Clinical outcomes and medical management of achondroplasia in Japanese children: A retrospective medical record review of clinical data.

Achondroplasia (ACH) is a rare, autosomal dominant skeletal dysplasia characterized by short stature, characteristic facial configuration, and trident hands. Before vosoritide approval in Japan, patients with ACH could start growth hormone (GH) treatment at age 3 years. However, ACH and its treatment in young Japanese children have not been studied. This retrospective, longitudinal, medical records-based cohort study (before vosoritide approval) summarized symptoms, complications, monitoring, surgery/interventions, and height with/without GH in Japanese patients with ACH <5 years. Complications were observed in 89.2% of all 37 patients; 75.7% required surgery or intervention. All patients were monitored by magnetic resonance imaging; 73.0% had foramen magnum stenosis, while 54.1% had Achondroplasia Foramen Magnum Score 3 or 4. Of 28 GH-treated patients, 22 initiating at age 3 years were generally taller after 12 months versus 9 non-GH-treated patients. Mean annual growth velocity significantly increased from age 2 to 3 versus 3 to 4 years in GH-treated patients (4.37 vs. 7.23 cm/year; p = 0.0014), but not in non-GH-treated patients (4.94 vs. 4.20 cm/year). The mean height at age 4 years with/without GH was 83.6/79.8 cm. These results improve our understanding of young patients with ACH in Japan and confirm that early diagnosis of ACH and monitoring of complications help facilitate appropriate interventions.

Efficacy of duloxetine by prior NSAID use in the treatment of chronic osteoarthritis knee pain: A post hoc subgroup analysis of a randomized, placebo-controlled, phase 3 study in Japan.

BACKGROUND: A previously conducted placebo-controlled, randomized, phase 3 study of 353 Japanese patients with knee osteoarthritis (OA) showed significant improvements for duloxetine vs placebo in pain and health-related quality of life (HRQoL) (ClinicalTrials.gov Identifier: NCT02248480). Reported here are post hoc subgroup analyses evaluating the efficacy of duloxetine according to the pattern of prior nonsteroidal anti-inflammatory drug (NSAID) use. METHODS: Patients with knee OA pain received once-daily duloxetine or placebo for 14 weeks. Pain was evaluated using the Brief Pain Inventory (BPI) and HRQoL was evaluated using the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC). Patients were divided into four subgroups based on their prior NSAID use: (i) no prior NSAID use; (ii) low-frequency NSAID use (<14 days/month); (iii) high-frequency transdermal NSAID use (transdermal NSAIDs only; ≥14 days/month for the 3 months before study entry); and (iv) high-frequency other NSAID use (eg, oral NSAIDs only, both oral and transdermal NSAIDs; ≥14 days/month for the 3 months before study entry). RESULTS: In each of the four prior NSAID use subgroups, there were greater reductions in BPI average pain severity score for duloxetine vs placebo at all timepoints during the 14-week treatment period; the treatment*prior NSAID use interaction was not statistically significant. In each subgroup, the proportion of patients achieving a ≥50% reduction in BPI average pain severity score was higher for duloxetine vs placebo. In each subgroup, there were greater reductions in WOMAC total score for duloxetine vs placebo at all timepoints; the treatment*prior NSAID use interaction was not statistically significant. In each subgroup, there were greater reductions at Week 14 in WOMAC pain, stiffness, physical function, and total scores for duloxetine vs placebo. CONCLUSIONS: Duloxetine was consistently effective with respect to pain relief and HRQoL in Japanese patients with knee OA pain, regardless of the pattern of prior NSAID use.

Predicting treatment outcomes of major depressive disorder by early improvement in painful physical symptoms: a pooled analysis of double-blind, placebo-controlled trials of duloxetine.

OBJECTIVE: We determined if early improvement in painful physical symptoms (PPS) can be a predictor of remission in the treatment of major depressive disorder (MDD). METHODS: We included randomized, double-blind, parallel-group clinical trials of duloxetine (40-60 mg/day) versus placebo for the acute treatment of MDD with associated PPS. Only those studies using the Montgomery-Åsberg Depression Rating Scale (MADRS) and the Brief Pain Inventory - Short Form (BPI-SF) were included. Three studies met all criteria and included male or female outpatients aged ≥18 years who met the diagnostic criteria for MDD, had a MADRS total score ≥20, and had at least moderate pain (BPI-SF average pain score ≥3). Positive predictive values (PPVs) and negative predictive values (NPVs) of early improvement in PPS for remission were analyzed. PPVs were the proportion of patients with remission (MADRS total score ≤10) at week 8 out of patients who experienced early improvement in BPI-SF average pain score (≥30% decrease from baseline at week 1, 2, or 4). NPVs were the proportion of patients without remission (MADRS total score >10) at week 8 out of patients who did not experience early improvement in PPS. RESULTS: Data from 1,320 patients were analyzed (duloxetine N=641 and placebo N=679). The overall remission (MADRS total score ≤10 at week 8) rate for the duloxetine group was significantly higher than the placebo group (38.5% vs 21.8%; P<0.0001). For both treatment groups, PPVs of early improvement in BPI-SF (30% improvement from baseline) were higher than the overall remission rate for all weeks examined (weeks 1, 2, and 4); in general, NPVs of early improvement in BPI-SF for nonremission were higher than the overall nonremission rate. CONCLUSION: Early improvement in PPS can be a useful clinical indicator of subsequent treatment outcome for MDD patients with associated PPS.

Associations among depression severity, painful physical symptoms, and social and occupational functioning impairment in patients with major depressive disorder: a 3-month, prospective, observational study.

PURPOSE: To investigate associations among depression severity, painful physical symptoms (PPS), and social and occupational functioning impairment in patients with major depressive disorder (MDD) who had achieved complete remission (CR) or partial remission (PR) after acute treatment. PATIENTS AND METHODS: This was a 12-week, multicenter, prospective, observational study. Patients with MDD treated with an antidepressant medication for the previous 12 weeks (±3 weeks) who had achieved CR (defined as a 17-item Hamilton Rating Scale for Depression [HAM-D17] score ≤7) or PR (HAM-D17 score ≥8 and ≤18) were enrolled. Depression severity, PPS, and impairment in social and occupational functioning were assessed using the HAM-D17, the Brief Pain Inventory (Short Form) (BPI-SF), and the Social and Occupational Functioning Assessment Scale (SOFAS), respectively, at enrollment (Week 12) and after 12 weeks (Week 24). RESULTS: Overall, 323 Japanese patients with MDD were enrolled (CR n=158, PR n=165) and 288 patients completed the study (CR n=139, PR n=149). HAM-D17 and SOFAS scores were strongly and negatively correlated at enrollment (Week 12; P<0.0001) and Week 24 (P<0.0001). A weak negative correlation between the BPI-SF and SOFAS was observed at Week 24 (P=0.0011), but not at enrollment (P=0.164). Remission status at enrollment (CR or PR) was associated with achieving normal social and occupational functioning (SOFAS score ≥80) at Week 24 in patients who had not achieved normal social and occupational functioning (SOFAS score <80) at enrollment (CR vs PR, OR=0.05 [95% CIs 0.01-0.18], P<0.0001). A greater proportion of patients with CR and no PPS at enrollment achieved SOFAS scores ≥80 at Week 24 than those with CR and PPS. CONCLUSION: Our results suggest that treating both depressive symptoms and PPS is important for achieving a normal level of functioning on a long-term basis in patients with MDD.

An observational study of duloxetine versus SSRI monotherapy in Japanese patients with major depressive disorder: subgroup analyses of treatment effectiveness for pain, depressive symptoms, and quality of life.

OBJECTIVE: To examine how clinical and demographic patient baseline characteristics influence effectiveness of duloxetine versus selective serotonin reuptake inhibitor (SSRI) treatment, in real-world Japanese clinical settings of patients with major depressive disorder (MDD) and associated painful physical symptoms (PPS). METHODS: This was a multicenter, 12-week, prospective, observational study in patients with MDD (Quick Inventory of Depressive Symptomatology ≥16) and at least moderate PPS (Brief Pain Inventory-Short Form [BPI-SF] average pain ≥3). Patients received duloxetine or SSRIs (escitalopram, sertraline, paroxetine, or fluvoxamine). Assessments were made by using BPI-SF average pain, 17-item Hamilton Rating Scale for Depression (HAM-D17), EuroQol 5-dimension questionnaire, Social Adaptation Self-Evaluation Scale, Global Assessment of Functioning, and ability to work. Predefined subgroups included the number of previous episodes of depression (0 vs ≥1), baseline BPI-SF average pain score (≤6 vs >6), baseline HAM-D17 total score (≤18 vs >18), baseline HAM-D17 retardation (≤7 vs >7) and anxiety somatic subscale scores (≤6 vs >6), and age (<65 vs ≥65 years). RESULTS: Treatment effectiveness was evaluated in 523 patients (duloxetine N=273, SSRIs N=250). Treatment with duloxetine was superior to SSRIs on most outcome measures in patients experiencing their first depressive episode, those with higher baseline PPS levels, and in patients with more severe baseline depression. This was also the case for older patients. In patients with less severe depression, SSRI treatment tended to show more improvements in depression and quality of life measures versus duloxetine treatment. CONCLUSION: These preplanned subgroup analyses of data from a prospective observational study suggest that, for Japanese MDD patients with PPS, duloxetine is more effective than SSRIs in patients with a first episode of MDD, with more severe depression, or more severe PPS.

An observational study of duloxetine versus SSRI monotherapy for the treatment of painful physical symptoms in Japanese patients with major depressive disorder: primary analysis.

OBJECTIVE: The objective of this study was to assess the effectiveness of duloxetine monotherapy, in comparison with selective serotonin reuptake inhibitor (SSRI) monotherapy, in the treatment of painful physical symptoms (PPS) in Japanese patients with major depressive disorder (MDD) in real-world clinical settings. METHODS: This was a multicenter, 12-week prospective, observational study. This study enrolled MDD patients with at least moderate PPS, defined as a Brief Pain Inventory-Short Form (BPI-SF) average pain score (item 5) ≥3. Patients were treated with duloxetine or SSRIs (escitalopram, sertraline, paroxetine, or fluvoxamine) for 12 weeks, and PPS were assessed by BPI-SF average pain score. The primary outcome was early improvement in the BPI-SF average pain score at 4 weeks post-baseline. RESULTS: A total of 523 patients were evaluated for treatment effectiveness (duloxetine N=273, SSRIs N=250). The difference in BPI-SF average pain score between the two groups was not statistically significant at 4 weeks post-baseline, the primary endpoint (least-squares mean change from baseline [95% confidence interval]: duloxetine, -2.8 [-3.1, -2.6]; SSRIs, -2.5 [-2.8, -2.3]; P=0.166). There was a numerical advantage for duloxetine in improvement from 4 to 12 weeks post-baseline, and the difference was statistically significant at 8 weeks post-baseline (least-squares mean change from baseline [95% confidence interval]: duloxetine, -3.6 [-3.9, -3.3]; SSRIs, -3.1 [-3.4, -2.8]; P=0.023). The 30% and 50% responder rates were significantly higher in patients treated with duloxetine at 4 and 8 weeks post-baseline. There were no serious adverse events experienced by duloxetine-treated patients. The rate of discontinuations due to adverse events was similar for duloxetine and the SSRIs (1.0% and 0.8% of patients, respectively). CONCLUSION: In this observational study, BPI-SF improvement was not significantly different at 4 weeks, the primary endpoint; however, patients treated with duloxetine tended to show better improvement in PPS compared to those treated with SSRIs.

Trajectories of depression symptom improvement and associated predictor analysis: An analysis of duloxetine in double-blind placebo-controlled trials.

BACKGROUND: In the treatment of major depressive disorder (MDD), it is not fully understood how individual symptoms improve over time (trajectory) in remitters. This study compared symptom improvement trajectories, as measured with the 17-item Hamilton Depression Rating Scale (HAM-D17), in remitters and nonremitters. METHODS: This analysis is based on 10 placebo-controlled, randomized, double-blind trials of duloxetine (40-60mg/day) for treatment of MDD from baseline up to week 8. Remission was defined as a HAM-D17 total score ≤7 at week 8 (last observation carried forward). Trajectories of HAM-D17 items were assessed by mixed model repeated measures analysis for treatment and remitter-nonremitter comparisons. Grouping of the trajectories was performed by factor analysis. Predictor analysis using HAM-D17 items was conducted by logistic regression. RESULTS: There were 1555 patients in the duloxetine group (489 [31.4%] remitters) and 1206 patients in the placebo group (290 [24.0%] remitters; P<.0001). For most items, the difference in trajectories between remitters and nonremitters appeared at early time points and increased over time. Treatment response trajectories were very similar for duloxetine and placebo remitters, while duloxetine nonremitters improved more than placebo nonremitters. For duloxetine remitters, we found 3 trajectory groups of HAM-D17 items. The predictor analysis showed that improvement in 6 individual items at week 1 or 2 was significantly associated with remission at week 8. LIMITATIONS: Generalizability of these results may be limited by the relatively short observation period used to define remission. CONCLUSIONS: Early monitoring of some symptoms of depression may prove useful in guiding treatment decisions.

Is duloxetine's effect on painful physical symptoms in depression an indirect result of improvement of depressive symptoms? Pooled analyses of three randomized controlled trials.

In treating Major Depressive Disorder with associated painful physical symptoms (PPS), the effect of duloxetine on PPS has been shown to decompose into a direct effect on PPS and an indirect effect on PPS via depressive symptoms (DS) improvement. To evaluate the changes in relative contributions of the direct and indirect effects over time, we analyzed pooled data from 3 randomized double-blind studies comparing duloxetine 60 mg/d with placebo in patients with major depressive disorder and PPS. Changes from baseline in Montgomery-Åsberg Depression Rating Scale total and Brief Pain Inventory-Short Form average pain score were assessed over 8 weeks. Path analysis examined the (1) direct effect of treatment on PPS and/or indirect effect on PPS via DS improvement and (2) direct effect of treatment on DS and/or indirect effect on DS via PPS improvement. At week 1, the direct effect of duloxetine on PPS (75.3%) was greater than the indirect effect through DS improvement (24.7%) but became less (22.6%) than the indirect effect (77.4%) by week 8. Initially, the direct effect of duloxetine on PPS was markedly greater than its indirect effect, whereas later the indirect effect predominated. Conversely, at week 1, the direct effect of treatment on DS (46.4%) was less than the indirect effect (53.6%), and by week 8 it superseded (62.6%) the indirect effect (37.4%). Thus, duloxetine would relieve PPS directly in the initial phase and indirectly via improving DS in the later phase.

Nurr1 expression is regulated by voltage-dependent calcium channels and calcineurin in cultured hippocampal neurons.

Nurr1 is an orphan nuclear transcription factor expressed in the brain. While Nurr1 is assumed to be an immediate early gene, it is not fully understood how Nurr1 expression is regulated in an activity-dependent manner in the central nervous system. Here, we investigated the molecular mechanisms underlying the regulation of Nurr1 expression in cultured hippocampal and cortical neurons. We found that upregulation of neural activity by high KCl and bicuculline enhances Nurr1 levels, while blockade of its activity by tetrodotoxin reduces Nurr1 levels. The induction of Nurr1 expression was mediated by voltage-dependent calcium channels (VDCCs), as shown by cadmium and VDCC-specific inhibitors. Furthermore, calcineurin, but not calcium/calmodulin-dependent protein kinase (CaMK) was critical for the induction. Thus, Nurr1 expression is regulated by VDCC and calcineurin in a cell-autonomous, neural activity-dependent manner.

Birth regulates the initiation of sensory map formation through serotonin signaling.

Although the mechanisms underlying the spatial pattern formation of sensory maps have been extensively investigated, those triggering sensory map formation during development are largely unknown. Here we show that the birth of pups instructively and selectively regulates the initiation of barrel formation in the somatosensory cortex by reducing serotonin concentration. We found that preterm birth accelerated barrel formation, whereas it did not affect either barreloid formation or barrel structural plasticity. We also found that serotonin was selectively reduced soon after birth and that the reduction of serotonin was triggered by birth. The reduction of serotonin was necessary and sufficient for the effect of birth on barrel formation. Interestingly, the regulatory mechanisms described here were also found to regulate eye-specific segregation in the visual system, suggesting that they are utilized in various brain regions. Our results shed light on roles of birth and serotonin in sensory map formation.

The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine hydroxylase level in the brain.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for dopamine synthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. BH4 deficiency leads to the loss of TH protein in the brain, although the underlying mechanism is poorly understood. To give insight into the role of BH4 in the developmental regulation of TH protein level, in this study, we investigated the effects of acute and subchronic administrations of BH4 or dopa on the TH protein content in BH4-deficient mice lacking sepiapterin reductase. We found that BH4 administration persistently elevated the BH4 and dopamine levels in the brain and fully restored the loss of TH protein caused by the BH4 deficiency in infants. On the other hand, dopa administration less persistently increased the dopamine content and only partially but significantly restored the TH protein level in infant BH4-deficient mice. We also found that the effects of BH4 or dopa administration on the TH protein content were attenuated in young adulthood. Our data demonstrate that BH4 and catecholamines are required for the post-natal augmentation of TH protein in the brain, and suggest that BH4 availability in early post-natal period is critical for the developmental regulation of TH protein level.

Compensatory regulation of dopamine after ablation of the tyrosine hydroxylase gene in the nigrostriatal projection.

The tyrosine hydroxylase (TH; EC 1.14.16.2) is a rate-limiting enzyme in the dopamine synthesis and important for the central dopaminergic system, which controls voluntary movements and reward-dependent behaviors. Here, to further explore the regulatory mechanism of dopamine levels by TH in adult mouse brains, we employed a genetic method to inactivate the Th gene in the nigrostriatal projection using the Cre-loxP system. Stereotaxic injection of adeno-associated virus expressing Cre recombinase (AAV-Cre) into the substantia nigra pars compacta (SNc), where dopaminergic cell bodies locate, specifically inactivated the Th gene. Whereas the number of TH-expressing cells decreased to less than 40% in the SNc 2 weeks after the AAV-Cre injection, the striatal TH protein level decreased to 75%, 50%, and 39% at 2, 4, and 8 weeks, respectively, after the injection. Thus, unexpectedly, the reduction of TH protein in the striatum, where SNc dopaminergic axons innervate densely, was slower than in the SNc. Moreover, despite the essential requirement of TH for dopamine synthesis, the striatal dopamine contents were only moderately decreased, to 70% even 8 weeks after AAV-Cre injection. Concurrently, in vivo synthesis activity of l-dihydroxyphenylalanine, the dopamine precursor, per TH protein level was augmented, suggesting up-regulation of dopamine synthesis activity in the intact nigrostriatal axons. Collectively, our conditional Th gene targeting method demonstrates two regulatory mechanisms of TH in axon terminals for dopamine homeostasis in vivo: local regulation of TH protein amount independent of soma and trans-axonal regulation of apparent L-dihydroxyphenylalanine synthesis activity per TH protein.

Biopterin levels in the cerebrospinal fluid of patients with PARK8 (I2020T).

PARK8 is the most common form of familial Parkinson's disease (PD). We measured biopterin and monoamine metabolite levels in the cerebrospinal fluids of 7 PARK8 patients (I2020T mutation in leucine-rich repeat kinase 2), 2 asymptomatic mutation carriers, and 21 sporadic PD patients. The biopterin levels in PARK8 patients were significantly higher than those in sporadic PD patients, although the symptoms were comparable in both groups, suggesting that PARK8 patients exhibit parkinsonian symptoms with higher biopterin levels than sporadic PD patients.

Partial biopterin deficiency disturbs postnatal development of the dopaminergic system in the brain.

Postnatal development of dopaminergic system is closely related to the development of psychomotor function. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of dopamine and requires tetrahydrobiopterin (BH4) as a cofactor. To clarify the effect of partial BH4 deficiency on postnatal development of the dopaminergic system, we examined two lines of mutant mice lacking a BH4-biosynthesizing enzyme, including sepiapterin reductase knock-out (Spr(-/-)) mice and genetically rescued 6-pyruvoyltetrahydropterin synthase knock-out (DPS-Pts(-/-)) mice. We found that biopterin contents in the brains of these knock-out mice were moderately decreased from postnatal day 0 (P0) and remained constant up to P21. In contrast, the effects of BH4 deficiency on dopamine and TH protein levels were more manifested during the postnatal development. Both of dopamine and TH protein levels were greatly increased from P0 to P21 in wild-type mice but not in those mutant mice. Serotonin levels in those mutant mice were also severely suppressed after P7. Moreover, striatal TH immunoreactivity in Spr(-/-) mice showed a drop in the late developmental stage, when those mice exhibited hind-limb clasping behavior, a type of motor dysfunction. Our results demonstrate a critical role of biopterin in the augmentation of TH protein in the postnatal period. The developmental manifestation of psychomotor symptoms in BH4 deficiency might be attributable at least partially to high dependence of dopaminergic development on BH4 availability.

Accumulation of phosphorylated tyrosine hydroxylase into insoluble protein aggregates by inhibition of an ubiquitin-proteasome system in PC12D cells.

Tyrosine hydroxylase (TH) is a rate-limiting enzyme for the biosynthesis of catecholamines including dopamine. The relationship between proteasomal dysfunction and the etiology of Parkinson's disease has been suggested, but it is unknown if TH protein is affected by proteasomal dysfunctions. Here, we examined the effect of inhibition of ubiquitin-proteasomal pathway on biochemical characteristics of TH protein in the neuronal cells. Inhibition of 20S or 26S proteasome by proteasome inhibitor I, or MG-132 in NGF-differentiated PC12D cells induced dot-like immunoreactivities with the anti-(40)Ser-phosphorylated TH (p40-TH) antibody. These dots were tightly co-localized with ubiquitin and positive to Thioflavine-S staining. These dot-like immunoreactivities were not obvious when immunostaining was performed against total-TH or choline acetyltransferase. Western blotting analysis showed time-dependent increase of p40-TH in the Triton-insoluble fractions. We also examined the effect of okadaic acid, an inhibitor of protein phosphatase 2A, which is a phosphatase acting on p40-TH. Okadaic acid increased the amount of insoluble p40-TH. These data suggest that p40-TH is prone to be insolubilized and aggregated by dysfunction of an ubiquitin-proteasome system in PC12D cells.

Activity-dependent coordination of presynaptic release probability and postsynaptic GluR2 abundance at single synapses.

The strength of an excitatory synapse depends on both the presynaptic release probability (p(r)) and the abundance of functional postsynaptic AMPA receptors. How these parameters are related or balanced at a single synapse remains unknown. By taking advantage of live fluorescence imaging in cultured hippocampal neurons where individual synapses are readily resolved, we estimate p(r) by labeling presynaptic vesicles with a styryl dye, FM1-43, while concurrently measuring postsynaptic AMPA receptor abundance at the same synapse by immunolabeling surface GluR2. We find no appreciable correlation between p(r) and the level of surface synaptic GluR2 under basal condition, and blocking basal neural activity has no effect on the observed lack of correlation. However, elevating network activity drives their correlation, which accompanies a decrease in mean GluR2 level. These findings provide the direct evidence that the coordination of pre- and postsynaptic parameters of synaptic strength is not intrinsically fixed but that the balance is tuned by synaptic use at individual synapses.

Myosin light chain kinase is not a regulator of synaptic vesicle trafficking during repetitive exocytosis in cultured hippocampal neurons.

The mechanism by which synaptic vesicles (SVs) are recruited to the release site is poorly understood. One candidate mechanism for trafficking of SVs is the myosin-actin motor system. Myosin activity is modulated by myosin light chain kinase (MLCK), which in turn is activated by calmodulin. Ca(2+) signaling in presynaptic terminals, therefore, may serve to regulate SV mobility along actin filaments via MLCK. Previous studies in different types of synapses have supported such a hypothesis. Here, we further investigated the role of MLCK in neurotransmitter release at glutamatergic synapses in cultured hippocampal neurons by examining the effects of two MLCK inhibitors, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl (ML-7) and wortmannin. Bath application of ML-7 enhanced short-term depression of EPSCs to repetitive stimulation, whereas it reduced presynaptic release probability. However, ML-7 also inhibited action potential amplitude and voltage-gated Ca(2+) channel currents. These effects were not mimicked by wortmannin, suggesting that ML-7 was not specific to MLCK in hippocampal neurons. When SV exocytosis was directly triggered by a Ca(2+) ionophore, calcimycin, to bypass voltage-gated Ca(2+) channels, ML-7 had no effect on neurotransmitter release. Furthermore, when SV exocytosis elicited by electrical field stimulation was monitored by styryl dye, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide], the unloading kinetics of the dye was not altered in the presence of wortmannin. These data indicate that MLCK is not a major regulator of presynaptic SV trafficking during repetitive exocytosis at hippocampal synapses.

Synaptotagmin in Ca2+ -dependent exocytosis: dynamic action in a flash.

Synaptotagmins have been the popular candidates for the Ca2+ sensor that couples local rise in Ca2+ to neurotransmitter release. Studies in worm, fly, and mouse corroborate the likely role for synaptotagmin I, the best-studied synaptotagmin prototype, as a Ca2+ trigger for synaptic vesicle exocytosis. Recent investigations have focused on structural domains of synaptotagmin that are critical for its function. Here we provide a brief overview of synaptotagmin I and discuss recent studies within the framework of neurotransmitter release mechanisms for fast synaptic transmission.

Regulation by glycogen synthase kinase-3beta of the arborization field and maturation of retinotectal projection in zebrafish.

The retinotectal projection is one of the best systems to study the molecular basis of synapse formation in the CNS because of the well characterized topographic connections and activity-dependent refinement. Here, we developed a presynaptic neuron-specific gene manipulation system in the zebrafish retinotectal projection in vivo using the nicotinic acetylcholine receptor beta3 (nAChRbeta3) gene promoter. Enhanced green fluorescent protein (EGFP) expression signals in living transgenic zebrafish lines carrying the nAChRbeta3 gene promoter-directed EGFP expression vector visualized the development of entire retinal ganglion cell (RGC) axon projection to the tectum. Microinjection of the nAChRbeta3 gene promoter-driven double-cassette vectors directing the expression of both dominant-negative glycogen synthase kinase-3beta (dnGSK-3beta) and EGFP enabled us to follow the development of individual RGCs and to examine the effect of the molecule on the axonal arborization and maturation of the same neurons in living zebrafish. We found that the expression of the dominant-negative form of zebrafish GSK-3beta suppressed the arborization field of RGC axon terminals in the tectum as estimated by the reduction of arbor branch length and arbor areas. Furthermore, the suppression of GSK-3beta activity increased the size of vesicle-associated membrane protein 2-EGFP puncta in RGC axon terminals at the early stage of innervation to the tectum. These results suggest that GSK-3beta regulates the arborization field and maturation of RGC axon terminals in vivo.