Bidirectional fear modulation by discrete anterior insular circuits in mice

Reviewer #1 (Public Review):

The authors sought to test whether anterior insular cortex neurons increase or decrease firing during fear behavior and freezing, bi-directionally control fear via separate, anatomically defined outputs. Using a fairly simple behavior where mice were exposed to tone-shock pairings, they found roughly equal populations that do indeed either increase or decrease firing during freezing. Next, they sought to test whether these distinct populations may also have distinct outputs. Using retrograde tracers they found that the anterior insular cortex contains non-overlapping neurons which project to the mediodorsal thalamus or amygdala. Mediodorsal thalamus-projecting neurons tended to cluster in deep cortical layers while amygdala-projecting neurons were primarily in more superficial layers. Stimulation of insula-thalamus projection decreased freezing behavior, and stimulation of insula-amygdala projections increased fear behavior. Given that the neurons that increased firing were located in deep layers, that thalamus projections occurred in deep layers, and that stimulation of insula-thalamus neurons decreased freezing, the authors concluded that the increased firing neurons may be thalamus projections. Similarly, given that decreased-firing neurons tended to occur in more superficial layers, that insula-amygdala projections were primarily superficial, and that insula-amygdala stimulation increased freezing behavior, authors concluded that the decreased firing cells may be amygdala projections. The study has several strengths though also some caveats.

Strengths:

The potential link between physiological activity, anatomy, and behavior is well laid out and is an interesting question. The activity contrast between the units that increase/decrease firing during freezing is clear.

It is nice to see the recording of extracellular spiking activity, which provides a clear measure of neural output, whereas similar studies often use bulk calcium imaging, a signal that rarely matches real neural activity even when anatomy suggests it might (see London et al 2018 J Neuro - there are increased/decreased spiking striatal populations, but both D1 and D2 striatal neurons increase bulk calcium).

Weaknesses:

The link between spiking, anatomy, and behavior requires assumptions/inferences: the anatomically/genetically defined neurons which had distinct outputs and opposite behavioral effects can only be assumed the increased/decreased spiking neurons, based on the rough area of the cortical layer they were recorded.

The behavior would require more control to fully support claims about the associative nature of the fear response (see Trott et al 2022 eLife) - freezing, in this case, could just as well be nonassociative. In a similar vein, fixed intertrial intervals, though common practice in the fear literature, pose a problem for neurophysiological studies. The first is that animals learn the timing of events, and the second is that neural activity is dynamic and changes over time. Thus it is very difficult to determine whether changes in neural activity are due to learning about the tone-shock contingency, timing of the task, simply occur because of time and independently of external events, or some combination of the above.

Reviewer #2 (Public Review):

In this study, the authors aim to understand how neurons in the anterior insular cortex (insula) modulate fear behaviors. They report that the activity of a subpopulation of insula neurons is positively correlated with freezing behaviors, while the activity of another subpopulation of neurons is negatively correlated to the same freezing episodes. They then used optogenetics and showed that activation of anterior insula excitatory neurons during tones predicting a footshock increases the amount of freezing outside the tone presentation, while optogenetic inhibition had no effect. Finally, they found that two neuronal projections of the anterior insula, one to the amygdala and another to the medial thalamus, are increasing and decreasing freezing behaviors respectively. While the study contains interesting and timely findings for our understanding of the mechanisms underlying fear, some points remain to be addressed.