Continuing the search for the engram: examining the mechanism of fear memories

Abstract

The goal of my research is to gain insight using rodent models into the fundamental molecular, cellular and systems that make up the base of memory formation. My work focuses on fear memories. Aberrant fear and/or anxiety may be at the heart of many psychiatric disorders. In this article, I review the results of my research group; these results show that particular neurons in the lateral amygdala, a brain region important for fear, are specifically involved in particular fear memories. We started by showing that the transcription factor CREB (cAMP/Ca(2+) response element binding protein) plays a key role in the formation of fear memories. Next, we used viral vectors to overexpress CREB in a subset of lateral amygdala neurons. This not only facilitated fear memory formation but also "drove" the memory into the neurons with relatively increased CREB function. Finally, we showed that selective ablation of the neurons overexpressing CREB in the lateral amygdala selectively erased the fear memory. These findings are the first to show disruption of a specific memory by disrupting select neurons within a distributed network.

Fig. 1

Schematic diagram depicting the strategy for selectively ablating neurons using inducible diphtheria toxin receptor (iDTR) transgenic mice. The mice express a simian DTR transgene. Expression of the DTR transgene is suppressed by a STOP cassette, which is floxed and removed when a neuron is infected with herpes simplex virus expressing cre recombinase (cAMP/Ca²⁺ response element binding protein [CREB]–cre vector or control-cre vector). Only neurons that have undergone cre-induced recombination will constitutively express DTR on the cell surface. At any point thereafter, systemic injection of diphtheria toxin (DT) induces apoptosis only in cells expressing DTR. In this way, only neurons infected with CREB-cre vector or Cntrl-cre vector will be ablated.

Fig. 2

Neurons with relatively increased CREB (cAMP/Ca²⁺ response element binding protein) function are essential for memory recall. (A) Schematic diagram of an experiment designed to test whether neurons with increased CREB function at the time of training are required for subsequent memory recall. (i) A subset of lateral amygdale (LA) neurons are infected with a CREB-cre vector (labelled green via green fluorescent protein [GFP]), which increases CREB in these neurons and induces them to undergo cre-induced recombination to express diphtheria toxin receptor (DTR). (ii) Mice are trained. A subset of LA neurons have high CREB levels, but because diphtheria toxin (DT) has not been administered, there is no cell death. (iii) Mice are tested. Neurons involved in the memory trace are depicted in red. Neurons with increased CREB out-compete their neighbours for inclusion in the fear memory trace (labelled yellow [green+red]). (iv) Mice receive systemic injections of DT to induce cell death only in the cells that express DTR (have undergone cre-induced recombination and, therefore, must have been infected with the CREB-cre vector). Mice are tested again. Neurons that previously had high CREB (infected) are now ablated. (B) Results from this experiment indicate that selectively ablating neurons with increased CREB at the time of training impairs subsequent memory recall. The CREB-cre group shows that increasing CREB function in a portion of neurons at the time of training enhances memory (Test 1) and subsequent ablation of these particular neurons completely reverses this enhancement (Test 2). The Cntrl-cre group show that GFP does not enhance memory and that selectively ablating these cells does not change this. The off-target group shows that increasing CREB function in neurons outside the LA does not enhance memory. The CREB alone (no cre) group shows that increasing CREB in the LA enhances memory (Test 1) and that DT injection (with no recombination) does not reverse this enhancement Test 2). The mice that received phosphate-buffered saline but no DT (PBS, no DT) show that CREB enhances memory in inducible DTR mice, and a systemic injection of PBS (rather than DT) does not change this on the second test day.