Part 2. A synaptic mechanism of memory allocation
3. General discussion and perspectives
In the present work, the use of rodents together with tools for the remote control of neuronal activity (chemogenetic and optogenetics) during specific behavioral paradigms, revealed mechanisms that critically contribute to cellular engram formation.
We defined a new role of inhibitory neurotransmission in the selection of cellular engrams during hippocampal-dependent memory formation. To address this question, we used two different approaches applied to the DG of the hippocampus:
1. We studied different neuronal types responsible for GABAergic transmission.
2. We investigated the role of synaptic contacts that sense inhibitory neurotransmission.
Altogether, how the present study contributes to the current state-of-the-art of engram theory?
We have seen in the introduction that the theory of engram seems to have found already substantial evidence that sustain it (Tonegawa et al., 2015; Josselyn et al., 2015;
Eichenbaum, 2016). However, we also anticipated that many questions remain unanswered and different aspects of the cellular engrams need to be established and experimentally demonstrated. Some very instructive works suggest that cells are selected in the cellular engram based on their level of excitability and network activity (Han et al., 2007; Han et al., 2009; Yiu et al., 2014). However, a precise mechanism explaining how memory is allocated among individual neurons has not yet been revealed. This was exactly the aim of the present study where we could reveal two mechanisms that perform such function. We first revealed a network mechanism, a dendritic lateral inhibition among GCs mediated by SST+ INs, that determine the size of the cellular engram and the stability of the associated memory. In this first part, our results suggest that GCs are in competition between each other to become part of the cellular engram where active cells trigger SST+
INs activity that in turn prevent neighboring GCs to become part of it. Second, we showed that GABAergic synapses affect the size of the cellular engram and memory stability. This second mechanism, which seems to act mainly post-synaptically, suggests that GABAergic synapses are critical determinants for the formation of the cellular engram and affect memory function.
Altogether, we revealed a common phenomenon: the alteration of the size of the cellular engram that affects memory stability that was assessed by manipulating two different components of the inhibitory system of the DG: INs and GABAergic synapses.
What concerns arise and what are the missing gaps regarding studies on cellular engrams and the present study?
One of the major concern in studies investigating cellular engrams is the use of IEGs as neuronal markers. IEGs are one of the most used tools to study this question and their use has been demonstrated to be technically relevant and functional. Nevertheless, we should address some concerns about them.
First, what type of neuronal activity drives IEGs expression? According to the literature, there are only a few studies showing what exact pattern of activity lead to IEGs expression such as c-fos or Arc/Arg3.1. For example, we know that c-fos expression highly correlates to natural (Dragunow and Faull, 1989; Rakhade et al., 2007) or optogenetic induced neuronal firing (Schoenenberger et al., 2009). However, these studies could not provide extensive and robust evidence for the kind of activity necessary to induce c-fos expression. In our work, we revealed that optogenetic-induced neuronal firing at 10 Hz could induce c-fos expression in around 80% of the ChR2 expressing cells independently of their type (Figure 19D and 22B) suggesting that neuronal firing at this frequency could reliably induce c-fos expression. We could however not exclude that optogenetic induced back propagating action potentials as well as optogenetic-induced dendritic and membrane depolarization could alone or together induce c-fos expression.
Second, are IEGs expressed in the same way in different neuronal types involved in memory? When IEGs are used to identify recently activated neurons that belong to the cellular engram it is necessary to understand that c-fos expression may differ in different type of cells. There is consensus that neuronal activity induces c-fos expression in most of glutamatergic cells in many brain regions (Kovacs, 2008). However, it is known that other type of cells, especially INs, may have different signaling and cellular cascades that may differ in the expression of c-fos (Cohen et al., 2016). Indeed, in the present work, detection of activity-dependent induction of c-fos in PV+ and SST+ INs resulted to be less clear compared to excitatory neurons. For this reason, we used a complementary tool described in the results, a synaptic activity reporter (SARE), that permitted us to confirm the detection of recently activated PV+ and SST+ INs (Figure S4).
Third, is the temporal resolution of IEGs adequate to label cellular engram? Many agree about the fact that these genes are rapidly transcribed and reflect very recent changes in neuronal activity (Guzowski et al., 2005). However, their expression time course profile range form minutes to hours (15 minutes to 1-2 hours or more). Accordingly, their temporal resolution do not faithfully reflect their real activity, but rather a trace of it. Other currently
available tools would be more appropriate to sense real-time activity such as Ca2+
indicators for example, which may tell us a very different story about the activity of cells belonging to cellular engrams.
The use of opsins and designed receptors to control neuronal activity
Many studies, as well as the present work, use opsins and other proteins to artificially stimulate cells or group of neurons belonging to the cellular engram. In our study, the stimulation protocol used with optogenetics was 10Hz. This stimulation protocol, at least for the hippocampus, is often based to what it is observed in freely moving animals where place cells frequently attain this firing frequency (Haggerty and Ji, 2015; Passecker et al., 2011). However, it is also true that place cells are group of cells that increase their firing when the animal is in a particular location of the environment. In our study and in all the above mentioned that use optogenetics, it must be pointed out that we artificially forced all the cells expressing the opsin (in our study around 13%) to fire synchronously and at a fixed firing rate of 10Hz. This type of stimulation try to mimic at best what it is observed in physiological conditions where group of place cells (around 9% of cells for the DG (GoodSmith et al., 2017), while between 30% to 70% for CA1, (Wilson and McNaughton, 1993; Gothard et al., 1996), synchronize and organize spatial information. It is however necessary to keep in mind that we are still far to represent faithfully what really happens to the network when the animal encode a new memory. Moreover, a recent study reported that place cells in addition to the well-known spatial information also code for continuous, tasks-relevant variables (Aronov et al., 2017). Although artificial stimulation allows the animal to recall the associated memory trace, more sophisticated ways to manipulate neuronal activity that best matches natural conditions are needed.
Future perspectives
The very first step needed in studies investigating cellular engrams are the refinement of the tools used to identify, label and manipulate these populations of cells. Moreover, with the advances in the technology that we are assisting nowadays, it is very likely that in the near future some of the questions raised in the precedent paragraphs could find their answers. According to engram definitions, a population of cells, to acquire engram properties must satisfy different criteria (Josselyn et al., 2015; Guan et al., 2016). Most of the studies as well as the present one have been able to reveal only one or two criteria.
However, to really demonstrate the theory of engram one need to prove that the neuronal population studied satisfy all the criteria at the same time.
Most of the studies on memory have focused their attention either on the “spatial domain” by looking at specific brain regions, or on the “temporal domain” by investigating one particular stage of memory (i.e. encoding, consolidation, storage or recall). Thus, it would be impossible to give an answer to the question: “where is memory located in the brain?” Memory is a complex phenomenon combining the spatial and temporal domains.
Each stage is intrinsically connected with each other and should be considered as a continuum rather than separate systems capable of independent functioning. Accordingly, memory does not occur in one specific brain area but rather requires the interaction of many different brain regions. Recent studies have shown that oscillatory activity can synchronize firing in very distal regions of the brain and controls specific behaviors (Karalis et al., 2016; Dejean et al., 2016). For these reasons, further research in the field should consider the spatial and temporal domains together. A very recent study precisely stared this investigation and for the first time revealed that different brain areas are simultaneously involved in episodic memory formation and that they reorganize the mnemonic trace over time (Kitamura et al., 2017).
Most of the studies, as well as the present work, have revealed that a group of cells that are active during a specific behavior become part of the cellular engram. But what is the contribution of each single cell to the cellular engram? What is the information bearing in each single cell? Moreover, what is the role of cells that are not active (i.e. cells that are silenced by the active ensemble and do not express IEGs)? In our study, we have seen that lateral inhibition excludes other cells to become part of the cellular engram, but nothing is known about what these cells do. Do they “passively” participate to the cellular engram? Is synaptic plasticity occurring as well in these cells? It would be of major relevance in future experiments to investigate the role of these “passive” cells and understand their contribution to the cellular engram.
Moreover, memory plays a pivotal role in different cognitive processes that include emotion, attention, consciousness, decision, etc., all eventually affecting our behavior.
Indeed, many neuronal circuits are involved in these cognitive processes and interact with memory function. Emerging evidence reveals that dysfunctions in these neural circuits may cause brain pathologies that interfere with correct memory function. For these reasons, future research on memory need to combine all these different aspects (i.e.
cognitive processes, distinct brain regions and brain pathologies). A first step could be the refinement of memory/cognitive tasks. Most of the studies, including the present, have been using a very old but robust model to study episodic memory, the fear conditioning paradigm. We should highlight that although this task allowed great discoveries in the field
of cellular engram, it is however a very limited approach. Indeed, episodic memories are not limited to the mere fear memory but rather include a vast diversity of memories which may be associated with other internal states or emotions. Future experiments should provide more precise and complementary tasks together with animal models of brain disorders to gain insight in the function of memory.
To conclude, memory is a complex phenomenon implicating multiple brain regions. For this reason, memory research requires a brain-wide approach that allows activity recordings, gene-expression profile, cell-type and activity dependent connectivity tracing over the entire brain. It would be of interest in the very near future to use a combination of in vivo calcium imaging for population activity recordings, whole-brain connectivity visualization using clearing-tissue techniques and neuronal ensemble-tagging transgenic approach, to strengthen our understanding in this field. Furthermore, inhibitory circuits are known to be a key element in regulating interconnected excitatory networks and synchronize different brain areas. Accordingly, the knowledge revealed by the present study about inhibitory circuits will be extremely helpful for this future approach to the study memory. I believe that this holistic approach integrating observations made at different levels will lead to decisive contributions in the study of the brain mechanisms of memory.
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