Tetanus toxin light chain (TeNT) silencing of PVH Gpr45 neurons

Stages

1. 1.
   Global Gpr45 loss-of-function phenotyping(functional_characterization)

   This stage establishes whether Gpr45 has a detectable role in energy balance before narrowing to specific cell types or brain regions.

   Selection: Assess whether whole-body Gpr45 disruption alters body weight, food intake, fat mass, energy expenditure, and body temperature.

2. 2.
   Cell-type-specific conditional deletion(secondary_characterization)

   This stage narrows the broad global phenotype to specific neuronal classes implicated in appetite regulation.

   Selection: Delete Gpr45 in Vglut2-positive, Vgat-positive, or Sim1-positive neurons and monitor body weight and food consumption.

3. 3.
   PVH-targeted regional deletion(secondary_characterization)

   This stage tests whether the PVH is a major anatomical locus for the obesity and hyperphagia phenotype.

   Selection: Inject AAV-Cre into the PVH of floxed Gpr45 mice and evaluate body weight and food intake.

4. 4.
   Direct manipulation of PVH Gpr45 neuronal activity(confirmatory_validation)

   This stage directly tests whether activity of PVH Gpr45 neurons can drive or suppress feeding phenotypes beyond receptor deletion alone.

   Selection: Use the Gpr45-CreERT2 knock-in to express chronic silencing, chronic activation, and acute chemogenetic actuators in the PVH and assess feeding and body-weight outcomes.

Steps

1. 1.
   Engineer and analyze global Gpr45 knockout mice

   Establish whether loss of Gpr45 produces an organism-level energy-balance phenotype.

   A whole-body loss-of-function test provides the broadest first-pass assessment of whether Gpr45 is relevant to obesity regulation.

2. 2.
   Breed floxed Gpr45 mice to Cre lines marking Vglut2, Vgat, or Sim1 populations

   Determine which neuronal populations mediate the obesity and hyperphagia phenotype.

   After a global phenotype is observed, cell-type-restricted deletion narrows the responsible neuronal classes.

3. 3.
   Inject AAV-Cre bilaterally into the PVH of floxed Gpr45 mice

   Test whether the PVH is a major anatomical site of Gpr45 action.

   Regional deletion follows cell-type narrowing to localize the phenotype to a specific hypothalamic nucleus.

4. 4.
   Use Gpr45-CreERT2 mice to express chronic and acute actuators in the PVHtargeting construct

   Directly manipulate PVH Gpr45 neuronal activity to test necessity and sufficiency for appetite control.

   After receptor deletion implicates PVH neurons, direct activity manipulation distinguishes circuit-activity effects from receptor-loss effects.

5. 5.
   Permanently silence PVH Gpr45 neurons with TeNTsilencing actuator

   Test whether PVH Gpr45 neuronal activity is required to restrain feeding and weight gain.

   A chronic silencing test follows targeting of the relevant neuronal population to assess necessity.

6. 6.
   Constitutively activate PVH Gpr45 neurons with NaChBacactivation actuator

   Test whether chronic activation of PVH Gpr45 neurons is sufficient to reduce feeding and body weight.

   A sufficiency test complements the necessity result from silencing.

7. 7.
   Acutely stimulate PVH Gpr45 neurons chemogenetically

   Test whether acute activation of PVH Gpr45 neurons can suppress feeding across motivational contexts.

   An acute perturbation can reveal immediate appetite-control capacity even when chronic constitutive activation shows limited effect.