTA and distilled water had no bacteria in 7 coronal sections, 6 middle sections and 9 apical sections, respectively. The results of Qmix, MTAD, CTR and CHX groups showed significant difference when compared with that of distilled water, EDTA and control groups(P<0.05). Residual antimicrobial resultsin EDTA, distilled water, no-irrigation groups showed significant differences compared with that of Qmix, MTAD, CTR, CHX groups according to pairwise comparison(P<0.05) on day 1, 2 and 3. There was no significant difference between the other two groups(P>0.05). Antimicrobial properties on the coronal 1/3 and apical 1/3, middle 1/3 and apical 1/3 showed significant difference(P<0.05) while middle 1/3 and coronal 1/3 showed no significant difference(P>0.05). CONCLUSIONS: Qmix, MTAD, CTR and CHX had an antimicrobial activity, but could not destroy Enterococcus faecalis completely. Antimicrobial activity in coronal 1/3 was better than in apical 1/3. Qmix, MTAD, CTR and CHX had a residual antimicrobial activity with various lasting times. The lasting time of residual antimicrobial activity was as follow: MTAD> CTR>Qmix>CHX. EDTA had no antibacterial and residual antimicrobial activities.

Protein kinase c increases the apparent affinity of the release machinery to Ca2+ by enhancing the release machinery downstream of the Ca2+ sensor.

Modulation of the release probability of releasable vesicles in response to Ca(2+) influx (Prob(Ca)) is involved in mediating several forms of synaptic plasticity, including short-term depression, short-term augmentation, and potentiation induced by protein kinases. Given such an important role, however, the mechanism underlying modulation of the Prob(Ca) is unclear. We addressed this question by investigating how the activation of protein kinase C modulates the Prob(Ca) at a calyx-type nerve terminal in rat brainstem. Various lengths of step depolarization were applied to the nerve terminal to evoke different amounts of Ca(2+) currents and capacitance jumps, the latter of which reflect vesicle release. The relationship between the capacitance jump and the Ca(2+) current integral was sigmoidal and was fit well with a Hill function. The sigmoidal relationship was shifted significantly to the left during the application of the PKC activator 12-myristate 13-acetate (PMA), suggesting that PMA increases the apparent affinity of the release machinery to Ca(2+). This effect was blocked in large part by the application of the PKC inhibitor bisindolylmaleimide, suggesting that the effect is mediated mainly by the activation of PKC. We also found that PMA increased the rate of miniature EPSCs evoked by the application of hypertonic sucrose solution, which triggers release downstream of the Ca(2+) influx. Taken together, our results suggest that PKC enhances the apparent affinity of the release machinery to Ca(2+) by a mechanism downstream of the binding between Ca(2+) and its sensor. These results have provided the first example of the mechanisms underlying modulation of the Prob(Ca).

Fast kinetics of exocytosis revealed by simultaneous measurements of presynaptic capacitance and postsynaptic currents at a central synapse.

The rate of release from nerve terminals depends on both the number of release sites and the rate of release at each site. The latter remains largely unknown at central synapses. We addressed this issue by simultaneously measuring the nerve terminal membrane capacitance and the postsynaptic current at single calyceal synapses in rat brainstem. We found that a 10 ms presynaptic step depolarization depleted a releasable pool containing 3300-5200 vesicles. Released vesicles were endocytosed with a time constant of a few seconds to tens of seconds. Release of only one third of this pool saturated both postsynaptic AMPA and NMDA receptors. A release site can release more than three vesicles in 10 ms (>300 vesicles per second). We conclude that both a large number of release sites and a fast release rate at each site enable synapses to release at a high rate.

Spatial variability in release at the frog neuromuscular junction measured with FM1-43.

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.

The reduced release probability of releasable vesicles during recovery from short-term synaptic depression.

Recovery from synaptic depression is believed to depend mainly on replenishment of the releasable pool of vesicles. We observed that during recovery from depression in a calyx-type synapse, part of the releasable pool was replenished rapidly. Half recovery occurred within 1 s, even in the absence of residual calcium. Vesicles that had recently entered the releasable pool had a 7- to 8-fold lower release probability than those that had been in the pool for more than 30 s. These results suggest that the reduction in the release probability of releasable vesicles contributes greatly to the level of depression. How synapses maintain transmission during repetitive firing is in debate. We propose that during repetitive firing, accumulation of intracellular Ca2+ may facilitate release of the rapidly replenished but reluctant vesicles, making them available for sustaining synaptic transmission.

Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses.

We studied how Ca2+ influx through different subtypes of Ca2+ channels couples to release at a calyx-type terminal in the rat medial nucleus of the trapezoid body by simultaneously measuring the presynaptic Ca2+ influx evoked by a single action potential and the EPSC. Application of subtype-specific toxins showed that Ca2+ channels of the P/Q-, N-, and R-type controlled glutamate release at a single terminal. The Ca2+ influx through the P/Q-type channels triggered release more effectively than Ca2+ influx through N- or R-type channels. We investigated mechanisms that contributed to these differences in effectiveness. Electrophysiological experiments suggested that individual release sites were controlled by all three subtypes of Ca2+ channels. Immunocytochemical staining indicated, however, that a substantial fraction of N- and R-type channels was located distant from release sites. Although these distant channels contributed to the Ca2+ influx into the terminal, they may not contribute to release. Taken together, the results suggest that the Ca2+ influx into the calyx via N- and R-type channels triggers release less effectively than that via P/Q-type because a substantial fraction of the N- and R-type channels in the calyx is localized distant from release sites.

Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.

Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.

We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable) pool. In addition, FM1-43 was used to measure the rate of membrane retrieval after tetanic stimulation, and the amount of membrane transferred to the surface membrane. This provided a measure of the rate of refilling of the reserve pool with recycled vesicles. The time courses of both synaptic depression and endocytosis were slowed by prolonged tetanic stimulation. This behavior could be fitted by a simple model, assuming a first-order kinetics for both vesicle endocytosis and mobilization. The results show that a nearly 20-fold decrease in the rate constant of endocytosis greatly delays refilling of the depleted reserve pool. However, to fully account for the slower recovery of depression, a decrease in the rate constant of vesicle mobilization from the reserve pool of about sixfold is also required.

R-type Ca2+ currents evoke transmitter release at a rat central synapse.

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and gamma-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Presynaptic inhibition of elicited neurotransmitter release.

Activation of presynaptic receptors for a variety of neurotransmitters and neuromodulators inhibits transmitter release at many synapses. Such presynaptic inhibition might serve as a means of adjusting synaptic strength or preventing excessive transmitter release, or both. Previous evidence showed that presynaptic modulators inhibit Ca2+ channels and activate K+ channels at neuronal somata. These modulators also inhibit spontaneous transmitter release by mechanisms downstream of Ca2+ entry. The relative contribution of the above mechanisms to the inhibition of elicited release has been debated for a long time. Recent evidence at synapses where the relationship between transmitter release and presynaptic Ca2+ influx has been well characterized suggests that inhibition of presynaptic voltage-dependent Ca2+ channels plays the major role in presynaptic inhibition of elicited neurotransmitter release. In addition, modulation of the release machinery might contribute to inhibition of elicited release.

Presynaptic calcium dynamics and transmitter release evoked by single action potentials at mammalian central synapses.

The relationship between presynaptic calcium transients ([Ca2+]t) and transmitter release evoked by a single stimulus was both investigated experimentally and modeled at a mammalian central synapse, the CA3 to CA1 pyramidal cell synapse in guinea pig hippocampal slices. In the present study, we compared the low-affinity calcium indicator furaptra with the higher-affinity indicator fura-2. The 10-90% rise time of the furaptra transient was 2.4 ms compared to 7.8 ms with fura-2; the half-decay time (tau 1/2) was 30 ms for furaptra, compared to 238 ms for fura-2. The half-width of the calcium influx was 1.8 ms with furaptra, which provides an upper limit to the duration of the calcium current (ICa) evoked by an action potential. Modeling the decay time course of the furaptra transients led to the conclusion that the predominant endogenous calcium buffer in these terminals must have relatively slow kinetics (kon < 10(5)/M.s), although the presence of small amounts of fast buffers cannot be excluded. The relationship between the [Ca2+]t measured with furaptra and the postsynaptic response was the same as previously observed with fura-2: the postsynaptic response was proportional to about the fourth power (m approximately 4) of the amplitude of either [Ca2+]t or calcium influx. Thus, although fura-2 may be locally saturated by the high local [Ca2+] responsible for transmitter release, the volume-averaged fura-2 signal accurately reflects changes in this local concentration. The result that both indicators gave similar values for the power m constrains the amplitude of calcium influx in our model: Ica < 1 pA for 1 ms.

Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction.

We used FM1-43 imaging and intracellular recordings of synaptic potentials to measure the time course of endocytosis in frog motor nerve terminals following tetanic nerve stimulation, and we used fura-2 imaging of intraterminal Ca2+ concentration to compare endocytic rate and [Ca2+]i. Following a 30 Hz tetanus, endocytosis declined exponentially with a time constant that depended on the duration of stimulation. The level of [Ca2+]i rose from a resting value of about 100 nM to more than 500 nM during 30 Hz stimulation, and rapidly declined to 200-250 nM after stimulation. [Ca2+]i returned to resting level with a time course that, like endocytosis, depended on the duration of tetanic stimulation. However, the rate of [Ca2+]i recovery was much slower than the rate of endocytosis, leading to the conclusion that endocytic rate is not determined solely by the instantaneous level of [Ca2+]i.

Synaptic transmission. Kinetics of synaptic-vesicle recycling.

The kinetics of different steps in synaptic-vesicle recycling, including exocytosis, internalization and repriming, have recently been estimated in various types of living cell.

GABAB receptor-mediated presynaptic inhibition in guinea-pig hippocampus is caused by reduction of presynaptic Ca2+ influx.

1. The hypothesis that activation of GABAB receptors inhibits evoked synaptic transmission by reducing the presynaptic Ca2+ influx was tested using a recently developed technique for simultaneously recording the presynaptic Ca2+ transient ([Ca2+]t) and the field excitatory postsynaptic potential (fEPSP) evoked by a single electrical stimulus at CA3 to CA1 synapses of guinea-pig hippocampus. 2. The GABAB receptor agonist baclofen reversibly blocked, in a dose-dependant manner, both the fEPSP and the presynaptic [Ca2+]t with similar time courses. During application of baclofen, the fEPSP was proportional to about the fourth power of the presynaptic [Ca2+]t, and the presynaptic fibre volley and the resting Ca2+ level did not change. These results are similar to those we previously observed following application of several voltage-dependent Ca2+ channel blockers, suggesting that baclofen inhibits the fEPSP by blocking the presynaptic Ca2+ influx. 3. The inhibition by baclofen of both the fEPSP and the presynaptic [Ca2+]t was blocked by the GABAB receptor antagonist CGP 35348, consistent with the causal relationship between the GABAB receptor-mediated presynaptic inhibition of the [Ca2+]t and the fEPSP. 4. The inhibition by baclofen of the [Ca2+]t was partially occluded by application of the voltage-dependent Ca2+ channel blocker omega-conotoxin-GVIA (omega-CgTX-GVIA), but not omega-agatoxin-IVA (omega-AgaTX-IVA), suggesting that baclofen reduces the presynaptic [Ca2+]t by blocking Ca2+ channels including the omega-CgTX-GVIA-sensitive type. 5. We conclude that baclofen inhibits evoked transmitter release by reducing presynaptic Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)