Synaptic Maps among Engram Cells underlie Memory

 

Science  27 Apr 2018: Vol. 360, Issue 6387, pp. 430-435

Interregional synaptic maps among engram cells underlie memory formation

Jun-Hyeok Choi, et.al.

School of Biological Sciences, Seoul National University, Gwanak-gu, Seoul 08826, South Korea.

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Memory resides in engram cells    distributed across the brain. However, the site-specific substrate within these engram cells remains theoretical, even though it is generally accepted that synaptic plasticity    encodes memories. We developed the dual-eGRASP (green fluorescent protein reconstitution across synaptic partners) technique to examine synapses between engram cells to identify the specific neuronal site for memory storage. We found an increased number and size of spines on CA1 engram cells    receiving input    from CA3 engram cells. In contextual fear conditioning, this enhanced connectivity between engram cells encoded memory strength. CA3 engram to CA1 engram projections strongly occluded long-term potentiation. These results indicate that enhanced structural and functional connectivity between engram cells across two directly connected brain regions forms the synaptic correlate for memory formation.

Memory storage and retrieval require specific populations of neurons that show increased neuronal activity during memory formation. Several studies identified these engram cells throughout various brain regions and demonstrated that activated engram cells can induce artificial retrieval of stored memories. To explain how memory is encoded in the engram, Hebb proposed a hypothetical mechanism, often paraphrased as “fire together, wire together”. This hypothesis suggests that synaptic strengthening between coactivated neurons forms the neural substrate of memory. However, it has not been possible to delineate whether memory formation enhances synapses between engram cells in connected brain regions because we could not distinguish presynaptic regions originating from engram cells and nonengram cells.

To compare two different presynaptic populations that project to a single postsynaptic neuron, we modified the green fluorescent protein (GFP) reconstitution across synaptic partners (GRASP) technique. GRASP uses two complementary mutant GFP fragments, which are expressed separately on presynaptic and postsynaptic membranes and reconstitute in the synaptic cleft to form functional GFP. This GFP signal indicates a formed synapse between the neuron expressing the presynaptic component and the neuron expressing the postsynaptic component. We developed an enhanced GRASP (eGRASP) technique, which exhibits increased GRASP signal intensity by introducing a weakly interacting domain that facilitates GFP reconstitution and a single mutation commonly found on most advanced GFP variants. We further evolved eGRASP to reconstitute cyan or yellow fluorescent protein. Placing the color-determining domain in the presynaptic neuron (cyan/yellow pre-eGRASP) and the common domain to the postsynaptic neuron (post-eGRASP) enabled visualization of the two synaptic populations that originated from two different presynaptic neuron populations and projected to a single postsynaptic neuron. We named this technique dual-eGRASP. We demonstrated that two colors reveal the contact interface in human embryonic kidney (HEK) 293T cells expressing the common domain with cells expressing either of the color-determining domains. We successfully applied this technique to synapses on dentate gyrus (DG) granule cells originating from either the lateral entorhinal cortex (LEC) or the medial entorhinal cortex (MEC) that projected to the outer and middle molecular layers of the DG, respectively.

Our finding that synaptic populations that fired together during memory formation showed the strongest connections demonstrates that classical Hebbian plasticity indeed occurs during the learning and memory process at CA3 to CA1 synapses. It is possible that cells with higher connectivity are allocated together into a memory circuit, in contrast to enhanced connectivity after learning. However, the allocated cell number remains constant regardless of the memory strength, whereas the connectivity is significantly enhanced with a stronger memory. This finding indicates a significant contribution of post-learning enhancement over the predetermined connectivity. The relationship between memory strength and synaptic connectivity suggests that these specific connections between engram cells across two directly connected brain regions form the synaptic substrate for memory.

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