The hippocampal dentate gyrus is critically involved in learning and memory. However, methods for imaging the activity of its principal neurons, the dentate gyrus granule cells, are missing. Here we demonstrate chronic two-photon imaging of granule cell population activity in awake mice using a cortical window implant that leaves the hippocampal formation intact and does not lead to obvious alteration of animal behavior. Using virus delivery, we targeted expression of genetically encoded calcium indicators specifically to dentate gyrus granule cells. Calcium imaging of granule cell activity 600 -800 m below the hippocampal surface was facilitated by using 1040 nm excitation of the red indicator R-CaMP1.07, but was also achieved using the green indicator GCaMP6s. We found that the rate of calcium transients was increased during wakefulness relative to an extremely low rate during anesthesia; however, activity still remained sparse with, on average, approximately one event per 2-5 min per cell across the granule cell population. Comparing periods of running on a ladder wheel and periods of resting, we furthermore identified state-dependent differences in the active granule cell population, with some cells displaying highest activity level during running and others during resting. Typically, cells did not maintain a clear state preference in their activity pattern across days. Our approach opens new avenues to elucidate granule cell function, plasticity mechanisms, and network computation in the adult dentate gyrus.

During navigation, rodents continually sample the environment with their whiskers. How locomotion modulates neuronal activity in somatosensory cortex, and how it is integrated with whisker-touch remains unclear. Here, we compared neuronal activity in layer 2/3 (L2/3) and L5 of barrel cortex using calcium imaging in mice running in a tactile virtual reality. Both layers increase their activity during running and concomitant whisking, in the absence of touch. Fewer neurons are modulated by whisking alone. Whereas L5 neurons respond transiently to wall-touch during running, L2/3 neurons show sustained activity. Consistently, neurons encoding running-with-touch are more abundant in L2/3 and they encode the run-speed better during touch. Few neurons across layers were also sensitive to abrupt perturbations of tactile flow during running. In summary, locomotion significantly enhances barrel cortex activity across layers with L5 neurons mainly reporting changes in touch conditions and L2/3 neurons continually integrating tactile stimuli with running.

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer’s disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase ( S -depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S -depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S -depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S -depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins.