Question 1

What is fear conditioning and how is freezing used to measure it?

Accepted Answer

Fear conditioning is a Pavlovian (classical) learning paradigm in which an initially neutral stimulus (the conditioned stimulus, CS — typically a tone) is paired with an aversive stimulus (the unconditioned stimulus, US — typically a mild foot shock). After one or more pairings, the CS alone elicits a defensive freezing response. Freezing — defined as the complete absence of all movement except respiration — is the primary behavioral measure of conditioned fear in rodents. The percent of time an animal spends frozen during a given epoch directly indexes the strength of the fear memory.

Question 2

What velocity threshold defines freezing?

Accepted Answer

Freezing is defined as movement velocity below a set threshold for a minimum continuous duration. The conventional threshold is 0.05 cm/s (i.e., the animal is essentially motionless) sustained for at least 1 second. These values come from validation studies comparing automated scoring to expert hand-scoring (Anagnostaras et al., 2000). Some systems use pixel-change thresholds instead of velocity, but the principle is the same: movement below a near-zero cutoff for a minimum bout duration. This calculator allows you to adjust both the velocity threshold and minimum bout duration to match your scoring system.

Question 3

What are epochs in a fear conditioning session?

Accepted Answer

A fear conditioning session is divided into epochs — defined time windows corresponding to different phases of the protocol. Common epochs include: Baseline (pre-CS habituation period, typically 120 s), CS (conditioned stimulus presentation, typically 20-30 s), ITI (inter-trial interval between CS-US pairings), and post-US recovery periods. During a context test, the entire session is typically one long epoch (e.g., 5 minutes in the training context with no CS). During a cued test, epochs include a baseline period in a novel context, then CS-on and CS-off periods. Analyzing freezing per epoch reveals whether fear is specific to the CS (cued fear) or the context (contextual fear).

Question 4

What is the difference between contextual and cued fear conditioning?

Accepted Answer

Contextual fear conditioning refers to freezing triggered by the training environment itself (the chamber, odors, lighting, floor texture), and is measured by returning the animal to the training context without any CS or US. It depends on the hippocampus and is sensitive to hippocampal lesions. Cued fear conditioning refers to freezing triggered by the specific CS (e.g., a tone), measured in a novel context where baseline freezing should be low. It depends on the amygdala and is relatively hippocampus-independent. A complete fear conditioning experiment typically tests both, allowing dissociation of hippocampal and amygdalar contributions to fear memory.

Question 5

What is a freezing bout and why does bout duration matter?

Accepted Answer

A freezing bout is a single continuous episode of freezing — the period from when the animal becomes immobile until it resumes movement. While percent freezing tells you the total proportion of time frozen, bout analysis reveals the temporal structure: many short bouts (fragmented freezing) vs. fewer long bouts (sustained freezing) can indicate different fear states. Mean bout duration, bout count, and latency to first freeze are standard bout-level metrics. A minimum bout duration (typically 1 second) is enforced to filter out brief pauses that are not true freezing responses.

Question 6

How do I set up epoch definitions for my protocol?

Accepted Answer

Define epochs by specifying a name and start/end times for each phase. For a standard 3-CS training protocol, epochs might be: Baseline (0-120 s), CS1 (120-150 s), ITI1 (150-240 s), CS2 (240-270 s), ITI2 (270-360 s), CS3 (360-390 s), Post-US (390-480 s). For a context test: Context (0-300 s). For a cued test: Pre-CS Baseline (0-120 s), CS-On (120-240 s). This calculator provides preset protocols for common designs, or you can define custom epochs. Epochs must not overlap and should cover the session duration.

Question 7

Can I use this calculator with pre-scored freezing data instead of velocity traces?

Accepted Answer

Yes. The calculator offers two input modes. In velocity trace mode, you upload a CSV with time and velocity columns, and the tool detects freezing frame-by-frame using your threshold settings. In pre-scored mode, you enter freezing time and total time for each epoch for each animal directly — useful when freezing has already been scored by hand or by another software system (e.g., Video Freeze, FreezeFrame, or manual scoring from video). Both modes compute percent freezing per epoch and group statistics.

Fear Conditioning % Freezing Calculator.

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Freezing Detection Settings

Session Epochs

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Calibrate your freezing threshold against manual scoring

Report epoch definitions explicitly in your methods

Pre-CS baseline in cued test should be low

Distinguish immediate vs. 24-hour fear memory

Account for locomotor effects of drug treatments

Resources

Fear Conditioning Calibration Checklist

Method

Validated

How to cite

How to Cite

What Is Fear Conditioning?

Measuring Freezing: From Manual Scoring to Automated Detection

Session Epoch Design: Training, Context, and Cued Tests

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Epoch	Start (s)	End (s)	Duration
Baseline	0	120	120s
CS1	120	150	30s
ITI1	150	240	90s
CS2	240	270	30s
ITI2	270	360	90s
CS3	360	390	30s
Post-US	390	480	90s