Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.     

Smoothing of the GB1 Hairpin Folding Landscape by Interfacial Confinement

We study the effects of confinement between planar walls on the folding thermodynamics of a β-hairpin, using large-scale replica-exchange molecular-dynamics simulations with an all-atom model and explicit solvent. We find that the folding free-energy landscape of this peptide observed in bulk is significantly modified when the peptide is confined between the walls. Most notably, the propensity of the peptide to form a misfolded state observed in the bulk solution becomes negligible under confinement. The absence of the misfolded state under confinement can be explained by an increased tendency of hydrophobic aromatic side chains to stay near the walls, because the misfolded state is characterized by a nonnative arrangement of aromatic side chains. These results from a simple confinement model may provide clues about the role of chaperonin confinement in smoothing folding landscapes by avoiding trapped intermediates.     

Local transmission processes and disease-driven host extinctions

Classic infectious disease theory assumes that transmission depends on either the global density of the parasite (for directly transmitted diseases) or its global frequency (for sexually transmitted diseases). One important implication of this dichotomy is that parasite-driven host extinction is only predicted under frequency-dependent transmission. However, transmission is fundamentally a local process between individuals that is determined by their and/or their vector’s behaviour. We examine the implications of local transmission processes to the likelihood of disease-driven host extinction. Local density-dependent transmission can lead to parasite-driven extinction, but extinction is more likely under local frequency-dependent transmission and much more likely when there is active local searching behaviour. Density-dependent directly transmitted diseases spread locally can therefore lead to deterministic host extinction, but locally frequency-dependent passive vector-borne diseases are more likely to cause extinctions. However, it is active searching behaviour either by a vector or between sexual partners that is most likely to cause the host to go extinct. Our work emphasises that local processes are essential in determining parasite-driven extinctions, and the role of parasites in the extinction of rare species may have been underplayed due to the classic assumption of global density-dependent transmission.     

A two-state model for Ca2+/CaM-dependent protein kinase II (alphaCaMKII) in response to persistent Ca2+ stimulation in hippocampal neurons

Persistent elevation of the intracellular free Ca(2+) concentration [Ca(2+)](i) is neurotoxic and therefore it is important to understand how it affects downstream components of the Ca(2+) signaling pathway. The response of calmodulin (CaM) and alphaCa(2+)/CaM-dependent protein kinase II (alphaCaMKII), to intracellular Ca(2+) overload in hippocampal neurons is studied by confocal imaging of fluorescently tagged proteins. Transient and persistent redistribution of CaM and alphaCaMKII together is seen from the cytosol to dendritic and somatic punctae. Typical persistent redistribution occurs following a lag of 138+/-(S.E.M.) 12 s and is complete at 460+/-(S.E.M.) 34 s (n=18), lack of Thr(286)-autophosphorylation of alphaCaMKII however promotes the formation of early transient punctae (peak at 40 s). In contrast, the T286D-mimick of phospho-Thr(286)-alphaCaMKII forms punctae with a delay >10 min, indicating that Thr(286)-autophosphorylation is antagonistic to CaMKII clustering. A two-state model is proposed in which phospho-Thr(286)-alphaCaMKII, formed immediately upon Ca(2+) stimulation, is primarily responsible for target interactions and memory functions of alphaCaMKII. However, a distinct clustering form denoted alphaCaMKII(c), generated upon persistent intracellular free Ca(2+) elevation, is deposited in the punctae which are made of self-interacting CaM/CaMKII complexes. Punctate deposition disables both the interactions and the activity of CaMKII.     

Sdmg1 is a conserved transmembrane protein associated with germ cell sex determination and germline-soma interactions in mice

In mammals, the supporting cell lineage in an embryonic gonad communicates the sex-determining decision to various sexually dimorphic cell types in the developing embryo, including the germ cells. However, the molecular nature of the sex-determining signals that pass from the supporting cells to the germ cells is not well understood. We have identified a conserved transmembrane protein, Sdmg1, owing to its male-specific expression in mouse embryonic gonads. Sdmg1 is expressed in the Sertoli cells of embryonic testes from 12.5 dpc, and in granulosa cells of growing follicles in adult ovaries. In Sertoli cells, Sdmg1 is localised to endosomes, and knock-down of Sdmg1 in Sertoli cell lines causes mis-localisation of the secretory SNARE Stx2 and defects in membrane trafficking. Upregulation of Sdmg1 appears to be part of a larger programme of changes to membrane trafficking pathways in embryonic Sertoli cells, and perturbing secretion in male embryonic gonads in organ culture causes male-to-female germ cell sex reversal. These data suggest that changes that occur in the cell biology of embryonic Sertoli cells may facilitate the communication of male sex-determining decisions to the germ cells during embryonic development.     

α4β2-Nicotinic Receptor Binding with 5-IA in Alzheimer’s Disease: Methods of Scan Analysis

Five patients with Alzheimer’s disease and five healthy volunteers were examined by SPECT with the nicotinic receptor ligand ¹²³I-5-IA-85380. Patients were scanned before and after 6 weeks of treatment with donepezil. Quantification by regions of interest was reliable and the optimal normalisation procedure used cerebellar ratios. We found relative reductions in 5-IA binding capacity in patients in thalamus, frontal and central regions of interest of approximately one standard deviation unit (Cohen’s d = 1). Reductions in binding after treatment with the acetylcholinesterase inhibitor donepezil of the same magnitude occurred in the brain stem. The study was clearly too small to confirm group differences, but it suggests that 5-IA can be used to examine both group differences and treatment effects in patients with Alzheimer’s disease. Special issue article in honor of George Fink.