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Little is understood about how engrams, sparse groups of neurons that store memories, are formed endogenously. Here, we combined calcium imaging, activity tagging, and optogenetics to examine the role of neuronal excitability and pre-existing functional connectivity on the allocation of mouse cornu ammonis area 1 (CA1) hippocampal neurons to an engram ensemble supporting a contextual threat memory. Engram neurons (high activity during recall or TRAP2-tagged during training) were more active than non-engram neurons 3 h (but not 24 h to 5 days) before training. Consistent with this, optogenetically inhibiting scFLARE2-tagged neurons active in homecage 3 h, but not 24 h, before conditioning disrupted memory retrieval, indicating that neurons with higher pre-training excitability were allocated to the engram. We also observed stable pre-configured functionally connected sub-ensembles of neurons whose activity cycled over days. Sub-ensembles that were more active before training were allocated to the engram, and their functional connectivity increased at training. Therefore, both neuronal excitability and pre-configured functional connectivity mediate allocation to an engram ensemble., Competing Interests: Declaration of interests S.A.J. is a member of the advisory board of Neuron., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The internal representation of an experience is thought to be encoded by long-lasting physical changes to the brain ("engrams") . Previously, we and others showed within the lateral amygdala (LA), a region critical for auditory conditioned fear, eligible neurons compete against one other for allocation to an engram. Neurons with relatively higher function of the transcription factor CREB were more likely to be allocated to the engram. In these studies, though, CREB function was artificially increased for several days before training. Precisely when increased CREB function is important for allocation remains an unanswered question. Here, we took advantage of a novel optogenetic tool (opto-DN-CREB) to gain spatial and temporal control of CREB function in freely behaving mice. We found increasing CREB function in a small, random population of LA principal neurons in the minutes, but not 24 h, before training was sufficient to enhance memory, likely because these neurons were preferentially allocated to the underlying engram. However, similarly increasing CREB activity in a small population of random LA neurons immediately after training disrupted subsequent memory retrieval, likely by disrupting the precise spatial and temporal patterns of offline post-training neuronal activity and/or function required for consolidation. These findings reveal the importance of the timing of CREB activity in regulating allocation and subsequent memory retrieval, and further, highlight the potential of optogenetic approaches to control protein function with temporal specificity in behaving animals.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Current approaches for optogenetic control of transcription do not mimic the activity of endogenous transcription factors, which act at numerous sites in the genome in a complex interplay with other factors. Optogenetic control of dominant negative versions of endogenous transcription factors provides a mechanism for mimicking the natural regulation of gene expression. Here we describe opto-DN-CREB, a blue-light-controlled inhibitor of the transcription factor CREB created by fusing the dominant negative inhibitor A-CREB to photoactive yellow protein (PYP). A light-driven conformational change in PYP prevents coiled-coil formation between A-CREB and CREB, thereby activating CREB. Optogenetic control of CREB function was characterized in vitro, in HEK293T cells, and in neurons where blue light enabled control of expression of the CREB targets NR4A2 and c-Fos. Dominant negative inhibitors exist for numerous transcription factors; linking these to optogenetic domains offers a general approach for spatiotemporal control of native transcriptional events., (Copyright © 2015 Elsevier Ltd. All rights reserved.)