Is your dog happy to see you when she wags her tail? Is your cat content because he is “making biscuits” or kneading your head? Is your bird annoyed because she just squawked?

Like Charles Darwin, most people have no trouble believing that animals have emotions. Darwin believed that emotions were universal, inherited, and reflective of internal physiological states, providing evidence of these ideas throughout his book, “The Expression of the Emotions in Man and Animals,” published in 1872. While Darwin’s peers did not necessarily support his initial ideas about emotions in non-human animals, his ideas have spurred numerous investigations in contemporary times.

From the “facial feedback hypothesis” (discussed in a previous post) to the influence of culture on emotional expression despite the shared evolutionary history of those expressions, research about emotions continues to be a hot topic across human and non-human animals. Although humans may have difficulty identifying the emotional state of their dogs at times, human adults and children can categorize different vocalizations (sounds of fear or positive emotion) by macaques (a small monkey) fairly accurately and birds (among many other animals, including dogs) can categorize emotional expressions on human faces appropriately. Rats, one of our most important animal models in behavioral neuroscience, “love” to be tickled and emit ultrasonic vocalizations similar to laughter, as the late Dr. Jaak Panksepp discovered and spent a large part of his career devoted to understanding this phenomenon.

It’s No Laughing Matter

In 1950, former American Psychological Association President, Dr. Frank Beach warned us to beware that “The Snark was a Boojum” and the field of comparative psychology, which is so critical to many aspects of psychology, would “softly and suddenly vanish[es] away” as Lewis Carroll wrote in his story, “Your snark be a boojum”. Although it has been 75 years (eek!) since Beach’s cautionary tale and 153 years since Darwin published his ideas, the best efforts of scientists from all backgrounds have yet to create a cohesive theory of emotion.

Disagreement remains on the definition of emotion, the etiology of emotions, the evolutionary history of basic emotions, the construct of basic emotions itself, etc. Yet despite the difficulties surrounding the measurement and framing of emotions, research progresses, albeit in small increments.

“Fears are nothing more than a state of mind.” – Napoleon Hill

Researchers from Fujian Normal University in China undertook the next challenge of distinguishing between the subjective feeling of fear and the objective behavioral and physiological changes associated with fear conditioning in rats. Grounded in translational science (using animal models to learn about human conditions), B. Zheng and his collaborators (pictured below) capitalized on findings from their previous research involving a meta-analysis derived from a systematic review of fear conditioning in rodents to develop their clever study published in the Psychonomic Society’s Learning & Behavior.

Some Critical Assumptions

In the current study, the researchers made two assumptions:

(1) typically measured fear responses in rodents, such as freezing in rats, may reflect a defensive response of behavior rather than a subjective “affective” experience, and

(2) ultrasonic vocalizations by rodents during different conditioning paradigms, such as fear conditioning, may represent cognitively complex, subjective “affective” responses, such as “fear” (i.e., 22 kHz) or “pleasure” (i.e., 50 kHz), depending on the acoustic properties.

The researchers hypothesized that these two indicators are distinguished by their thresholds – the physiological freezing response is triggered at lower stimulus thresholds (e.g., shock). In contrast, the “affective” vocalization is triggered at a higher intensity and greater specificity threshold.

Working from these assumptions, the researchers designed two experiments using a classical conditioning-based fear conditioning paradigm. In the first experiment, the researchers manipulated the characteristics of the two stimuli being associated: the Unconditioned Stimulus (US), which was a foot shock, and the stimulus to be conditioned (CS), a 9 kHz tone.

As seen in the top part of the figure below, the researchers manipulated both the intensity of the foot shock (0.5-0.9 mA) given and the duration of the tone (5-80 sec), creating 8 different conditions (control, standard fear conditioning, weak fear conditioning, strong fear conditioning, short learning, long learning, short test, and long test).

The bottom part of the figure illustrates the sequence of experimental phases received by each of the groups of rats.

Males are Predictable

As an aside, the researchers fear conditioned 128 male Sprague-Dawley (SD) rats for Experiment 1 and 40 male SD rats categorized into high and low anxiety groups from an Open Field Test for Experiment 2. Despite the current practice of including males and females in experimental studies, the current study only assessed male rats. While this choice was unexpected, the researchers justified their protocol with existing research. Research had shown that female rats were much more variable in their vocalization use during fear conditioning than male rats. Thus, excluding female rats from the current study limited the possibility of a floor effect and a potential confound.

Mismatches are the Key

The results of the first experiment supported the expected results – shock intensity and duration of CS affected the intensity of fear conditioning, but more importantly, the expression of freezing behavior and vocalizations were dependent on the conditions during cue tests. A mismatch in US intensity and CS duration between the fear learning acquisition phase and the cue testing phase resulted in a decoupling of the two measured responses – freezing and 22 kHz vocalizations, which the researchers proposed was evidence for two different responses.

The figure below summarizes the responses of the rats to each test trial (CS1, CS2, CS3) over the course of the fear learning trials (top panel A & B) and during testing (bottom panel C & D). Each line represents a different shock intensity (i.e., green is weak fear conditioning with the lowest level of shock, yellow is typical shock level, and red is strong shock levels).

As shown in panel A, freezing occurred most frequently during the last test trial (CS3) and differed from the control group (black line). In contrast, under the strongest shock conditions during fear conditioning, 22 kHz vocalizations differed between the weak conditioning and control groups for CS1 and CS2 trials (panel B). The true test was during the cue test, in which freezing (panel C) behavior was significantly higher for the strongest shock group (red line), while the standard (yellow line) and the strongest shock group (red line) showed significantly more ultrasonic vocalizations.

As the researchers stated in their conclusion, 22-kHz USVs were more responsive to manipulations of intensity and duration of threat than freezing, but the USVs were slower to emerge in periods of ambiguous information.

Level of Anxiety Matters

These findings were replicated in Experiment 2 with one difference – high anxiety rats (HA rows in the figure below) emitted more 22-kHz USVs (green lines in the figure below) than low anxiety rats (LA rows). This pattern was observed for all CS presentations (thick vertical gray segments). That is, both HA and LA groups showed elevated freezing levels (orange lines) following all CS presentations across each shock group (designated by the borders) and between fear learning and final cue tests, but differed in their USVs.

Responses over time for low anxiety (LA) and high anxiety (HA) rat groups under different conditions.

One intriguing follow-up finding the researchers mentioned was that the fear conditioning in this study corresponded to a reduction in 50-kHz ultrasonic vocalizations (USVs), which are pleasure sounds for rats, over time. This suppression of these sounds may be indicative of an alternative affective measure of fear, with its absence being the meaningful measure.

As the researchers stated:

“Our study reveals that in rodent fear conditioning paradigms, while freezing reflects an automatic defensive reaction in rodents, 22‑kHz ultrasonic vocalizations capture a subjective fear component—offering a new, multi‐dimensional perspective for improving the translational relevance of rodent fear models.”

Although further work is needed on the neural circuitry underlying these two different mechanisms of fear expression before the findings can be translated to human conditions of anxiety and fear, this study offers a novel paradigm to begin separating the behavioral and affective responses to fear. We are also reminded to continue to beware of snarks that are boojums so that critical studies such as these translational studies continue and do not “softly and suddenly vanish[es] away.”

Featured Psychonomic Society Article

Zheng, B., Rao, J., Bao, L., Yu, D., & Yin, B. (2025). Differential modulation of freezing and 22-kHz USVs by shock intensity, tone-duration matching, and anxiety levels in rodent fear-conditioning paradigms. Learning & Behavior, 1-23. https://doi.org/10.3758/s13420-025-00671-6