One of the fundamental questions of neuroscience is how the brain can create, store,
and retrieve memories. In the early part of the twentieth century, Karl Lashley attempted
to shed light on this question using cortical lesion studies. He concluded that complex
behavior relies on both local and distributed storage and retrieval mechanisms in
the brain. Lashley (1931) acknowledged that multiple, dispersed brain regions were
necessary to enable complex behavior, a principle he referred to as mass action of
cerebral function. The search for the mechanistic substrates of memory, what Richard
Semon called the “engram” has continued into the present day (Semon, 1921). Later,
Hebb (1949). pioneered the idea of neuronal ensembles, which he referred to as “cell
assemblies,” or small populations of sparsely distributed neurons active in response
to a specific salient stimulus. Another theory put forth by Hebb was the idea that
learning occurs via strengthening of synaptic connections between neurons, or synaptic
(Hebbian) plasticity. Hebb, Lashley, and their contemporaries had access to a limited
toolkit and relied mostly on lesion studies and clinical case studies to test their
theories. Fortunately, in the past two decades there has been a renaissance in tools
and technology available to identify, characterize, and manipulate neuronal ensembles
and engrams (Koya et al., 2009; Kim et al., 2011; Choi et al., 2018; DeNardo et al.,
2019; Matos et al., 2019). The advent of these tools has led to an explosion of research
that is beginning to uncover the cellular and molecular mechanisms by which memories
are encoded and retrieved. This Research Topic contains five papers that further our
understanding, both empirically and theoretically, of the cellular and molecular mechanisms
within neuronal ensembles that support the engram, using both established methods
and cutting-edge technology, as well as incorporating new statistical approaches.
Josselyn et al. previously demonstrated that neurons made more excitable prior to
fear conditioning are more likely to be allocated into a fear memory ensemble and
are preferentially reactivated during memory retrieval (Han et al., 2007; Yiu et al.,
2014). In this topic, Cho et al. demonstrate that neurons in the lateral amygdala
that are active during the formation of a fear memory ensemble are less likely to
be reactivated during memory recall and required for memory expression when retrieval
is preceded by retraining mice 24 h after initial fear conditioning. Interestingly,
when retraining occurred only 6 h after fear conditioning, the initial fear memory
ensemble remained essential for subsequent memory recall, suggesting a form of “co-allocation.”
The effect was specific to retraining; recall alone did not have the same effect.
Their findings imply that there may be separate allocation mechanisms that drive initial
learning and relearning. Sortman et al. use the Daun02 inactivation method in Fos-LacZ
transgenic rats to demonstrate that Fos-expressing neuronal ensembles in the prelimbic
cortex mediate both recently acquired and well-trained cocaine self-administration.
These findings build on their previous work showing a critical role for the self-administration
ensemble in cocaine seeking (Kane et al., 2021).
Koutlas et al. used a method known as targeted recombination in activated populations
(TRAP) by means of the TRAP2 transgenic mouse line (DeNardo et al., 2019) to identify
a neuronal ensemble within the VTA that is activated by social stress. The social
stress ensemble was a relatively small population (~11%) of cells, and heterogeneous
in terms of cell types. Furthermore, the authors discovered that the social stress-activated
neurons are more excitable than surrounding non-ensemble neurons, even when recorded
a month after the initial exposure to stress. Such persistent changes in excitability
are remarkable given that stress exposure was acute. Just one excellent example of
what we can begin to uncover with the availability of tools such as the TRAP2 system.
An interesting question for future research is to identify the molecular mechanism
by which excitability is heightened in the social stress ensemble neurons. Murthy
et al. employed the mammalian GFP reconstitution across synaptic partners (mGRASP)
system (Kim et al., 2011) to identify synaptic connections between activated CA1 and
CA3 ensemble neurons. They find that the Arc-TRAP system allows efficient synaptic
labeling compared to the Fos-tTA system, most likely driven by the difference in permanent
vs. transient labeling, respectively. The authors suggest that the possibility to
combine mGRASP technology with two-photon microscopy will enable longitudinal monitoring
of synapses between ensemble cells, bringing us closer to identifying the “synaptic
engram.” This advance allows ensemble and engram researchers to empirically test theories
about Hebbian synaptic plasticity and associative learning that have been part of
theoretical dogma for decades.
Finally, Körber and Sommer review studies on neuronal ensembles active in pursuit
of drug and non-drug rewards, identified using the immediate early gene c-fos. A particular
emphasis is placed on studies of alcohol-related seeking ensembles. The authors discuss
the use of graph theory-based network science to identify ensembles and to infer which
brain regions form higher order neuronal ensembles, or meta-ensembles. This approach
represents a major advance in our overall understanding of brain-wide ensemble dynamics
because it helps us to overcome the limitation of studying ensembles by characterizing
a single brain region at a time.
The broad range of brain regions, sensory modalities, types of learning and various
engram labeling methodologies displayed in this topic show that there are many approaches
to answering the essential question of how the brain can learn, store, and retrieve
information. An important topic for future research is to investigate the identity
and stability of the neurons that harbor engrams. For instance, are memories allocated
to dedicated neurons that are meant to act as information storage units from birth
onwards? If true, it will be relevant to determine which proportion of neurons in
the brain is dedicated for this purpose and whether these cells can be distinguished
based on their genetic, physiological, and morphological properties. Recent studies
demonstrate that after memory allocation, memory-supporting neuronal ensembles can
be stable over time. In particular, cortical ensembles remain essential for memory
expression several weeks after memory acquisition (Kitamura et al., 2017; Matos et
al., 2019; Visser et al., 2020). Insight into the molecular and cellular processes
that occur within these cells to consolidate and retrieve the memory is crucial for
understanding mechanisms that contribute to memory persistence. Vice versa, it is
of relevance to investigate which processes within neuronal ensembles mediate fading,
or forgetting, of memories. Enhanced synaptic strength has recently been described
between cortical ensemble neurons and disruption of this connectivity impaires remote
memory expression (Lee et al., 2023). This indicates that a lasting synaptic engram
supports memory retention, however, the molecular pathways supporting a synaptic engram
remain to be elucidated.
Furthermore, longitundinal imaging techniques, such as two-photon microscopy and calcium
imaging in freely moving animals, provide a plethora of possibilities to increase
our understanding of the spatiotemporal dynamics of neuronal ensembles and the relationship
between learning-induced neuronal activity and immediate early gene-based labeling
methods. For technical reasons, most of these studies have thus far been performed
in superficial brain structures, leaving important questions about deep brain structures
involved in information storage largely unaddressed. For instance, it is poorly understood
how reward-associated information is real-time processed by neuronal ensembles in
the striatum. Finally, although opto- and chemogenetic manipulation techniques have
provided invaluable evidence for the causal involvement of neuronal ensembles in memory
processing using animal models, it remains a challenge to translate ensemble interventions
to the clinic. With this objective in mind, it is also of utmost importance to acquire
detailed insight into the learning-induced physical adaptations (i.e., the engram)
that develop in memory-encoding neuronal ensembles. Specifically, identification of
neurobiological substrates that are unique to a specific engram, e.g., drug reward
or traumatic memory, holds promise for development of therapeutic intervention options.
Author contributions
LR, EK, and MO contributed to the writing of this editorial. All authors contributed
to the article and approved the submitted version.