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Label-free flavoprotein imaging of the mouse cortex as a behaviour-independent assay for touch, pain and itch (iced)

Can label-free, through-skull flavoprotein autofluorescence imaging of the mouse cortex measure brain responses to touch, capsaicin-induced pain and histamine-induced itch, as an alternative to mouse behavioural assays?

Source: Label-free neuroimaging in mice captures sensory activity in response to tactile stimuli and acute pain (doi:10.57844/arcadia-b963-15ac). Path record: arcadia-neuroimaging-path.json. Headline outcome partial. Touch produced localized contralateral somatosensory signals and capsaicin produced widespread oscillatory cortical activation absent in vehicle trials; histamine produced activation indistinguishable from saline and inconsistent between trials, so the itch application was iced. Abandonment reasons: technical-gap. All graphs · Back to the pilot

How to read these graphs

A discourse graph breaks a piece of research into typed one-sentence nodes and draws how they bear on one another. The grammar is that of Discourse Graphs (Chan and Akamatsu). Read each graph left to right: a question, the experiments that addressed it, the results they produced, and the hypothesis every result is drawn against.

QUE Question: What the work set out to answer.
HYP Hypothesis: The answer the authors expected. Every result earns one edge against it.
EXP Experiment: One attempt at the question: a trial arm, an assay, a survey, an analysis.
RES Result: One finding from an experiment, with where in the source it comes from.
ISS Issue: An open question or untried branch the work leaves for someone else.

addresses: An experiment addresses the question.
produces: An experiment produces a result.
supports: A positive result that backs the hypothesis.
opposes: A negative result that counts as evidence against the hypothesis, or a refutation of an earlier claim.
informs: A partial or mixed result that bears on the hypothesis without settling it.
consistent with: A negative or inconclusive result that does not count either way: no positive control passed, or there is no sensitivity or power statement, so the absence of an effect cannot be told from a measurement that failed.
relation: A thin dotted line: a link the source itself states between two experiments, such as one being derived from another.

A dashed box marks a result the rule above counts as uninformative, or an issue. In the drawing, click a node to jump to its text below.

The graph

Read left to right. The question (QUE) is addressed by 3 experiments (EXP), each of which produces results (RES), and each result bears on the hypothesis (HYP) with one of four edges. 6 support it, 0 oppose it, 0 inform it, and 3 are merely consistent with it because the informativeness rule would not count them as evidence either way. Click a node to jump to its full text below.

supports positive finding
opposes informative negative, or refutation
informs partial finding
consistent with inconclusive or uninformative: no positive control passed, or no sensitivity statement
thin grey a relation the record itself states between two attempts
dashed box a result the informativeness rule marks uninformative, or an issue

The grammar is that of Discourse Graphs (Chan and Akamatsu): QUE, HYP, EXP, RES and ISS nodes with supports, opposes and informs edges. Every node and edge is derived from fields already in the Negative-Results Record; the edge a finding earns is decided by its outcomeClass and informativeness. Issues (ISS) hang below the graph as a strip: the open questions and untried branches the path leaves for someone else to pick up.

Nodes

Question and hypothesis

QUE: Can label-free, through-skull flavoprotein autofluorescence imaging of the mouse cortex measure brain responses to touch, capsaicin-induced pain and histamine-induced itch, as an alternative to mouse behavioural assays?
HYP: Determine whether in vivo flavoprotein autofluorescence imaging can measure brain responses to touch, capsaicin-induced pain and histamine-induced itch, as a behaviour-independent alternative to mouse behavioural assays.

Experiments (3 attempts)

Node	Attempt	Type	Headline	JSON
EXP	Intradermal capsaicin produces widespread oscillatory cortical activation absent from paired vehicle trials widespread, high-intensity cortical activation for ~10 minutes after injection, absent in vehicle trials; complex and oscillatory; nape injection produced briefer activity confined to the somatosensory area of one hemisphere	`wet-lab`	`positive`	record
EXP	Histamine-induced itch produces no cortical signal separable from saline injection (iced for a technical gap) Histamine and saline both produced widespread bilateral activation of similar magnitude, inconsistent between trials and not localized. The positive control passed; the negative control did not stay clean, so the experiment cannot say whether an itch signal exists.	`wet-lab`	`inconclusive-controls-failed`	record
EXP	Vibrotactile stimulation produces localized contralateral somatosensory autofluorescence (assay validation) Contralateral, somatotopically appropriate responses for right hindlimb, left hindlimb and right forelimb stimulation; a 1-3% increase in autofluorescence above baseline after averaging 50 stimulations.	`wet-lab`	`positive`	record

Results (9 findings)

Node	Finding	Id	Outcome and informativeness	Edge	JSON
RES	Does intradermal capsaicin in the left hindlimb produce cortical activity distinguishable from its paired vehicle injection? target: capsaicin; whole-brain dF/F after injection versus paired vehicle trial = widespread high-intensity oscillatory cortical activation for ~10 min (n = 1)	`capsaicin-lhl` in arcadia-neuroimaging-capsaicin	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does intradermal capsaicin in the right hindlimb produce cortical activity distinguishable from its paired vehicle injection? target: capsaicin; whole-brain dF/F after injection versus paired vehicle trial = widespread oscillatory cortical activation less intense than the previously vehicle-injected limb (n = 1)	`capsaicin-rhl` in arcadia-neuroimaging-capsaicin	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does intradermal capsaicin in the nape of the neck produce cortical activity distinguishable from its paired vehicle injection? target: capsaicin; whole-brain dF/F after injection versus paired vehicle trial = brief oscillatory activation confined to the somatosensory area of one hemisphere (n = 1)	`capsaicin-nape` in arcadia-neuroimaging-capsaicin	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does intradermal histamine in the left hindlimb produce cortical activity distinguishable from its paired saline injection? target: histamine dihydrochloride; whole-brain dF/F after injection versus paired saline trial = widespread bilateral autofluorescence above baseline; magnitude similar to saline; not localized; inconsistent between trials (n = 1)	`histamine-lhl` in arcadia-neuroimaging-histamine	`inconclusive-controls-failed` uninformative negative control not clean; no sensitivity or power statement	consistent-with HYP	record
RES	Does intradermal histamine in the right hindlimb produce cortical activity distinguishable from its paired saline injection? target: histamine dihydrochloride; whole-brain dF/F after injection versus paired saline trial = widespread bilateral autofluorescence above baseline; magnitude similar to saline; not localized; inconsistent between trials (n = 1)	`histamine-rhl` in arcadia-neuroimaging-histamine	`inconclusive-controls-failed` uninformative negative control not clean; no sensitivity or power statement	consistent-with HYP	record
RES	Does histamine-induced itch produce cortical activity detectable by label-free flavoprotein autofluorescence imaging in anaesthetized mice? target: histamine-induced itch, cortical response; consistency and localisation of cortical activation across histamine trials = no consistent or localized activation; magnitude similar to saline control (n = 2)	`histamine-overall` in arcadia-neuroimaging-histamine	`inconclusive-controls-failed` uninformative negative control not clean; no sensitivity or power statement	consistent-with HYP	record
RES	Does vibrating stimulation of the right hindlimb produce a localized autofluorescence increase in the expected contralateral somatosensory area? target: right hindlimb vibrotactile stimulation; peak dF/F above pre-stimulus baseline, stimulus-triggered average over 50 stimulations = 1-3% % (n = 1)	`tactile-rhl` in arcadia-neuroimaging-tactile	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does vibrating stimulation of the left hindlimb produce a localized autofluorescence increase in the expected contralateral somatosensory area? target: left hindlimb vibrotactile stimulation; peak dF/F above pre-stimulus baseline, stimulus-triggered average over 50 stimulations = 1-3% % (n = 1)	`tactile-lhl` in arcadia-neuroimaging-tactile	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record
RES	Does vibrating stimulation of the right forelimb produce a localized autofluorescence increase in the expected contralateral somatosensory area? target: right forelimb vibrotactile stimulation; peak dF/F above pre-stimulus baseline, stimulus-triggered average over 50 stimulations = 1-3% % (n = 1)	`tactile-rfl` in arcadia-neuroimaging-tactile	`positive` not-applicable rule applies to negative and inconclusive findings only	supports HYP	record

Relations between attempts

arcadia-neuroimaging-capsaicin wasDerivedFrom arcadia-neuroimaging-tactile the tactile response gated entry to these trials
arcadia-neuroimaging-histamine wasDerivedFrom arcadia-neuroimaging-tactile the tactile response gated entry to these trials
arcadia-neuroimaging-histamine comparedWith arcadia-neuroimaging-capsaicin the same method and animals detected capsaicin-induced pain

Issues (8 open questions and untried branches)

ISS Why did we see no reproducible activity in response to itch? (open-question)
ISS How could we improve the methodology to enable this measurement? (open-question)
ISS Repeat the itch experiments with a sensitive genetically encoded calcium indicator such as GCaMP to increase signal, testing the hypothesis that too few itch-responsive neurons produce too little autofluorescence to detect above noise. (proposed-by-authors)
ISS Perform the experiments on non-anesthetized animals, because anesthesia is known to suppress cortical activity (ref 33). (proposed-by-authors)
ISS Repeat the capsaicin experiments to distinguish activity patterns inherent to the brain from those caused by experimental variability, and determine what injection location, dose and prior-injection factors contribute to the oscillations. (proposed-by-authors)
ISS Test analgesic drugs to see whether they alleviate pain-related sensory activity in the brain, as a check on drug-screening applicability. (proposed-by-authors)
ISS Perform this imaging on chronic mouse pain and itch models rather than the acute models used here. (proposed-by-authors)
ISS Imaging session 2024-03-06 is deposited on Zenodo (20.2 GB, the largest single raw session) but is not cited by any figure or result in the pub; no analysis of it is reported. (data-deposited-not-analysed)