Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

The hippocampus is assumed to retrieve memory by reinstating patterns of cortical activity that were observed during learning. To test this idea, we monitored the activity of individual cortical neurons while simultaneously inactivating the hippocampus. Neurons that were active during context fear conditioning were tagged with the long-lasting fluorescent protein H2B-GFP and the light-activated proton pump ArchT. These proteins allowed us to identify encoding neurons several days after learning and silence them with laser stimulation. When tagged CA1 cells were silenced, we found that memory retrieval was impaired and representations in the cortex (entorhinal, retrosplenial, perirhinal) and the amygdala could not be reactivated. Importantly, hippocampal inactivation did not alter the total amount of activity in most brain regions. Instead, it selectively prevented neurons that were active during learning from being reactivated during retrieval. These data provide functional evidence that the hippocampus reactivates specific memory representations during retrieval.

Retrieving a recently formed context fear memory reactivates neurons in the hippocampus, amygdala, and cortex. Several weeks after learning, the degree of reactivation is altered in hippocampal and amygdala networks but remains stable in the cortex.

Episodic memories are encoded by a sparse population of hippocampal neurons. In mice, optogenetic manipulation of this memory engram established that these neurons are indispensable and inducing for memory recall. However, little is known about their in vivo activity or precise role in memory. We found that during memory encoding, only a fraction of CA1 place cells function as engram neurons, distinguished by firing repetitive bursts paced at the theta frequency. During memory recall, these neurons remained highly context specific, yet demonstrated preferential remapping of their place fields. These data demonstrate a dissociation of precise spatial coding and contextual indexing by distinct hippocampal ensembles and suggest that the hippocampal engram serves as an index of memory content.