Fura-2 Calcium Imaging Is a Valid Method of Investigating Signalling & Integrative Properties of the Subfornical Organ
Bibliographic record
Abstract
Research into the integration of signalling molecules at the subfornical organ (SFO) is typically conducted through in vitro electrophysiology methods on dissociated SFO neurons. We performed Fura-2 calcium imaging on dissociated SFO neurons and compared our observations to past electrophysiology data to determine if our imaging protocol can be used as an alternative, high-throughput method to investigate the integrative properties of the SFO. Sequential bath-application of 10-8 M cholecystokinin (CCK), a signalling molecule involved in energy balance, and 10-8 M Angiotensin-II (ANG), an excitatory signalling molecule involved in fluid balance, was performed on dissociated SFO neurons in hypoglycemic (1 mM glucose), normoglycemic (5 mM glucose), and hyperglycemic (10 mM glucose) conditions. ANG caused an increase in intracellular calcium ([Ca2+]i) in 32.8% (n = 20/61), 53.7% (n = 29/54) and 69.8% (n = 30/43) of SFO neurons at 1, 5 and 10 mM glucose, respectively, which is similar to the proportion of SFO neurons which depolarized to ANG in electrophysiology experiments [33.3% (n = 4/12) at 1 mM, 50.0% (n = 16/32) at 5mM, and 72.7% (n = 8/11) at 10 mM glucose]. These results suggest that ANG-related depolarizations and detectable [Ca2+]i increases are associated with each other. CCK caused an increase in [Ca2+]i in 31.1% (n = 19/61), 35.2% (n = 19/54), and 44.2% (n = 19/43) of SFO neurons at 1, 5 and 10 mM glucose, respectively, which is similar to the overall proportion of CCK-responsive neurons in electrophysiology experiments [41.2% (n = 7/17) at 1mM, 48.9% (n = 22/45) at 5 mM, and 47.6% (n = 10/21) at 10 mM glucose]. CCK elicits both excitatory and inhibitory responses, so our results suggest that increases in [Ca2+]i may be associated with both types of events. Lastly, we observed four subpopulations of SFO neurons: those who respond exclusively to ANG (n = 48/158), those who respond exclusively to CCK (n = 26/158), those who respond to both peptides (n = 31/158), and those who respond to neither peptide (n = 53/158). The observation of four subpopulations aligns with past electrophysiological data. Overall, our results suggest that SFO neurons respond to ANG and CCK similarly in imaging and electrophysiology trials and that Fura-2 calcium imaging is a reliable high-throughput method for researching signal transduction and the integrative properties of the SFO.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".