In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice

Previous studies found that the hippocampus is a brain structure essential for processing and retrieving memories^1,2. Since the 1950s, the neural circuits of the hippocampus in rodents have been a focus in studying memory formation, storage, and retrieval³. The anatomical structures within the hippocampus include the subregions of dentate gyrus (DG), CA1, CA2, CA3, CA4, and subiculum⁴. Complex bidirectional connections exist among these subregions, of which the DG, CA1, and CA3 form a prominent trisynaptic circuit that consists of granule cells and pyramidal cells⁵. This circuit receives its primary input from the entorhinal cortex (EC) and has been a classic model for studying synaptic plasticity. Prior in vivo research on the hippocampus function has mostly concentrated on the CA1^6,7 due to its easier access. While CA1 neurons serve important roles in memory formation, consolidation, and retrieval, particularly in place cells for spatial memory, other subregions of the hippocampus are also vital^8,9. In particular, recent studies have highlighted the functions of DG in memory formation. Place cells in DG have been reported to be more stable than those in CA1¹⁰, and their activities reflect context-specific information¹¹. Further, activity-dependent labeling of DG granule cells can be reactivated to induce memory-related behaviors¹². Therefore, to gain a deeper understanding of the information coding in DG, it is crucial to investigate the activities of the DG subregion while the animal is carrying out memory-dependent tasks.

Prior studies of DG activities have mostly used in vivo electrophysiology¹³. However, this technique has some drawbacks: First, in electrical recordings, it may be difficult to directly identify the various types of cells that are generating the signal. The recorded signals are from both inhibitory and excitatory cells. Therefore, further data processing methods are required to separate these two cell types. Moreover, it is difficult to combine other cell type information, such as projection-specific subgroups or activity-dependent labeling, with electric recordings. In addition, due to the anatomical morphology of DG, the recording electrodes are often implanted in an orthogonal direction, which greatly limits the number of neurons that can be recorded. Thus, it is difficult for electric recordings to achieve monitoring hundreds of individual neurons from the DG structure in the same animal¹⁴.

A complementary technique of recording neuron activities in DG is to use in vivo calcium imaging¹⁵. Calcium ions are fundamental to cellular signaling processes in organisms, playing a crucial role in many physiological functions, especially within the mammalian nervous system. When neurons are active, the intracellular calcium concentration increases rapidly, reflecting the dynamic nature of neuronal activity and signal transmission. Therefore, recording the real-time changes in intracellular calcium levels in neurons provides important insights into the neural coding mechanisms.

Calcium imaging technology utilizes specialized fluorescent dyes or genetically engineered calcium indicators (GECIs) to monitor calcium ion concentrations in neurons by detecting changes in fluorescence intensity, which can then be captured through microscopic imaging¹⁶. Commonly, the GCaMP family of calcium indicator genes, comprising green fluorescent protein (GFP), calmodulin, and M13 polypeptide sequences, are employed. GCaMP can emit green fluorescence when it binds to calcium ions¹⁷, allowing the fluctuations in green fluorescence to be recorded via imaging¹⁸. Additionally, to obtain clear images of the target brain region, a Gradient Index Lens (GRIN lens) is typically implanted above the region of interest. The GRIN lens enables imaging of the deep brain region that cannot be accessed directly from the surface.

This technique is relatively easy to combine with other genetic tools to label different cell types. Moreover, as the imaging plane is parallel to the cells' orientation in DG, hundreds of neurons are accessible for imaging with each successful surgery. In this work, we present a complete and detailed surgery protocol for in vivo calcium imaging in the dentate gyrus in mice (Figure 1). The procedure involves two major operations. The first one is to inject the AAV-CaMKIIα-GCaMP6f virus into the DG. The second operation is to implant a GRIN lens above the virus injection site. These two procedures are conducted in the same sitting. After recovery from these surgeries, the next step is to check the imaging quality with miniaturized microscopes (miniscopes). If the imaging field has hundreds of active cells, the subsequent procedure is to attach the miniscope base plate onto the mouse skull using dental cement; the mouse can then be used for imaging experiments. We also present a MATLAB-based preprocessing pipeline for streamlining the analysis of the collected calcium data.