Comparing behaviours induced by natural memory retrieval and optogenetic reactivation of an engram ensemble in mice

Abstract

Memories are thought to be stored within sparse collections of neurons known as engram ensembles. Neurons active during a training episode are allocated to an engram ensemble ('engram neurons'). Memory retrieval is initiated by external sensory or internal cues present at the time of training reactivating engram neurons. Interestingly, optogenetic reactivation of engram ensemble neurons alone in the absence of external sensory cues is sufficient to induce behaviour consistent with memory retrieval in mice. However, there may exist differences between the behaviours induced by natural retrieval cues or artificial engram reactivation. Here, we compared two defensive behaviours (freezing and the syllable structure of ultrasonic vocalizations, USVs) induced by sensory cues present at training (natural memory retrieval) and optogenetic engram ensemble reactivation (artificial memory retrieval) in a threat conditioning paradigm in the same mice. During natural memory recall, we observed a strong positive correlation between freezing levels and distinct USV syllable features (characterized by an unsupervised algorithm, MUPET (Mouse Ultrasonic Profile ExTraction)). Moreover, we observed strikingly similar behavioural profiles in terms of freezing and USV characteristics between natural memory recall and artificial memory recall in the absence of sensory retrieval cues. Although our analysis focused on two behavioural measures of threat memory (freezing and USV characteristics), these results underscore the similarities between threat memory recall triggered naturally and through optogenetic reactivation of engram ensembles. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Keywords: amygdala; engram; mouse; optogenetic reactivation; threat memory; ultrasonic vocalizations.

Figure 1.

Natural (CS-induced) and artificial (optogenetic reactivation of neurons in an engram ensemble) conditioned threat memory retrieval induce similar freezing and USV behaviour. To gain access to neurons in an engram ensemble, we used an allocation strategy that took advantage of a viral vector (HSV-NpACY) encoding both a blue light (BL)-sensitive excitatory (ChR2) and red light (RL)-sensitive inhibitory (eNpHR3.0) opsin and enhanced yellow fluorescent protein (eYFP). This allowed us to use BL to increase the excitability of NpACY+ neurons to bias their allocation to an engram ensemble. We could then manipulate the activity of these engram neurons with either BL or RL. (a) Schematic of the viral construct (left) and sparse virus expression (eYFP) (right) in principal (excitatory) neurons in the lateral nucleus of the amygdala (LA). (b) Allocation and subsequent optogenetic activation of engram neurons. Immediately before threat training (tone CS + footshock), NpACY+ neurons were photostimulated with BL to increase their excitability and bias their allocation into the engram ensemble supporting this memory. Memory (artificial and natural) was assessed over 2 days. First, artificial memory retrieval was assessed by placing mice in a novel context and reactivating engram (NpACY+) neurons with BL (opto reactivation). Second, natural memory retrieval was assessed by placing mice in a second novel context during which the CS tone was presented twice, first when engram ensembles were silenced with RL and then without RL (tone test). This allowed us to assess whether the activity of NpACY+ neurons was indeed critical for natural memory retrieval. Ultrasonic vocalizations (USVs) were recorded with a cage mate for a 5 min period 1 day before training (pre-training recording), immediately after training (post-training recording) and after each test (post-test tone recording, post-test opto recording). (c ) Comparison of freezing behaviour of the same mice during artificial and natural memory retrieval. During opto reactivation test, mice froze at low levels in a novel context but froze at high levels when neurons in the engram ensemble were reactivated with BL. In the tone test, mice froze at high level to the tone but at low levels when engram neurons were silenced with RL, showing the necessity of engram neurons to natural memory retrieval. (d) High positive correlation between freezing behaviour induced by natural memory retrieval (tone test) and optogenetic reactivation of neurons in the engram ensemble (opto reactivation) in individual mice. (e) Comparison of USVs across sessions (pre-training, post-training, post-test opto and post-test tone) of the same mice: (i) power spectral density (PSD, measurement of the power content of the USVs over different frequencies from 25 kHz to 125 kHz) over sessions. Inset depicts higher magnification and heat map over 20–30 kHz range showing differences between sessions; (ii) time to the first call (s) across sessions; (iii) number of USV syllables across sessions; (iv) total syllable activity (s) across sessions; and (v) syllable duration (ms) across sessions. (f) Correlation between each USV characteristic and freezing behaviour during natural (test tone, grey) or artificial memory (opto test, blue) test: (i) and (ii) high positive correlation between PSD in 20–30 kHz range (i) and time to make the first call (ii) and tone freezing in natural memory test; (iii), (iv) and (v) negative correlation between the number of syllables (iii) and time in syllables (iv) and syllable duration (v) and tone freezing; (vi) and (vii) high positive correlation between PSD in 20–30 kHz range (vi) and time to make the first call (vii) and optogenetic reactivation of engram neuron-induced freezing; (viii), (ix), (x) negative correlation between the number of syllables (viii) and time in syllables (ix) and syllable duration (x) and optogenetic reactivation of engram neuron-induced freezing. Importantly, we observed a similar pattern of behaviours with natural and artificial memory across these two different behaviours.