New insights from 22-kHz ultrasonic vocalizations to characterize fear responses: relationship with respiration and brain oscillatory dynamics

Abstract

Fear behavior depends on interactions between the medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA), and the expression of fear involves synchronized activity in theta and gamma oscillatory activities. In addition, freezing, the most classical measure of fear response in rodents, temporally coincides with the development of sustained 4-Hz oscillations in prefrontalamygdala circuits. Interestingly, these oscillations were recently shown to depend on the animal's respiratory rhythm, supporting the growing body of evidence pinpointing the influence of nasal breathing on brain rhythms. During fearful states, rats also emit 22-kHz ultrasonic vocalizations (USV) which drastically affect respiratory rhythm. However, the relationship between 22-kHz USV, respiration and brain oscillatory activities is still unknown. Yet such information is crucial for a comprehensive understanding of how the different components of fear response collectively modulate rat's brain neural dynamics. Here we trained male rats in an odor fear conditioning task, while recording simultaneously local field potentials in BLA, mPFC and olfactory piriform cortex, together with USV calls and respiration. We show that USV calls coincide with an increase in delta and gamma power and a decrease in theta power. In addition, during USV emission in contrast to silent freezing, there is no coupling between respiratory rate and delta frequency, and the modulation of fast oscillations amplitude relative to the phase of respiration is modified. We propose that sequences of USV calls could result in a differential gating of information within the network of structures sustaining fear behavior, thus potentially modulating fear expression/memory. Significance StatementWhile freezing is the most frequently used measure of fear, it is only one amongst the different components of rodents' response to threatening events. USV is another index that gives additional insight into the socioemotional status of an individual. Our study is the first to describe the effects of USV production on rat's brain oscillatory activities in the fear neural network, and to relate some of them to changes in nasal breathing. A better knowledge of the impact of USV calls on brain neural dynamics is not only important for understanding the respective weight of the different components of fear response, but is also particularly relevant for rodent models of human neuropsychiatric disorders, for which socio-affective communication is severely impaired.

Respiration and brain neural dynamics associated with interval timing during odor fear learning in rats

“…However, others have found that a 4Hz oscillation develops in the mPFC during freezing behavior and that this oscillation was driven by respiration (Moberly et al, 2016;Karalis et al, 2016;Bagur and Benchenane, 2018). In line with these studies, we recently showed that respiratory frequency and delta activity frequency were highly correlated during freezing (Dupin et al, 2019). It could be hypothesized that the respiration-triggered 4Hz oscillation, when it occurs, is particularly well suited to modulate the amplitude of theta and higher frequency oscillations.…”

“…At the behavioral level, Bad Timers showed more USV emissions during the two training sessions. USV emission is known to narrow and lower the respiratory frequency range (Frysztak and Neafsey, 1991;Hegoburu et al, 2011;Dupin et al, 2019), which might have impeded the development of clear-cut temporal patterns. In the present study, all the animals except two (out of 30) produced USVs albeit x x at different rates.…”

The deep and slow breathing characterizing rest favors brain respiratory-drive

“…Individual data of LFP-respiration coupling index as a function of inhalation amplitude or duration (Fig.7) emphasized the strong and systematic coupling of this structure with respiration. PFC has been previously shown to be very well tuned to the 4 Hz respiratory frequency during animal freezing [38][39][40][41] . We show here, as we evoked in Dupin et al (2019), that the strong tuning of PFC with respiratory rhythm holds whatever the respiratory frequency.…”

“…PFC has been previously shown to be very well tuned to the 4 Hz respiratory frequency during animal freezing [38][39][40][41] . We show here, as we evoked in Dupin et al (2019), that the strong tuning of PFC with respiratory rhythm holds whatever the respiratory frequency. This suggests that PFC could function as a hub structure, able to tune its activity to that of different areas according to the respiration frequency.…”

“…To study frequency-frequency coupling between LFP signals and respiration, we designed a homemade method that allowed tracking instantaneous frequency synchrony 41 . To do so, for each detected respiratory cycle, the frequency was estimated as 1/cycle duration and the time course of the instantaneous respiratory frequency was extracted.…”