Retrograde targeting of CA1-Projecting Neurons in Rat Medial Entorhinal Cortex Superficial Layers

Angel Lopez1, Boris Zemelman1, Laura Colgin1

1 University of Texas at Austin, Center for Learning and Memory, Department of Neuroscience

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Cells in medial entorhinal cortex (MEC) layer III send direct projections to stratum lacunosum-moleculare in hippocampal subregion CA1. Previous studies have provided evidence that these direct MEC projections to CA1 are facilitated by fast gamma coupling between the regions. We are developing a method to drive fast gamma coupling between MEC and CA1 to investigate its potential functional significance.  A previous study in mouse brain slices showed that optogenetically stimulating MEC layer II/III neurons at theta frequency induced theta-nested fast gamma oscillations in MEC (Pastoll et al., 2013). We set out to develop analogous methods in vivo to determine whether optogenetically stimulating MEC at theta frequency drives theta-nested fast gamma in CA1 and improves memory operations in freely behaving rats. In order to label layer III MEC neurons that project to CA1, we injected a recently developed variant of an adeno-associated virus that has retrograde functionality into CA1. We then examined local and MEC virus-encoded fluorescent protein expression patterns. We found that more superficial injections produced local expression in CA1 stratum oriens and stratum pyramidale and also labeled presumed MEC layer Va neurons, as well as producing labeling in other unanticipated brain regions. In contrast, deeper injections that produced local expression in CA1 stratum lacunosum-moleculare and dentate gyrus resulted in more localized and robust labeling of neurons in MEC superficial layers. These data suggest that MEC superficial layer neurons are more effectively labeled by deep rAAV2R injections into the hippocampus than by more superficial hippocampal injections, and therefore deep injections will be used in our study testing optogenetic manipulation of MEC rhythms.