Table 1 |.

Approaches used to define itch-specific neuropeptides and neurons

Manipulation	Criteria	Advantages	Disadvantages
Assessment of behavioural correlates of itch
Loss of neuropeptide function in mice (via genetic KO or siRNA-mediated knockdown)	Impaired itch behaviour and normal pain behaviour	Direct evidence Sensory neuron-specific KO is feasible	Compensation: multiple related neuropeptides and receptors Time consuming to generate double-KO or triple-KO mice and CKO mice Spinally restricted CKO may not be feasible
Chemogenetic or optogenetic inhibition or viral, chemical or intersectional genetic ablation of neurons	Impaired itch behaviour and normal pain behaviour	Spinally restricted inhibition/ablation possible Sensory neuron-specific inhibition/ablation possible Ablation can be fast and efficient	Low level of fidelity of a Cre line Neurotoxin approach may have off-target effects Most acute pain behaviour cannot be appropriately evaluated after cervical inhibition or ablation Genetic compensation Indirect effects
Loss of neuropeptide function via pharmacological approaches	Impaired itch behaviour and normal pain behaviour	Acute and convenient Agonists may be available Dose-dependent scratching behaviour can be assessed	Off-target effects Potential presynaptic effects originating in primary afferents Pharmacological artefacts
Gain of neuropeptide function via pharmacological approaches	Induced itch behaviour and normal pain behaviour	Acute and convenient Agonists may be available Dose-dependent scratching behaviour can be assessed	Off-target effects Potential presynaptic effects originating in primary afferents Pharmacological artefacts Ceiling or floor effects
Chemogenetic or optogenetic activation of neurons	Induced itch behaviour and normal pain behaviour	Spinal cervical cord-restricted activation Validation using BB-sap possible Sensory neuron-specific activation of central or peripheral fibres possible	Low level of fidelity of a Cre line Chemogenetic or optogenetic artefacts Artificial behaviour can only suggest the capacity of fibres and is not necessarily an indicator of an endogenous function of manipulated neurons
Assessment of neural correlates of itch
Electrophysiological recording of neuronal responses to chemical itch, pain, cooling or scratching	Inhibited by pain stimuli	Single-cell resolution Cell-type recording	Low level of fidelity of Cre or eGFP mouse lines Lack of specificity of some chemicals (e.g. capsaicin) May not simulate neuropeptide release in freely behaving animals

BB-sap, bombesin–saporin; CKO, conditional knockout; eGFP, enhanced GFP; KO, knockout; siRNA, small interfering RNA.