A contextual fear conditioning paradigm in head-fixed mice exploring virtual reality

Contextual fear conditioning is a classical laboratory task that tests
associative memory formation and recall. Techniques such as multi-photon
microscopy and holographic stimulation offer tremendous opportunities to
understand the neural underpinnings of these memories. However, these
techniques generally require animals to be head-fixed. There are few
paradigms that test contextual fear conditioning in head-fixed mice, and
none where the behavioral outcome following fear conditioning is freezing,
the most common measure of fear in freely moving animals. To address this
gap, we developed a contextual fear conditioning paradigm in head-fixed
mice using virtual reality (VR) environments. We designed an apparatus to
deliver tail shocks (unconditioned stimulus, US) while mice navigated a VR
environment (conditioned stimulus, CS). The acquisition of contextual fear
was tested when the mice were reintroduced to the shock-paired VR
environment the following day. We tested three different variations of
this paradigm and, in all of them, observed an increased conditioned fear
response characterized by increased freezing behavior. This was especially
prominent during the first trial in the shock-paired VR environment,
compared to a neutral environment where the mice received no shocks. Our
results demonstrate that head-fixed mice can be fear conditioned in VR,
discriminate between a feared and neutral VR context, and display freezing
as a conditioned response, similar to freely behaving animals.
Furthermore, using a two-photon microscope, we imaged from large
populations of hippocampal CA1 neurons before, during, and following
contextual fear conditioning. Our findings reconfirmed those from the
literature on freely moving animals, showing that CA1 place cells undergo
remapping and show narrower place fields following fear conditioning. Our
approach offers new opportunities to study the neural mechanisms
underlying the formation, recall, and extinction of contextual fear
memories. As the head-fixed preparation is compatible with multi-photon
microscopy and holographic stimulation, it enables long-term tracking and
manipulation of cells throughout distinct memory stages and provides
subcellular resolution for investigating axonal, dendritic, and synaptic
dynamics in real-time.