Linking ultradian Growth Hormone secretion to the molecular structure of excitatory synapses in the vole hypothalamus
Notice bibliographique
Résumé
Growth hormone (GH) is a pituitary hormone that is secreted in a pulsatile manner multiple times within a 24-hour period, establishing an ultradian secretion rhythm. GH release is controlled by two hypothalamic neuropeptides, GH-releasing hormone (GHRH) produced in the arcuate nucleus (ARC) and somatostatin (SOM) produced in the anterior periventricular nucleus (PeVN). The former works to stimulate GH secretion while the latter works to inhibit GH secretion. Our laboratory has found that the number of clusters of PSD-95 increase at bodies of GHRH cells during peaks of GH secretion and decrease when the hormone levels go down (GH troughs). However, the mechanisms that orchestrate these rapid changes in the ARC of the hypothalamus remain largely unknown. Previous studies on GH circuitry in our laboratory have used mice as the experimental animal. However, due to inconsistencies in their GH secretion patterns, we found it difficult to obtain tissue samples that corresponded to the peak of GH secretion. Specifically, since peaks last only around 40 minutes and are followed by 2-hour trough periods, the ability to predict the timing of GH secretion becomes crucial to minimize the numbers of animals used. Keeping in line with with the 3R’s formulated by Drs. William Russell and Rex Burch to improve the welfare of animals in research, specifically in this case, reduction in the animals used to obtain information, we propose using the common vole (Microtus arvalis) as an experimental animal. Studies done on this species have shown evidence of group synchronicity in behaviour and show promise for their use in neuroendocrine research. We hypothesized that voles might exhibit synchronous ultradian secretion patterns in which peaks and troughs would occur at similar time points between individuals. During a pilot experiment, voles were unhabituated to the blood collection procedure and GH rhythms were detected for all 4 animals indicating a resiliency of GH secretion to stress that has not typically been observed in other rodents. Furthermore, our data suggests that group synchronicity in voles does not occur by chance with 67% - 100% of animals being synchronous in each group tested. However, when testing hormone pattern predictability, only 1 out of 4 voles was found to be in a GH peak, indicating that predictability of secretion patterns may still be more difficult than hypothesized.Previous studies in our lab have tested antibody compatibility in M. arvalis with great success in synaptic markers (e.g., VGAT, VGLUT2, PSD-95, and Gephyrin). Thus we decided to use single-domain antibodies (also known as nanobodies) procured from the Camelidae family which allows for better tissue penetration due to their small size (12-14 kDa compared to 150 kDa in traditional antibodies). Also, due to their small size, nanobodies directly labeled with fluorophores exhibit a 1:1:1 conjugation ratio. This allows for more accurate PSD-95 quantification, as well as more accurate counting of synaptic clusters. Since our lab had never used nanobodies before, we needed to set up a working protocol to get the best possible signal-to-noise-ratio (SNR) and resolution. From this, we found the best working concentration for the PSD-95 nanobody to be 1:1000 (2.5 μg/ml). Next, we optimized STED imaging parameters to achieve the highest possible resolution which allowed for clearer identification of synaptic puncta and allowed us to count PSD-95 clusters in GHRH cells using a strong analysis tool: Statistical Object Distance Analysis (SODA) which uses strong statistical analysis such as complete spatial randomness, to assess coupling of synaptic partners in a more accurate and biologically significant way
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,001 |
| Communication savante | 0,001 | 0,001 |
| Science ouverte | 0,001 | 0,001 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».