Authors :
Presenting Author: Emmie Banks, BA – Emory University
Claire-Anne Gutekunst, PhD – Department of Neurosurgery – Emory University; Anna Eaton, High School Diploma – Department of Biomedical Engineering – Georgia Institute of Technology; Geoffrey Vargish, PhD – Section on Cellular and Synaptic Physiology – National Institute of Health; Kenneth Pelkey, PhD – Section on Cellular and Synaptic Physiology – National Institute of Health; Chris McBain, PhD – Section on Cellular and Synaptic Physiology – National Institute of Health; James Zheng, PhD – Department of Cell Biology – Emory University; Viktor Olah, PhD – Department of Cell Biology – Emory University; Matthew Rowan, PhD – Department of Cell Biology – Emory University
Rationale:
The mammalian brain contains the most diverse array of cell types of any organ, including dozens of neuronal subtypes with distinct anatomical and functional characteristics. Through the use of Cre lines, access to specific neuron types has steadily improved over past decades. Despite their extraordinary utility, development and cross-breeding of Cre lines is time-consuming and expensive, presenting a significant barrier to entry for many investigators. Furthermore, cell-based therapeutics developed in Cre mice are not clinically translatable. To overcome these limitations, several adeno-associated viral (AAV) vectors utilizing neuron-type-specific regulatory transcriptional sequences (enhancer-AAVs) were recently developed to target specific excitatory neuron types in the cortex (Graybuck et al., 2021). However, no such vector tool has been investigated which allows for selective targeting of distinct excitatory neuron types in the hippocampus.
Methods:
Using a publicly available RNAseq dataset, we first evaluated the potential of several recently identified regulatory enhancer sequences for neuron-type-specific targeting in the hippocampus. We identified a candidate regulatory enhancer with apparent selectivity for one particular excitatory neuron type in the hippocampus, dentate gyrus (DG) granule cells, and packaged this sequence into an AAV. We then stereotaxically injected this enhancer-AAV into the DG of C57BL/6J mice to drive fluorophore expression in a neuron-type-selective manner.
Results: We found that this novel enhancer-driven AAV targeting method selectively targeted DG granule cells, including their mossy fiber axons and dendritic processes, without the need for a Cre-based genetic model system. Other nearby excitatory cell types (e.g., CA3 pyramidal cells and hilar mossy cells) were not targeted despite their close proximity to the DG.
Conclusions: Here we identified a promising enhancer-AAV for targeting DG granule cells in the hippocampus, and validated its selectivity in wild-type adult mice. As the DG has been proposed as a locus for seizure activity, incorporating functional constructs (e.g., opsins, DREADDs) into this enhancer-AAV to manipulate DG granule cell activity may be a promising area of future research as an intervention for epilepsy. Thus, this enhancer-AAV approach should allow for rapid deployment of different constructs to independently modulate DG granule cell activity in the hippocampus across different mouse disease models. This method also has the translational potential for human use, as the cell-type-specificity of several enhancer-viral approaches has been shown to scale well from rodent models to mature human brain tissue.
Funding: The current work was supported by these grants: 1R56AG072473 (MJR), 1RF1AG079269 (MJR).