Exploring Memory Representations with Activity-Based Genetics

Abstract

The brain is thought to represent specific memories through the activity of sparse and distributed neural ensembles. In this review, we examine the use of immediate early genes (IEGs), genes that are induced by neural activity, to specifically identify and genetically modify neurons activated naturally by environmental experience. Recent studies using this approach have identified cellular and molecular changes specific to neurons activated during learning relative to their inactive neighbors. By using opto- and chemogenetic regulators of neural activity, the neurons naturally recruited during learning can be artificially reactivated to directly test their role in coding external information. In contextual fear conditioning, artificial reactivation of learning-induced neural ensembles in the hippocampus or neocortex can substitute for the context itself. That is, artificial stimulation of these neurons can apparently cause the animals to "think" they are in the context. This represents a powerful approach to testing the principles by which the brain codes for the external world and how these circuits are modified with learning.

Figure 1.

Two systems for the genetic manipulation of active neural ensembles. (A) In this tetracycline (TET)-based system, two transgenes are required, a cfos promoter-driven tetracycline transactivator (tTA) and a tetracycline-responsive element (TRE) promoter-driven gene of interest. In the presence of doxycycline (Dox) the tTA is expressed in electrically active (cfos⁺) neurons but is prevented from activating expression of the gene of interest by the presence of Dox. In the absence of Dox, a window is opened during which active neurons that express tTA drive expression of the gene of interest from the TRE promoter. (B) This Cre-based system also uses two transgenes, a cfos promoter-driven CreER^t2 and a gene of interest that is flanked by loxP sites and positioned in an inverted orientation to any neuronal promoter (Pr). The loxP sites are arranged such that Cre activity will lead to a single inversion event of the flanked DNA. In the absence of tamoxifin (TAM) the Cre recombinase is inactive so that no recombination takes place even in active neurons. On administration of TAM, any active (cfos⁺) neurons will express the Cre, which is now active, and inverts the orientation of the gene of interest. This gene is then constitutively and permanently expressed from the neurons specific promoter.

Figure 2.

Manipulating contextual memory representations. This figure shows the arrangement of two sets of experiments to test the role of distributed neural ensembles in the coding of contextual memory. Both experiments use the cfos-tTA system discussed in Figure 1A to label neurons that are activated by exploration of a context (ctxA). In A, the active neurons express hM3Dq and in B, the neurons express ChR2. The animals are then fear conditioned in a distinct context (ctxB), whereas the ctxA neurons are activated with either clozapine N-oxide (CNO) or light, panels A and B, respectively. Memory retrieval is then tested in ctxA and ctxB. In panel A, the mice only show a fear response when the ctxA neurons are artificially activated while the animal is in ctxB, suggesting formation of a hybrid representation. In panel B, the animals show fear in ctxA even though they never received a shock in that context, suggesting that the artificial stimulation of the ChR2 positive neurons tagged in ctxA was able to substitute for (represent) that context. (Red circles) Neural ensembles expressing the genetic effector hM3Dq in A and ChR2 in B. (Blue circles) Neural ensembles naturally active by sensory input during training in ctxB.