Developmental milestones assessment in newborn mice (P1–P21)

Sarah van Veen; Emily Meeus

Apr 21, 2026

Developmental milestones assessment in newborn mice (P1–P21)

DOI

https://dx.doi.org/10.17504/protocols.io.rm7vz4d1rlx1/v1

Sarah van Veen^1,2,
Emily Meeus¹

¹KU Leuven;
²Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815

Sarah van Veen

KU Leuven

DOI: https://dx.doi.org/10.17504/protocols.io.rm7vz4d1rlx1/v1

Protocol Citation: Sarah van Veen, Emily Meeus 2026. Developmental milestones assessment in newborn mice (P1–P21). protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4d1rlx1/v1

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: April 21, 2026

Last Modified: April 21, 2026

Protocol Integer ID: 315402

Keywords: ASAPCRN, sensorimotor, neurobehavioral, reflex, developmental milestones, mouse, developmental milestones assessment in newborn mice, movements in newborn mice, mouse models of human neurodevelopmental disorder, newborn mice, developmental milestones assessment, human neurodevelopmental disorder, developmental sign, evaluating mouse model, assessing weight gain, p1 to p21, battery of behavioral test, behavioral test, p21, weight gain, p1

Funders Acknowledgements:

Aligning Science Across Parkinson's

Grant ID: ASAP-000458

Abstract

A battery of behavioral tests assessing weight gain, reflexes, and coordinated movements in newborn mice from P1 to P21. Designed for fast throughput of multiple litters daily. Measures focus on day of first appearance of each developmental sign. No specialized equipment required. Suitable for evaluating mouse models of human neurodevelopmental disorders.

Guidelines

Experimental cohorts are generated from heterozygous breedings. Wild-type (WT) and knockout (KO) littermates are analyzed within the same litters to control for maternal and environmental effects.

Test procedures
Each pup is evaluated on the tests appropriate for its postnatal age. Tests are repeated daily until the pup meets the acquisition criterion on two consecutive days. All behavioral testing and scoring is performed by experimenters blinded to genotype.

Materials

Top-loading balance
60 W lamp (supplemental overhead heat)
Stopwatch
Small bowl
Laboratory bench paper (test surface, cleaned between litters)
45° inclined mesh screen (negative geotaxis)
Pipette tip box (cliff aversion)
Cotton applicator sticks (rooting, ear twitch) — pull tip and twist into a fine filament
Small horizontal rod suspended over cage (forelimb grasp)
Plastic sheet with 13 cm diameter circle drawn (open field traversal)
Data recording sheet (one per pup; see Hill et al. for template)
Two observers (primary + secondary) per session

Safety warnings

Use litter means (not individual pup values) as the unit of statistical analysis to account for litter effects.

Ethics statement

Prior ethics approval should be obtained before performing these experiments.

Before start

Setup — daily testing
     Move the home cage into the testing room. Allow 1 hour of habituation before testing begins.
     Perform all testing at the same time each day to control for circadian influences.
     Place pups with a small amount of home cage nesting material in a small bowl, position a 60 W lamp overhead  to supplement heat from above.
     Weigh each pup individually before behavioral testing begins (record daily weight).
     Assign one observer to manipulate the pup and one to operate the stopwatch and record data.
     Tests are performed on laboratory bench paper (smooth side); replace or clean between litters.

Identification

On P1, weigh each pup and genotype it. 

Use a non-toxic marker or a small identification tattoo to reliably distinguish individual pups.

Determine and record sex of each pup.     

Surface righting

 Sensorimotor domain: Reflex — labyrinthine & body righting, strength, coordination

AB
SetupSmooth plastic sheet
StimulusHold pup supine (two fingers at head, two at hindquarters); release
ResponsePup flips onto abdomen with all four paws contacting the surface
MeasureLatency to righting (s)
Timeout / retry30 s
Acquisition criterionSuccessful righting within 30 s on two consecutive days
Start dayP1 onwards
 

Negative geotaxis

Sensorimotor domain: Reflex — labyrinthine, coordination
 
AB
Setup45° inclined mesh screen
StimulusPlace pup head-down on screen; release
ResponsePup rotates 180° to a head-up position
MeasureLatency to complete turn (s)
Timeout / retry30 s (repeat once if pup slips)
Acquisition criterionCompletion within 30 s on two consecutive days
Start dayP1 onwards
 

Cliff aversion

 Sensorimotor domain: Reflex — labyrinthine, strength, coordination

AB
SetupSmall box with smooth surface
StimulusPosition pup with forepaws and snout over platform edge; release
ResponsePup withdraws and turns away from the edge, both forepaws away from the edge
MeasureLatency to withdrawal (s)
Timeout / retry30 s (repeat once if pup slips)
Acquisition criterionSuccessful withdrawal within 30 s on two consecutive days
Start dayP1 onwards

Rooting reflex

Sensorimotor domain: Tactile reflex, motor coordination

AB
SetupCotton applicator — pull tip and twist into a fine filament
StimulusGently stroke filament front-to-back along the side of the head, 3x per side; repeat on contralateral side if no response
ResponseDirected head turn toward the stimulus
MeasurePresence/absence of response
Timeout / retryN/A
Acquisition criterionClear response on two consecutive days
Start dayP1 onwards
 

Forelimb grasp

Sensorimotor domain: Strength
 
AB
SetupSmall horizontal rod 
StimulusHold pup with forepaws resting on rod until it grasps; release
ResponsePup maintains grip on rod
MeasureLatency to release (s)
Timeout / retryRepeat once if pup falls immediately
Acquisition criterionMaintains grip for ≥1 s on two consecutive days
Start dayP4 onwards
 

Auditory startle

Sensorimotor domain: Auditory reflex
 
AB
SetupLaboratory bench
StimulusSingle sharp hand clap at ~10 cm from the pup
ResponseWhole-body startle/jump
MeasurePresence/absence of response
Timeout / retryN/A
Acquisition criterionConsistent startle response on two consecutive days
Start dayP8 onwards
 

Ear twitch reflex

Sensorimotor domain: Tactile reflex
 
AB
SetupCotton applicator — fine filament (as for rooting)
StimulusLightly brush filament against tip of ear 3×
ResponseEar flattening or twitching against side of head
MeasurePresence/absence of response
Timeout / retryN/A
Acquisition criterionReproducible response on two consecutive days
Start dayP8 onwards
 

Open field traversal

Sensorimotor domain: Locomotion — extinguishment of pivoting, limb coordination
 
AB
SetupPlastic sheet with 13 cm diameter circle drawn
StimulusPlace pup in center of circle; release
ResponsePup moves completely outside the circle
MeasureLatency to exit circle (s)
Timeout / retry30 s
Acquisition criterionExits circle within 30 s on two consecutive days
Start dayP10 onwards
 

Eye opening

Sensorimotor domain: Developmental milestone
 
AB
SetupVisual inspection under adequate lighting
StimulusN/A
ResponseEyes open
MeasureScore: 0 = no eyes open; 1 = one eye open; 2 = both eyes open
Timeout / retryN/A
Acquisition criterionScore 2 (both eyes open) recorded as day of milestone
Start dayP12 onwards
 

Air righting

Sensorimotor domain: Reflex — labyrinthine & body righting, coordination
 
AB
SetupLab bench with plastic sheet
StimulusHold pup upside down ~10–12 cm above lab bench (two fingers at head, two at hindquarters); release
ResponsePup rights mid-air and lands on all four paws
MeasurePresence/absence of correct landing
Timeout / retryN/A
Acquisition criterionCorrect landing on two consecutive days
Start dayP8 onwards
 

Statistical analysis

The unit of analysis is the litter mean, not the individual pup. For each milestone test, calculate the mean postnatal day of acquisition separately for WT and KO pups within each litter. Use these litter means as independent observations in all group comparisons.

Apply a nested two-tailed t test. Adjust p-values for multiple comparisons across all nine milestone tests using Bonferroni correction (multiply each raw p-value by 9; significance threshold α = 0.0056).

Protocol references

Hill JM, Lim MA, Stone MM. Developmental milestones in the newborn mouse. In: Gozes I, editor. Neuropeptide Techniques (Neuromethods, Vol. 39). Totowa, NJ: Humana Press; 2008. p. 131–149.

	A	B
	Setup	Smooth plastic sheet
	Stimulus	Hold pup supine (two fingers at head, two at hindquarters); release
	Response	Pup flips onto abdomen with all four paws contacting the surface
	Measure	Latency to righting (s)
	Timeout / retry	30 s
	Acquisition criterion	Successful righting within 30 s on two consecutive days
	Start day	P1 onwards

	A	B
	Setup	45° inclined mesh screen
	Stimulus	Place pup head-down on screen; release
	Response	Pup rotates 180° to a head-up position
	Measure	Latency to complete turn (s)
	Timeout / retry	30 s (repeat once if pup slips)
	Acquisition criterion	Completion within 30 s on two consecutive days
	Start day	P1 onwards

	A	B
	Setup	Small box with smooth surface
	Stimulus	Position pup with forepaws and snout over platform edge; release
	Response	Pup withdraws and turns away from the edge, both forepaws away from the edge
	Measure	Latency to withdrawal (s)
	Timeout / retry	30 s (repeat once if pup slips)
	Acquisition criterion	Successful withdrawal within 30 s on two consecutive days
	Start day	P1 onwards

	A	B
	Setup	Cotton applicator — pull tip and twist into a fine filament
	Stimulus	Gently stroke filament front-to-back along the side of the head, 3x per side; repeat on contralateral side if no response
	Response	Directed head turn toward the stimulus
	Measure	Presence/absence of response
	Timeout / retry	N/A
	Acquisition criterion	Clear response on two consecutive days
	Start day	P1 onwards

	A	B
	Setup	Small horizontal rod
	Stimulus	Hold pup with forepaws resting on rod until it grasps; release
	Response	Pup maintains grip on rod
	Measure	Latency to release (s)
	Timeout / retry	Repeat once if pup falls immediately
	Acquisition criterion	Maintains grip for ≥1 s on two consecutive days
	Start day	P4 onwards

	A	B
	Setup	Laboratory bench
	Stimulus	Single sharp hand clap at ~10 cm from the pup
	Response	Whole-body startle/jump
	Measure	Presence/absence of response
	Timeout / retry	N/A
	Acquisition criterion	Consistent startle response on two consecutive days
	Start day	P8 onwards

	A	B
	Setup	Cotton applicator — fine filament (as for rooting)
	Stimulus	Lightly brush filament against tip of ear 3×
	Response	Ear flattening or twitching against side of head
	Measure	Presence/absence of response
	Timeout / retry	N/A
	Acquisition criterion	Reproducible response on two consecutive days
	Start day	P8 onwards

	A	B
	Setup	Plastic sheet with 13 cm diameter circle drawn
	Stimulus	Place pup in center of circle; release
	Response	Pup moves completely outside the circle
	Measure	Latency to exit circle (s)
	Timeout / retry	30 s
	Acquisition criterion	Exits circle within 30 s on two consecutive days
	Start day	P10 onwards

	A	B
	Setup	Visual inspection under adequate lighting
	Stimulus	N/A
	Response	Eyes open
	Measure	Score: 0 = no eyes open; 1 = one eye open; 2 = both eyes open
	Timeout / retry	N/A
	Acquisition criterion	Score 2 (both eyes open) recorded as day of milestone
	Start day	P12 onwards

	A	B
	Setup	Lab bench with plastic sheet
	Stimulus	Hold pup upside down ~10–12 cm above lab bench (two fingers at head, two at hindquarters); release
	Response	Pup rights mid-air and lands on all four paws
	Measure	Presence/absence of correct landing
	Timeout / retry	N/A
	Acquisition criterion	Correct landing on two consecutive days
	Start day	P8 onwards