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described in this pioneering work fromJohn Landers and his colleagues aregoing to drive the identification of novel
ALS genes in the future. Translatingthese findings to disease risk for individual patients will however be a tremendous challenge and caution should betaken before any individual variant identified using this approach can be implicated in ALS.
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DeJesus-Hernandez, M., Mackenzie, I.R., Boeve,B.F., Boxer, A.L., Baker, M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adamson, J.,et al. (2011). Neuron 72, 245–256.

Johnson, J.O., Mandrioli, J., Benatar, M., Abramzon, Y., Van Deerlin, V.M., Trojanowski, J.Q.,
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Kim, H.J., Kim, N.C., Wang, Y.D., Scarborough,E.A., Moore, J., Diaz, Z., MacLea, K.S., Freibaum,B., Li, S., Molliex, A., et al. (2013). Nature 495,467–473.
Ling, S.C., Polymenidou, M., and Cleveland, D.W.
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Renton, A.E., Majounie, E., Waite, A., Simón-Sánchez, J., Rollinson, S., Gibbs, J.R., Schymick,J.C., Laaksovirta, H., van Swieten, J.C., Myllykangas, L., et al.; ITALSGEN Consortium (2011).
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Chasing the Trace
Adam Santoro1,2 and Paul W. Frankland1,2,3,4,*
1Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada
2Institute of Medical Science, University of Toronto, Toronto, ON M5S 1A8, Canada
3Department of Psychology, University of Toronto, Toronto, ON M5S 3GM, Canada
4Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada*Correspondence: paul.frankland@sickkids.ca
http://dx.doi.org/10.1016/j.neuron.2014.10.001

Event memories are stored in hippocampal-cortical networks. In this issue of Neuron, two studies, Cowansage et al. (2014) and Tanaka et al. (2014), tag active cells during memory encoding and optogenetically manipulate the activity of these ‘‘engram’’ cells during subsequent recall to reveal how hippocampal and corticalcell ensembles interact during retrieval.
While there is plenty of debate in thememory field, the classical view is thatmemory traces for events are laid downin cell ensembles across distributed hippocampal-cortical networks. The hippocampus is considered necessary, at least
temporarily after encoding, for successful
retrieval of these event memories via
reinstatement of the patterns of activity
within these cortical ensembles. According to this view, the hippocampus contains indices or pointers to cortical cell
assemblies that collectively represent agiven event (e.g., Eichenbaum, 2000).
Observations of retrograde amnesia
following hippocampal damage in humanpatients (such as H.M.), as well as in experimental animals, provide broad support
for this view (Squire et al., 2004). However,they tell little about how hippocampal
and cortical cell ensembles interact tosupport memory retrieval. Two studies
published in the current issue of Neuron,
Cowansage et al. (2014) and Tanaka
et al. (2014), shed light on this interaction.
Both studies used a genetic strategy
to tag active cells at the time of memory
encoding with light-sensitive opsins andthen optogenetically manipulate the activity of these ‘‘engram’’ cells during retrieval.
In the first study, Tanaka et al. (2014)
used a Fos-driven reporter mouse to tag

active cells as mice learned an association between a shock and a context. Theformation and maintenance of contextual
fear memories engages distributed networks, and, as expected, training tagged
ensembles of cells throughout the hippocampus and cortex. Usually, when mice
return to the original training context,
they exhibit conditioned fear responses,including freezing behavior, indicatingthat they recognize this as the place inwhich they previously received a shock.
By expressing the inhibitory opsin (ArchT)
in tagged cells in the CA1 region of thehippocampus, Tanaka et al. (2014) examined the impact of silencing engram cells

Neuron 84, October 22, 2014 ª2014 Elsevier Inc. 243

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just in this region (rather than the wholehippocampal-cortical network) on fearmemory retrieval (Figure 1). When thesetagged CA1 cells were silenced, mice nolonger froze in the conditioning context.
Using a comparable tag and manipulate
approach, a recent study showed thatsilencing engram cells in either the dentate gyrus or CA3 regions of the hippocampus similarly impaired retrieval of acontextual fear memory (Denny et al.,2014).
While these data suggest that reactivating ensembles of hippocampal cells
that were active at the time of encoding
is required for successful retrieval, there
is an alternative explanation. Perhaps
silencing any population of hippocampal
cells (and not necessarily the engramcells) produces a more general disruptionof hippocampal function and impairs
retrieval. Tanaka et al. (2014) designed
a clever experiment to test this idea.
They first tagged CA1 cells with the inhibitory opsin when exposing mice to adifferent environment. They subsequently
fear conditioned these mice in the regular training context while silencing thispopulation of tagged cells (and presumably preventing these tagged cells frombecoming part of the contextual fear
memory engram). They then placed
mice back into the fear conditioning
context and silenced this population of‘‘excluded’’ cells. Silencing had no effecton the retrieval of the fear memory, indicating that their disruption was specificto the target memory and did not affectother contextual memories presumably
stored in the hippocampus.
Why don’t the mice remember? Whilefear memories like these are thought tobe represented in distributed hippocampal-cortical networks, silencing just theengram neurons in the CA1 was sufficient
to impair retrieval. According to the classical view, preventing reactivation of ahippocampal index should prevent reactivation of the entire hippocampal-corticalnetwork. The tagging system used byTanaka et al. (2014) allowed them toaddress this issue in ways not possible
previously. After CA1 silencing on theretrieval test, they evaluated the likelihoodof reactivation for tagged engram cells in
the cortex. While overall activation levels(measured by expression of the immediate-early gene Fos) were similar with orwithout hippocampal silencing, silencing
CA1 encoding cells led to a selective
reduction in reactivation of tagged cells
in the cortex.
Therefore, these data suggest thatsilencing CA1 engram cells induces
retrieval failure because, at least in part,
it prevents the reactivation of engram
cells in the cortex. If so, is it possible tobypass the hippocampus (and its index)
and artificially express a fear memory bydirectly driving these cortical engram
cells? This is the question addressed by
Cowansage et al. (2014). Using a similar
tag-and-manipulate approach, they firstfear conditioned mice and, as Tanaka
et al. (2014) observed, found that engram
cells were tagged throughout the hippocampus and cortex. Now, instead ofturning off tagged cells in the CA1, Cowansage et al. (2014) directly reactivatedengram cells in the retrosplenial cortex
after placing mice in a neutral context(where freezing isn’t normally observed).
Artificial reactivation of this componentof the memory trace induced freezing.
This suggests that reactivation of tagged
retrosplenial cells alone was sufficient to
artificially express the fear memory.
Focal stimulation of a population ofengram cells in the retrosplenial cortex
was effective in retrieving the memory
possibly because it led to activation of amuch broader network of engram cells
in the cortex. Indeed, the retrosplenialcortex reciprocally connects with many
hippocampal and cortical regions and,during contextual fear memory retrieval,
activity of the retrosplenial cortex is coordinated with activity in many other corticalregions (Wheeler et al., 2013). Consistentwith the idea that the retrosplenial cortex
acts as a hub within a broader memory
network, Cowansage et al. (2014) foundthat ‘‘natural’’ and ‘‘artificial’’ expressionof a fear memory activated similar populations of cells in subregions of the amygdala and entorhinal cortex. It is worth
noting that the artificially expressed memory is weaker than naturally expressedfear memories, with freezing levels hovering between 15%–20% following activation of retrosplenial engram cells. This is
not surprising given that it is unlikely that
all engram cells were reactivated. Furthermore, optical stimulation cannot recapitulate the precise temporal patterningobserved during the natural retrieval of

244 Neuron 84, October 22, 2014 ª2014 Elsevier Inc.

fear memories, leading to further information loss.
While the hippocampus is necessary for
retrieval of contextual fear memories soonafter encoding, eventually these types ofmemories may be expressed independently of the hippocampus (Franklandand Bontempi, 2005). Cowansage et al.
(2014) next asked whether hippocampal
activity was necessary for artificial recallof a fear memory, when tested just a
few days after encoding. To do this, they
pharmacologically inhibited the hippocampus while mice ‘‘naturally’’ expressed
a contextual fear memory or ‘‘artificially’’
expressed a contextual fear memory(induced by driving retrosplenial engram
cells). As expected, shutting down thehippocampus blocked natural recall of
the memory. However, shutting down
hippocampal activity had no effect on
artificial expression of the memory. Thissurprising finding suggests that driving
cortical engram cells alone is sufficientfor memory expression, even soon aftertraining. Presumably, whereas undernormal conditions the only way to reactivate cortical engram cells is via reactivating the appropriate indices or pointersin the hippocampus, accessing corticalengram cells directly circumvents thisrequirement.
The creative application of this ‘‘tagand-manipulate’’ approach allowed thesestudies to interrogate this classical modelof consolidation in ways not previously
possible. In particular, the ability to selectively manipulate cells that were active
during encoding allowed Tanaka et al.
(2014) to provide support for the widely
held view that the successful retrievaldepends upon reinstatement of cortical
patterns of activity that occurred at thetime of learning via activation of hippocampal indices. Complementing this
discovery, Cowansage et al. (2014)
showed that it is nonetheless possible
to completely bypass the hippocampus
and artificially express a fear memory bytargeting engram cells in the cortex
directly.
However, as elegant as these studies
are, one major issue remains unresolved.
What is the nature of the index? Thisis a fundamental issue that divides
opinion. At one extreme, hippocampal
cell ensemble activity is thought to provide little more than an index, withNeuronPreviews

ABCFigure 1. Opsin-Mediated Reactivation or Inhibition of Memory Engram Cells(A) Tanaka et al. (2014) demonstrate that reactivation of tagged CA1 cells is necessary for the recapitulation of cortical engram cells. When the tagged CA1 cells
are inhibited, a different population is recruited in the cortex, impairing memory retrieval.
(B) A memory successfully expresses via optogenetic reactivation of a distinct cortical ensemble (i.e., the retrosplenial cortex), even when pharmacologicallyinhibiting the hippocampus (Cowansage et al., 2014). Importantly, the engram cells in other cortical regions reactivate during this artificial retrieval.
(C) Previous results demonstrate that reactivation of tagged hippocampal cells is sufficient for successful memory retrieval, presumably via reactivation of corticalengram cells (Liu et al., 2012).

Neuron 84, October 22, 2014 ª2014 Elsevier Inc. 245

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memory content stored in the cortex (Teyler and DiScenna, 1986). According to thisaccount, memories expressed via activation of hippocampal indices or, artificially,
via direct activation of cortical engram
cells should not differ in quality. That is,
the same content is being accessed,
albeit via different routes. Alternatively,
others argue that, along with containing
an index, the information in the hippocampus necessarily includes at least somecontent that is not present in the cortex(for example, contextually dense or highlyspatial details) (Winocur and Moscovitch,2011). Therefore, according to this account, whether or not the hippocampus
contributes to expression does make adifference in the quality of the retrieved
memory. A fear memory expressed via
activation of hippocampal indices should
retain its contextually rich and detailed
nature. In contrast, direct activation ofcortical engram cells will lead to expres-sion of a fear memory that is necessarily
less detailed and more gist-like in quality.
In fear conditioning studies, memory quality has most often been assessed bycomparing freezing levels in trained
versus similar contexts. However, since
the artificial recall is already assessed ina neutral context in the Cowansage et al.
(2014) study, these types of context
generalization experiments are notpossible here. This particular debate is
destined to continue, and it is our hopethat the creative application of new tools
will also shed light on this question.
REFERENCES
Cowansage, K.K., Shuman, T., Dillingham, B.C.,
Chang, A., Golshani, P., and Mayford, M. (2014).
Neuron 84, this issue, 432–441.
Denny, C.A., Kheirbek, M.A., Alba, E.L., Tanaka,K.F., Brachman, R.A., Laughman, K.B., Tomm,N.K., Turi, G.F., Losonczy, A., and Hen, R. (2014).
Neuron 83, 189–201.

Eichenbaum, H. (2000). Nat. Rev. Neurosci. 1,41–50.
Frankland, P.W., and Bontempi, B. (2005). Nat.
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Teyler, T.J., and DiScenna, P. (1986). Behav. Neurosci. 100, 147–154.
Wheeler, A.L., Teixeira, C.M., Wang, A.H., Xiong,X., Kovacevic, N., Lerch, J.P., McIntosh, A.R., Parkinson, J., and Frankland, P.W. (2013). PLoS Comput. Biol. 9, e1002853.
Winocur, G., and Moscovitch, M. (2011). J. Int.
Neuropsychol. Soc. 17, 766–780.

Keeping an Eye on Cortical States
James F.A. Poulet1,2,*
1Department of Neuroscience, Max-Delbrück Center for Molecular Medicine (MDC), Berlin-Buch, Robert-Rössle-Str. 10, 13092 Berlin,Germany
2Cluster of Excellence NeuroCure, Neuroscience Research Center, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany*Correspondence: james.poulet@mdc-berlin.de
http://dx.doi.org/10.1016/j.neuron.2014.10.005

Membrane potential recordings in awake mice have correlated cortical state with locomotion and whisker
movements. In this issue of Neuron, Reimer et al. (2014) now reveal that pupil dilation in stationary miceequally signals a change in cortical state and an enhancement of visual processing.
Sensory processing and perception are
not simply a passive detection of stimuli
by the nervous system; in animals thatare awake and behaving, it is an active
process and a highly integrative one. The
peripheries of our sensory systems areconstantly engaged, whether we realize
it or not: eyes scan, hands manipulate,noses sniff, tongues roll. Although werarely use them, we even have muscles
to move our ears—maybe the vestige ofsome ancient mechanism to reposition
them and capture more sound. When sensory input reaches the CNS, it is integrated

with sensory signals of other modalities
and a wide range of internally generated
signals including copies of motor commands, memories, arousal, and attention.
Understanding where, how, and why sensory integration occurs in the brain is a
grand challenge for neuroscience.
Nowhere is the integration of external
and internal neural signals more apparent
than in the mammalian neocortex. Thevery first electroencephalogram (EEG) recordings of electrical activity from awake
animals and the human brain revealed patterns of spontaneous activity that corre-246 Neuron 84, October 22, 2014 ª2014 Elsevier Inc.

lated to different behavioral states but
seemed unrelated to direct sensory input.
This suggested that the neocortex would
be a good place to study changes in brain
states and their relation to sensory integration, in the hope of finding cellular correlates possibly in identified populations ofneurons. This was theoretically possible,
but anesthesia was typically used toimmobilize the animal. It was a dilemma if
you were interested in waking brain states.
The head-restrained mouse preparation
came to the rescue and is now in widespread use. This provides the stability