Using Immunohistology to Investigate Cholinergic Fiber Density across Layers in the Medial Entorhinal Cortex

Acetylcholine is a neuromodulator that plays a key role in spatial navigation, learning and memory by modulating neurons in the hippocampus and medial entorhinal cortex (MEC). Acetylcholine released in the MEC is produced by cholinergic projection neurons located in the medial septum and diagonal band of Broca (MS). Acetylcholine is released at synaptic terminals in the MEC and via volume transmission to modulate activity of neurons at the individual and population level. Yet, the exact density of cholinergic projection fibers in different layers of the MEC has not been quantified. To address this question, we used a ChAT-Cre mouse and stereotaxic virus injection to transfect the cholinergic neurons in the MS with a conditional recombinant adeno- associated virus (AAV) coding for the green fluorescent protein (GFP). The GFP was only expressed in the cholinergic cells. After 14 days, we euthanized the mouse and performed a transcardiac perfusion procedure to preserve the brain in formalin. Then, we sliced the MS (coronally) and the MEC (sagittally). We processed the brain slices using immunohistology with anti-GFP antibodies. Following the staining, we imaged the brain slices using confocal microscopy and measured the fluorescence intensity in each of the six layers. The rationale for this approach was that layers with the highest fluorescence likely have the highest amount of cholinergic innervation. The results of this study will help us identify which neuron types and compartments in the MEC are preferentially targeted by acetylcholine. This is particularly interesting in the context of grid cells, a type of neurons in the MEC which fire in a grid-like pattern when an animal is navigating its space, providing a neuronal base for memory-guided spatial navigation.