FSH Stimulated Inhibin B in Disorders of Puberty

Background: Predicting puberty is a clinical challenge. Available tests include basal gonadotropins, GnRH and GnRH analogue stimulation test, hCG stimulation test and basal inhibin B(INHB). Unlike GnRH and GnRH analogue stimulated LH, no study has investigated for possibility of rapidly releasable pool of inhibin B from gonads so far. Therefore, in quest of a better diagnostic test present study was undertaken to explore stimulability of inhibin B and if found stimulable, potential role of FSH stimulated inhibin B(FSH-INHB) as marker of entry into puberty. Methods: A total of forty-two subjects fulfilling eligibility criteria were enrolled into this prospective interventional study. Study cohort was divided into Cohort A (Healthy children in puberty; n=26) and Cohort B (Patients of hypogonadotropic hypogonadism; n=16). All participants were subjected to FSH stimulation test and GnRHa stimulation test as per study protocol. Data was analysed for male and female separately. Results: Mean delta change between INHB and FSH-INHB in cohort A (Male; n=18) was 188.8 pg/ml (p value-0.002) while in cohort B(Male; n=8) was 16.64 pg/ml (p value-0.076). Mean delta change in cohort A(Female; n=8) was 1065 pg/ml (p value- 0.023) while in cohort B (Female; n=8) was 9.8 pg/ml (p value-0.128). On ROC analysis, INHB of 68.88 pg/ml in male had 94.4 % sensitivity and 87.5% specificity while 51.47 pg/ml in female had 75% sensitivity and 100% specificity for entry into puberty. Cut off for FSH-INHB were 116.14 pg/ml and 116.50 pg/ml for male and female respectively (100% sensitivity/100% specificity). Conclusion: Inhibin B was stimulable in both male and female after entry into puberty, highlighting the presence of rapidly releasable pool in ovaries and testes. FSH stimulated inhibin B may emerge as promising tool for entry into puberty.

The mammalian rod synaptic ribbon is essential for Cav channel facilitation and ultrafast fusion of the readily releasable pool of vesicles

Rod photoreceptors (PRs) use ribbon synapses to transmit visual information. To signal ‘no light detected’ they release glutamate continually to activate post-synaptic receptors, and when light is detected glutamate release pauses. How a rod’s individual ribbon enables this process was studied here by recording evoked changes in whole-cell membrane capacitance from wild type and ribbonless (RIBEYE-ko) rods. Wild type rods created a readily releasable pool (RRP) of 92 synaptic vesicles (SVs) that emptied as a single kinetic phase with a τ < 0.4 msec. Lowering intracellular Ca2+-buffering accelerated Cav channel opening and facilitated release kinetics, but RRP size was unaltered. In contrast, ribbonless rods created an RRP of 24 SVs, and lacked Cav channel facilitation; however, Ca2+ channel-release coupling remained tight. The release deficits caused a sharp attenuation of rod-driven light responses measured from RIBEYE-ko mice. We conclude that the synaptic ribbon facilitates Ca2+-influx and establishes a large RRP of SVs.

Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

The readily-releasable pool dynamically regulates multivesicular release

The number of neurotransmitter-filled vesicles released into the synaptic cleft with each action potential dictates the reliability of synaptic transmission. Variability of this fundamental property provides diversity of synaptic function across brain regions, but the source of this variability is unclear. The prevailing view is that release of a single (univesicular release, UVR) or multiple vesicles (multivesicular release, MVR) reflects variability in vesicle release probability, a notion that is well-supported by the calcium-dependence of release mode. However, using mouse brain slices, we now demonstrate that the number of vesicles released is regulated by the size of the readily-releasable pool, upstream of vesicle release probability. Our results point to a model wherein protein kinase A and its vesicle-associated target, synapsin, dynamically control release site occupancy to dictate the number of vesicles released without altering release probability. Together these findings define molecular mechanisms that control MVR and functional diversity of synaptic signaling.