Early changes in the properties of CA3 engram cells explored with a novel viral tool
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eLife Assessment
This important study characterizes and validates a new activity marker - fast labelling of engram neurons (FLEN) - which is transiently active and driven by cFos, allowing the monitoring of intrinsic and synaptic properties of engram neurons shortly after the learning experience. The results convincingly demonstrate the utility of this novel viral tool for studying early changes in the properties of engram cells. However, the study would benefit from exploring how accurately FLEN reflects endogenous cFos activity, how this labelling technique compares to previous versions, and from careful consideration of alternative explanations such as changes in release probability.
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Abstract
Forming new memories after a one-time experience requires initial encoding then consolidation over time. During learning, multimodal information converges onto the hippocampus, activating sparse neuronal assemblies which are thought to form a memory representation through concerted activity and synaptic interconnectivity. In this work, we use a novel tool for fast-labeling of engram neurons (FLEN). FLEN is based on c-Fos activity-dependent transient expression of a destabilized fluorescent marker ZsGreen1 rapidly after one-trial learning. With FLEN, we explore the electrophysiological properties of c-Fos activated CA3 pyramidal neurons a few hours following one-trial learning of an episodic-like memory. In parallel, we employ the Robust Activity Marker (RAM) system, which provides activity-dependent labelling 24 hours following a novel experience. Comparing FLEN+ and RAM+ neurons allows to characterize how the properties of neuronal assemblies evolve during an initial phase of consolidation. Whereas no difference was observed in the excitability of FLEN+ vs. FLEN-neurons, RAM+ neurons were more excitable than RAM-neurons. This suggests that CA3 pyramidal neurons recruited in an engram progressively acquire increased excitability as compared to neurons which were not activated by the one-trial contextual memory task. In contrast, FLEN+ CA3 neurons show an increased number of excitatory inputs. Overall, with the FLEN strategy, we can show that both the intrinsic excitability and the synaptic properties of CA3 pyramidal neurons undergo progressive plastic changes over the first day following a one-trial memory task.
Article activity feed
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eLife Assessment
This important study characterizes and validates a new activity marker - fast labelling of engram neurons (FLEN) - which is transiently active and driven by cFos, allowing the monitoring of intrinsic and synaptic properties of engram neurons shortly after the learning experience. The results convincingly demonstrate the utility of this novel viral tool for studying early changes in the properties of engram cells. However, the study would benefit from exploring how accurately FLEN reflects endogenous cFos activity, how this labelling technique compares to previous versions, and from careful consideration of alternative explanations such as changes in release probability.
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Reviewer #1 (Public review):
Summary:
The manuscript by Cupollilo et al describes the development, characterization, and application of a novel activity labeling system; fast labelling of engram neurons (FLEN). Several such systems already exist but this study adds additional capability by leveraging an activity marker that is destabilized (and thus temporally active) as well as being driven by the full-length promoter of cFos. The authors demonstrate the activity-dependent induction and time course of expression, first in cultured neurons and then in vivo in hippocampal CA3 neurons after one trial of contextual fear conditioning. In a series of ex vivo experiments, the authors perform patch clamp analysis of labeled neurons to determine if these putative engram neurons differ from non-labelled neurons using both the FLEN system as well …
Reviewer #1 (Public review):
Summary:
The manuscript by Cupollilo et al describes the development, characterization, and application of a novel activity labeling system; fast labelling of engram neurons (FLEN). Several such systems already exist but this study adds additional capability by leveraging an activity marker that is destabilized (and thus temporally active) as well as being driven by the full-length promoter of cFos. The authors demonstrate the activity-dependent induction and time course of expression, first in cultured neurons and then in vivo in hippocampal CA3 neurons after one trial of contextual fear conditioning. In a series of ex vivo experiments, the authors perform patch clamp analysis of labeled neurons to determine if these putative engram neurons differ from non-labelled neurons using both the FLEN system as well as the previously characterized RAM system. Interestingly the early labelled neurons at 3 h post CFC (FLEN+) demonstrated no differences in excitability whereas the RAM-labelled neurons at 24h after CFC had increased excitability. Examination of synaptic properties demonstrated an increase in sEPCS and mEPSC frequencies as well as those for sIPSCs and mIPSCs which was not due to a change in the mossy fiber input to these neurons.
Strengths:
Overall the data is of high quality and the study introduces a new tool while also reassessing some principles of circuit plasticity in the CA3 that have been the focus of prior studies.
Weaknesses:
No major weaknesses were noted.
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Reviewer #2 (Public review):
Summary:
Cupollilo et al. investigate the properties of hippocampal CA3 neurons that express the immediate early gene cFos in response to a single foot shock. They compare ex-vivo the electrophysiological properties of these "engram neurons" labeled with two different cFos promoter-driven green markers: Their new tool FLEN labels neurons 2-6 h after activity, while RAM contains additional enhancers and peaks considerably later (>24 h). Since the fraction of labeled CA3 cells is comparable with both constructs, it is assumed (but not tested) that they label the same population of activated neurons at different time points. Both FLEN+ and RAM+ neurons in CA3 receive more synaptic inputs compared to non-expressing control neurons, which could be a causal factor for cFos activation, or a very early consequence …
Reviewer #2 (Public review):
Summary:
Cupollilo et al. investigate the properties of hippocampal CA3 neurons that express the immediate early gene cFos in response to a single foot shock. They compare ex-vivo the electrophysiological properties of these "engram neurons" labeled with two different cFos promoter-driven green markers: Their new tool FLEN labels neurons 2-6 h after activity, while RAM contains additional enhancers and peaks considerably later (>24 h). Since the fraction of labeled CA3 cells is comparable with both constructs, it is assumed (but not tested) that they label the same population of activated neurons at different time points. Both FLEN+ and RAM+ neurons in CA3 receive more synaptic inputs compared to non-expressing control neurons, which could be a causal factor for cFos activation, or a very early consequence thereof. Frequency facilitation and E/I ratio of mossy fiber inputs were also tested, but are not different in both cFos+ groups of neurons. One day after foot shock, RAM+ neurons are more excitable than RAM- neurons, suggesting a slow increase in excitability as a major consequence of cFos activation.
Strengths:
The study is conducted to high standards and contributes significantly to our understanding of memory formation and consolidation in the hippocampus. Modifications of intrinsic neuronal properties seem to be more salient than overall changes in the total number of (excitatory and inhibitory) inputs, although a switch in the source of the synaptic inputs would not have been detected by the methods employed in this study
Weaknesses:
With regard to the new viral tool, a direct comparison between the new tool FLEN and existing cFos reporters is missing.
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