Neuron

ArticleNeurons Are Recruited to a Memory Trace
Based on Relative Neuronal Excitability
Immediately before Training
Adelaide P. Yiu,1,2,3,4,8 Valentina Mercaldo,1,2,3,4,8 Chen Yan,1,2,3,4 Blake Richards,1,2,3,4,5 Asim J. Rashid,1,2,3,4
Hwa-Lin Liz Hsiang,1,2,3,4 Jessica Pressey,6 Vivek Mahadevan,6 Matthew M. Tran,1 Steven A. Kushner,1,7Melanie A. Woodin,6 Paul W. Frankland,1,2,3,4 and Sheena A. Josselyn1,2,3,4,*
1Program in Neurosciences & Mental Health, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8, Canada
2Department of Psychology
3Institute of Medical Sciences
4Department of Physiology
University of Toronto, Toronto, ON M5G 1X8, Canada
5Department of Biological Sciences, University of Toronto Scarborough, Toronto, ON M1C 1A4, Canada
6Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5G 1X8, Canada
7Department of Psychiatry, Erasmus Medical Center, 3000 CA Rotterdam, the Netherlands
8Co-first author*Correspondence: sheena.josselyn@sickkids.ca
http://dx.doi.org/10.1016/j.neuron.2014.07.017

SUMMARY

Memories are thought to be sparsely encoded inneuronal networks, but little is known about why agiven neuron is recruited or allocated to a particularmemory trace. Previous research shows that in
the lateral amygdala (LA), neurons with increased
CREB are selectively recruited to a fear memory
trace. CREB is a ubiquitous transcription factor implicated in many cellular processes. Which process
mediates neuronal memory allocation? One hypothesis is that CREB increases neuronal excitability tobias neuronal recruitment, although this has notbeen shown experimentally. Here we use several
methods to increase neuronal excitability and show
this both biases recruitment into the memory traceand enhances memory formation. Moreover, artificial
activation of these neurons alone is a sufficient
retrieval cue for fear memory expression, showingthat these neurons are critical components of thememory trace. These results indicate that neuronalmemory allocation is based on relative neuronal
excitability immediately before training.

INTRODUCTION
Although different brain regions may specialize in storing
different types of memories, computational and observational
findings suggest that only a small portion of neurons within agiven region is necessary to encode any particular memory (Guzowski et al., 1999; Kanerva, 1988; Reijmers et al., 2007; Rolls
and Treves, 1990; Wilson and McNaughton, 1993). For instance,
it is generally agreed that the amygdala, in particular the lateral
nucleus of the amygdala (LA), is important for auditory fear (Da722 Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc.

vis, 1992; Duvarci and Pare, 2014; Fanselow and Gale, 2003;
Maren, 2003) or threat (LeDoux, 2014) conditioning, in which atone is paired with an aversive shock. However, while over
70% of pyramidal/principal neurons in the rodent LA respondto both tone and shock presentation (suggesting these neurons
are ‘‘correctly wired’’ and therefore eligible to become part of thememory trace), only a small portion (10%–30%) of these
eligible neurons seem to be recruited into any one fear memory
trace (Han et al., 2007; Reijmers et al., 2007; Repa et al., 2001;
Rumpel et al., 2005). Similarly, there is evidence for a stable
sparse fear memory trace in the human amygdala (Bach et al.,2011). Here we examine the mechanisms that help determine
which particular neurons are selected or allocated (Zhou et al.,2009) to a sparsely encoded fear memory trace in the mouse LA.
Previously, we and others showed that LA neurons compete
against one another for allocation to a fear memory trace, andfurthermore that it was possible to bias the outcome of thiscompetition by manipulating CREB (cAMP/Ca2+ responsive
element binding protein) function in individual LA neurons. Neurons with relatively higher CREB function were more likely to be
included, whereas neurons with relatively lower CREB function
were more likely to be excluded from the memory trace (Hanet al., 2007, 2009; Zhou et al., 2009). Importantly, the overall
size of the LA memory trace remained stable despite thesevarious CREB manipulations, and indeed did not vary with thestrength of the fear memory. This suggests that there is a limit
or constraint on the overall size of the LA fear memory trace.
Interestingly, decreasing CREB function in a small populationof random LA neurons did not disrupt memory formation. This
result is likely because the small population of neurons withdecreased CREB function was excluded from the memory trace(which was composed of nonmanipulated neurons). In contrast,
increasing CREB function in a similar small portion of LA
neurons was sufficient to enhance memory formation (Hanet al., 2007). Together, these results suggest that neuronalcompetition is important for neuronal allocation and memory formation, and that neurons with relatively higher CREB are

NeuronIncreasing Excitability in LA Enhances Memorycompetitively advantaged (and therefore, more likely to ‘‘win’’
this competition).
CREB is a ubiquitous transcription factor implicated in many
diverse cellular processes, including proliferation, survival, apoptosis, differentiation, metabolism, glucose homeostasis, andneuronal excitability (Lonze and Ginty, 2002). For instance,
CREB bidirectionally modulates neuronal excitability (increasing
CREB function increases the propensity of neurons to fire action
potentials, while decreasing CREB function decreases neuronal
excitability) (Dong et al., 2006; Viosca et al., 2009; Zhou et al.,2009). Which of these CREB-mediated processes is importantfor neuronal allocation during memory formation? One plausible
mechanism is that neurons with high levels of CREB are preferentially recruited to a memory trace because these neurons are
more excitable than their neighbors (a postsynaptic neuron
that is more excitable than its neighbor would be ‘‘primed’’ forallocation into a given memory trace). Although an increase inexcitability has been proposed as a mechanism mediatingneuronal allocation during memory formation (Kim et al., 2013;
Zhou et al., 2009), this idea has not been directly tested experimentally. Here we used three different methods to determine
whether relatively higher excitability before training confers acompetitive advantage for neuronal allocation to a fear memory.
RESULTSHSV Microinjected into the LA Preferentially Infects
Excitatory Pyramidal/Principal Neurons
To manipulate excitability in a small, arbitrarily chosen subpopulation of LA neurons, we used replication-defective herpes simplex
viral (HSV) vectors. To phenotype the type of LA cells infected by
HSV, we microinjected mice with HSV expressing GFP (to allow
for easy visualization of infected cells) into the LA and performed
immunohistochemistry for different cell markers (NeuN, neuronal
nuclei, as a marker of neurons; aCaMKII, alpha Ca2+/calmodulin-dependent protein kinase II, as a marker of excitatory pyramidal/principal neurons; GFAP, glial fibrillary acidic protein, as amarker of astrocytes; and GAD67, glutamate decarboxylase 67,
as a marker of inhibitory neurons). We observed complete overlap
between LA cells infected by HSV (GFP+) and cells expressing aneuronal marker (NeuN+), but no overlap between infected cells
and cells expressing GFAP, confirming that HSV is neurotropic(Cole et al., 2012; Fink et al., 1996). Moreover, HSV predominantly(98%) infected pyramidal/principal excitatory (aCaMKII+) neurons in the LA, with only a very small number (<2%) of infected
neurons costaining with the inhibitory neuronal marker GAD67(Figures 1A and 1B). Therefore, consistent with previous reports,
we found that following microinjection into the LA, HSV preferentially infects pyramidal/principal neurons (Cole et al., 2012).
Because of this tropism, we used HSV to manipulate excitability
in a portion of pyramidal/principal neurons in the LA.
Manipulating Neuronal Excitability Using
Voltage-Dependent K+ Channels
Neuronal excitability is determined by the composition, distribution, and properties of ion channels (e.g., Na+, K+, and Ca2+) inthe plasma membrane. Increasing CREB function increases
neuronal excitability, in part, by decreasing voltage-gated K+

currents (Dong et al., 2006; Lopez de Armentia et al., 2006;
Viosca et al., 2009; Zhou et al., 2009). This, in turn, inhibits the
postburst afterhyperpolarization (AHP, a hyperpolarizing current
which mediates the ‘‘undershoot phase’’ or refractory period
following an action potential). Two members of the voltagedependent K+ family of channels, KCNQ2 and related KCNQ3,
help mediate the AHP and function as molecular brakes onneuron firing (Delmas and Brown, 2005; Gu et al., 2005). Expression of a dominant-negative KCNQ2 mutant (hQ2-G279S;
dnKCNQ2) coassembles with native KCNQ2/3 subunits, disrupts their function, and thereby increases neuronal excitability(Peters et al., 2005; Schroeder et al., 1998; Wuttke et al.,2007). We observed that LA neurons in adult mice endogenously
express KCNQ2-containing channels (Figure 1C), suggestingthat expression of the dnKCNQ2 construct could be a viable
method for increasing excitability in LA neurons. To test this,
we first transfected primary hippocampal neurons with the
dnKCNQ2 construct. The dnKCNQ2 construct was also
observed near the axon initial segment (Figure 1D), consistent
with the notion that dnKCNQ2 coassembles with, and blocks
the function of, endogenous KCNQ2/3 channels. Therefore, as
our first method to increase excitability without directly manipulating CREB function, we used HSV vectors to express
dnKCNQ2. To decrease excitability without directly decreasing
CREB function, we used HSV to express Kir2.1, an inwardly
rectifying K+ channel, which reduces neuronal input resistanceand decreases evoked action potential firing (Dong et al., 2006).
To verify that expression of our excitability constructs indeed
manipulated neuronal excitability, we assessed excitability (asmeasured by firing rate) of cultured hippocampal neurons transfected with GFP, CREB, dnKCNQ2, Kir2.1, and CREB+Kir2.1
constructs. Consistent with previous reports, we found that
expression of dnKCNQ2 (Peters et al., 2005) or CREB (Donget al., 2006; Han et al., 2006; Lopez de Armentia et al., 2006;
Viosca et al., 2009; Zhou et al., 2009) increased excitability, while
Kir2.1 decreased (Dong et al., 2006) excitability. Moreover, the
CREB-induced increase in excitability was blocked by coexpression with Kir2.1 in the same neurons (Figure 2A; one-way
ANOVA on firing rate of transfected/not-transfected neurons
with between-group factor Transgene [GFP, CREB, dnKCNQ2,
Kir2.1, and CREB+Kir2.1], F4,28 = 7.18, p < 0.001, posthoc Newman-Keuls tests showed that dnKCNQ2 and CREB increased
excitability over GFP, whereas CREB+Kir2.1 condition was notdifferent from GFP). To confirm that HSV-CREB and HSVdnKCNQ2 increased excitability in infected LA neurons, we
also assessed excitability in ex vivo LA slices following microinjection of HSV expressing GFP, CREB, or dnKCNQ2 into mice.
LA neurons overexpressing dnKCNQ2 or CREB showed higher
firing rates than LA neurons expressing GFP (Figure 2B; oneway ANOVA conducted on firing rate of infected/not-infected
neurons with between-group factor Vector (GFP, CREB, and
dnKCNQ2), F2,21 = 4.15, p < 0.05, LSD posthoc tests showedthat both neurons with dnKCNQ2 or CREB fired more action potentials than neurons expressing GFP and did not differ fromeach other). Therefore, although we did not directly determine
excitability following our manipulations in vivo, results from cell
culture and ex vivo LA neuron slices confirm previous reportsthat dnKCNQ2 and CREB enhance excitability.
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NeuronIncreasing Excitability in LA Enhances Memory

Figure 1. Using Replication-Defective HSV Viral Vectors to Manipulate Neuronal Excitability(A and B) Following microinjection into the LA, HSV vectors preferentially infect pyramidal/principal neurons. Mice were microinjected with HSV expressing GFP
into the LA, and 4 days later infected cells were phenotyped using immunohistochemistry for various cell markers. Example images from 4 days postinjection(dpi). (A) DAPI (blue, nuclear stain), GFP (green, infected cell), various cell markers (red). Top: NeuN was used as a marker of neurons. All infected cells (GFP+) were
neurons (also NeuN+). Bottom: aCaMKII was used as a marker of excitatory pyramidal/principal neurons. The vast majority of infected cells (roughly 98%) were
excitatory pyramidal neurons (also aCaMKII+). (B) Top: we found no (0%) HSV-infected cell that coexpressed endogenous markers typical of astrocytes (GFAP,
glial fibrillary acidic protein) and (bottom) only very rare HSV-infected cells that coexpressed inhibitory neurons (GAD67, glutamate decarboxylase 67) (roughly1.7%). Detailed quantification cell phenotype infected by HSV-GFP in six mice shows that following microinjection into the LA, HSV overwhelmingly infects
excitatory pyramidal neurons.
(C) Endogenous KCNQ2 is widely expressed in adult mouse brain neurons (including LA) and localized to cell body and proximal parts of neurites (near axon initial
segment). Left: low-power (upper panel) and high-power (lower panel) immunofluorescence images of adult mouse brain stained with antibody directed against
KCNQ2 (red). Middle (upper and lower panels): MAP2 (microtubule-associated protein 2, neuronal cytoskeletal protein, green) used to visualize dendritic processes. Right (top panel): Sections were counterstained with DAPI (blue) to visualize nuclei. Lower panels: higher-power images showing KCNQ2 (upper, red) and
MAP2 (middle, green) staining. Bottom panel: triple labeling shows KCNQ2 localized to cell body and proximal parts of neurites, as expected.
(D) Left: primary hippocampal neurons transfected with dnKCNQ2 vector that also expresses GFP (GFP, gray; KCNQ2, red). Right: magnification of transfected
neuron shows increased KCNQ2 protein (KCNQ2, red) in the cell body and proximal parts of neurites, as expected. Red arrow points to transfected neuron,depicting that dnKCNQ2 is localized to correct neuronal region. White arrows point to representative nontransfected neurons and the endogenous KCNQ2
expression. Data presented are mean ± SEM.

Memory-Enhancing Effects of Increasing CREB Levels
in a Small Portion of LA Neurons Are Mimicked by
dnKCNQ2 and Blocked by Coexpression of Kir2.1
We previously showed that increasing CREB function in a small,random population of LA neurons by microinjecting HSV-CREB
was sufficient to enhance conditioned fear memory formation,and that these neurons overexpressing CREB were selectively
allocated to the fear memory trace (Han et al., 2007, 2009).
To examine if the memory-enhancing effects of CREB overexpression in these experiments could be attributed to CREB
increasing neuronal excitability, we microinjected HSV vectors
expressing GFP (as a control), CREB (to increase excitability),
dnKCNQ2 (to increase excitability), Kir2.1 (to decrease excit724 Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc.

ability), or CREB+Kir2.1 (to counteract the increase in excitability
produced by CREB) into the LA 2 days before weak auditory fear
conditioning (in which a tone conditioned stimulus [CS] waspaired with low-intensity, 0.3 mA shock). Importantly, following
microinjection, these viral microinjections infect roughly 10% of
LA principal neurons (as determined by stereological counting),
and the pattern of infection appears random (Figure 2C; see Supplemental Experimental Procedures). In agreement with previous findings (Han et al., 2007, 2009; Rexach et al., 2012; Zhou
et al., 2009), we observed that increasing CREB in a small portionof LA neurons enhanced memory formation (Figure 2D).
Similarly, expressing dnKCNQ2 in roughly 10% of LA neurons
produced a memory enhancement that was strikingly similar toNeuronIncreasing Excitability in LA Enhances Memory

Figure 2. Increasing Intrinsic Excitability by Overexpressing CREB or dnKCNQ2 in a Small, Random Portion of LA Principal/Pyramidal Neurons Enhances Fear Memory Formation; Neurons with Relatively Higher Excitability Are Preferentially Allocated to this Fear Memory Trace(A) Primary hippocampal neurons transfected with CREB or dnKCNQ2 construct show increased firing rates (relative to not-transfected neighboring neurons),
whereas neurons transfected with GFP show no increase in firing. Neurons expressing Kir2.1 show decreased firing rates, whereas neurons coexpressing both
CREB and Kir2.1 are not different from neurons expressing GFP. Left: example traces from injecting 120 pA current. Right: quantification. GFP n = 7 neurons,
CREB n = 6 neurons, dnKCNQ2 n = 12 neurons, Kir2.1 n = 6 neurons, CREB+Kir2.1 n = 2 neurons.
(B) Ex vivo LA neurons from mice microinjected with CREB or dnKCNQ2 vector show increased firing rates relative to not-infected neurons (or neurons expressing
GFP control vector, which do not show differences from not-infected neighbors). Left: example traces. Right: quantification. GFP n = 9 neurons, CREB n = 7
neurons, dnKCNQ2 n = 8 neurons.
(C) Microinjecting HSV vector infects 10% of LA principal neurons (based on stereological counting). Example image from mouse brain 4 days postinjection(dpi). DAPI (blue, nuclear stain), GFP (green, infected neuron). LA outlined with dotted line.
(D) Expressing CREB or dnKCNQ2 vector in a small portion of LA principal neurons 2 days before weak auditory fear conditioning enhances fear memory formation. Expressing Kir2.1 vector in a similar portion of neurons does not affect fear memory. However, the memory enhancement produced by CREB vector wasprevented by coexpressing Kir2.1 in the same neurons. GFP vector n = 7 mice, CREB n = 7 mice, dnKCNQ2 n = 15 mice, Kir2.1 n = 10 mice, CREB+Kir2.1 n = 8
mice.
(E) Neurons are allocated to the memory trace based on relative excitability. In mice microinjected with CREB or dnKCNQ2 vector, infected neurons were more
likely to be part of the memory trace (active arc+ neurons assessed 5 min after the memory test) than their noninfected neighbors. In mice microinjected with GFP
vector, infected neurons were equally likely to be arc+, while in mice microinjected with Kir2.1 vector, infected neurons were less likely to be arc+ than theirnoninfected neighbors. Left: example images. DAPI (blue, nuclear stain), arc+ (red, active neuron following memory test), GFP (green, infected neuron). Right:
quantification of images. GFP vector n = 8 sections from 5 mice, CREB n = 8 sections from 5 mice, dnKCNQ2 n = 14 sections from 6 mice, Kir2.1 n = 4 sections
from 4 mice. Data presented are mean ± SEM. n.s., not statistically different.

that observed following CREB overexpression. However, coexpression of CREB and Kir2.1 in the same LA neurons blocked
the memory enhancement produced by CREB vector alone(one-way ANOVA, F4,42 = 14.75, p < 0.001, mice microinjectedwith CREB or dnKCNQ2 vector froze more than mice with GFP
vector, but were not different from each other; mice microinjected with CREB+Kir2.1 vector froze less than mice with
CREB vector alone, but were not different from mice with GFP
vector [posthoc Newman-Keuls]).
The Memory-Enhancing Effects of Increasing CREB or
Blocking KCNQ2 Function Are Behaviorally Specific
These results suggest that increasing excitability in a small
portion of LA neurons before fear conditioning enhanced theformation of a fear memory. To more thoroughly examine thiseffect, we conducted a series of control experiments. First, we
examined the phase of memory which was important in thememory enhancement produced by CREB or dnKCNQ2 expression. To determine whether the higher freezing during the tone
test observed in mice microinjected with HSV-CREB or HSVdnKCNQ2 was due to an effect on acquisition or expression of
conditioned fear memory, we microinjected our viral vectors
1 day after training (rather than 2 days before training). As a control for the possible effects of surgery, we compared freezing
levels on the test day to no-surgery control mice. We observedthat microinjecting our viral vectors 1 day after either weak (0.3mA shock) or strong (0.6 mA shock) training did not affectfreezing levels during the tone test (Figure 3A; strong training,
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NeuronIncreasing Excitability in LA Enhances Memory

Figure 3. The Memory Enhancement Produced by Overexpressing CREB or dnKCNQ2 in the LA Prior to Fear Conditioning Is Specific(A) Microinjecting CREB or dnKCNQ2 vector after strong or weak training has no effect on expression of a previously acquired fear memory. Strong training; GFP
n = 10 mice, CREB n = 11, dnKCNQ2 n = 11, no surgery n = 8. Weak training; GFP n = 8, CREB n = 8, dnKCNQ2 n = 8, no surgery n = 8.
(B) Microinjecting CREB or dnKCNQ2 vector 11 days before weak auditory fear conditioning (such that mice were trained after transgene expression dissipated)
does not enhance memory. n = 8 mice per group. Right: little to no LA transgene expression 10 days postinjection (dpi, bottom) compared to high levels oftransgene expression 4 dpi (top).
(C) Microinjecting CREB or dnKCNQ2 vector 2 days before weak auditory fear conditioning produces an enduring memory enhancement that outlasts transgene
expression. n = 8 mice per group.
(D) Microinjecting CREB vector into the central amygdala (CeA) or basal amygdala (BA) 2 days before weak auditory fear conditioning fails to enhance memory,
showing that the memory enhancement is anatomically specific. LA, GFP n = 7; CeA, CREB n = 8; and BA, CREB n = 8. Data presented are mean ± SEM. n.s., notstatistically different.

F3,36 = 0.52, p > 0.05; weak training, F3,28 = 0.81, p > 0.05). Thesefindings indicate that increasing excitability in a small subset of
LA neurons did not increase freezing by enhancing the expression of a previously acquired fear memory. Instead, these results
are consistent with the interpretation that CREB and dnKCNQ2
enhanced the formation of a fear memory.
Second, we asked when an increase in neuronal excitability
resulted in enhanced fear memory. Transgene expression using
this HSV system begins hours following microinjection, peaks
within 2–3 days later, and dissipates within 7–12 days (Figure 3B,
right) (Sekeres et al., 2012). We took advantage of this transgene
expression time course by microinjecting CREB or dnKCNQ2
vectors 11 days before training (such that mice were trained ata time when the transgenes were no longer expressed). Microin726 Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc.

jecting dnKCNQ2 or CREB vector 11 days before training had noeffect on subsequent memory formation, suggesting that theenhanced memory was dependent on increased neuronal excitability at the time of training (Figure 3B, left; F2,20 = 0.09, p > 0.05).
Third, we examined the duration of memory enhancement
produced by CREB or dnKCNQ2 vector. We found that the
memory enhancement produced by high levels of CREB or
dnKCNQ2 at the time of training (mice were microinjected with
vectors 2 days before training) was enduring and even continued
beyond transgene expression (14 days after microinjection) (Figure 3C; F2,21 = 8.44, p < 0.05, mice microinjected with CREB or
dnKCNQ2 vector continued to freeze higher than mice microinjected with GFP vector [but did not differ from each other] even
14 days following microinjection [Newman-Keuls]). Finally, weNeuronIncreasing Excitability in LA Enhances Memory

Figure 4. Overexpressing CREB or dnKCNQ2 in the LA Does Not Increase Overall Anxiety-like Behavior(A–G) Increasing excitability by overexpressing CREB or dnKCNQ2 in a random small portion of LA neurons does not impact basal anxiety-like behavior as
assessed in the open field (A–C) or elevated plus maze (D–G). We observed no effect of microinjecting CREB or dnKCNQ2 vectors into LA on open-field behavior(either total distance traveled [B] or time spent in outer, middle, and inner zone [C]). GFP vector n = 9 mice, CREB n = 8 mice, dnKCNQ2 n = 9 mice. We observed
no effect of microinjecting excitability vectors into the LA on elevated plus maze behavior (time in open versus closed arms [E], crossings in open versus closed
arms [F], or total distance traveled [G]). GFP vector n = 8 mice, CREB n = 5 mice, dnKCNQ2 n = 6 mice. Data presented are mean ± SEM. n.s., not statisticallydifferent.

examined the anatomical specificity of the memory enhancement induced by overexpression of CREB. To this end, we microinjected CREB vector into the neighboring basal amygdala(BA) or central amygdala (CeA) 2 days prior to training. We
observed no memory enhancement with CREB overexpression
in these neighboring amygdala nuclei (Figure 3D; one-way
ANOVA conducted on three groups, GFP vector in LA, CREB
vector in CeA, or CREB vector in BA, F2,20 = 2.58, p > 0.05), suggesting that the memory-enhancing effects of CREB overexpression are anatomically specific. Together, these results
indicate memory formation was enhanced if, and only if, CREB
or dnKCNQ2 was expressed in a small population of LA neurons
at the time of training. Once formed, however, this enhanced
memory endured and no longer required continued overexpression of CREB or dnKCNQ2.
Although we observed no difference in baseline (pretone)
freezing in our experiments (Figures S1A–S1D available online),
we next asked whether increasing excitability in a small portionof LA neurons enhanced freezing during the tone test by nonspecifically increasing overall basal anxiety-like behavior. We microinjected CREB or dnKCNQ2 vector into the LA 2 days before
testing in the open field (Figure 4A) or on an elevated plus
maze (Figure 4D). In the open field, a decrease in total distancetraveled and/or an increase in time spent in the periphery of theopen field are commonly used phenotypic markers of anxietylike behavior in rodents (Crawley et al., 1997; Prut and Belzung,2003). Neither CREB nor dnKCNQ2 vector decreased total distance traveled (Figure 4B; F2,23 = 0.07, p > 0.05) or increased
the amount of time spent in the periphery of the open field (Figure 4C; no significant effect of Vector X Zone [time spent in outer,
middle, and inner zones of the open field], F4,46 = 0.03, p > 0.05;
or Vector, F2,23 = 0.89, p > 0.05; but as expected, significant effect of Zone, F2,46 = 72.96, p < 0.001, with all groups of mice tending to spend more time in the outer zone). Anxiety-like behavior in
the elevated plus maze is often inferred from a decrease in thetime spent in the open arm and/or a decrease in the number ofopen arm crossings (Pellow and File, 1986; Rodgers and Dalvi,1997). Consistent with our findings using the open-field test, microinjecting CREB or dnKCNQ2 vector did not increase theamount of time spent in the open versus closed arms (Figure 4E;
no significant Vector X Arm interaction, F2,16 = 1.32, p > 0.05; oreffect of Vector alone, F2,16 = 1.30, p > 0.05; as expected, significant effect of Arm with all groups tending to spend more time inthe closed arm, F1,66 = 214.12, p < 0.001), crossings in open
versus closed arms (Figure 4F; no significant Vector X Arm interaction, F2,16 = 1.25, p > 0.05; or effect of Vector alone, F2,16 =
1.69, p > 0.05; significant effect of Arm only, F1,16 = 29.12, p <0.001), or total distance traveled (Figure 4G; F2,16 = 0.25, p >0.05) in the elevated plus maze. These results verify that the increase in freezing observed following microinjection of CREB
or dnKCNQ2 vectors in auditory fear conditioning cannot be
attributed to a nonspecific increase in anxiety-like behavior.
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NeuronIncreasing Excitability in LA Enhances Memory

Neurons with Relatively Increased Excitability at theTime of Training Are Preferentially Recruited/Allocatedto a Memory Trace
We observed robust memory enhancement despite infecting asmall population of LA neurons (10%, roughly 16,000–20,000
neurons; see Supplemental Experimental Procedures) with
CREB or dnKCNQ2 vector, suggesting that these infected neurons (with increased excitability) were preferentially allocatedto the memory trace. To examine this, we assessed whether neurons with CREB or dnKCNQ2 vector were overrepresented in thememory trace. To visualize neurons that may be part of the fear
memory trace, we used cellular compartment analysis of temporal activity by fluorescent in situ hybridization (catFISH), a technique that takes advantage of the unique transcriptional timecourse of the activity-dependent gene arc (activity-regulated
cytoskeleton-associated protein) (Guzowski et al., 1999, 2001).
Under basal, quiet conditions, neurons typically show no (orvery low levels of) arc RNA. Neural activity induces a rapid but
transient burst of arc transcription such that arc RNA is observedin the nucleus within 5 min of neuronal activity. However, this arc
RNA is transported to dendrites such that roughly 40 min after
neuronal activity, neurons again show low levels of arc in the nucleus. In this way, the spatial localization of arc RNA in a neuron(nucleus, cytoplasm, both, or neither) may serve as a molecular
activity time stamp for any particular neuron, with arc localized
to the nucleus serving as a visual marker of a recently active
neuron. To identify infected neurons, we used the GFP expressed by our viral vectors.
Specifically, we examined the brains of mice 5 min following
a conditioned fear (tone) memory test and assessed the overlapof active (arc+, neurons with arc localized to the nucleus) andinfected (GFP+) versus noninfected (GFP ) neurons in mice
microinjected with GFP, CREB, dnKCNQ2, or Kir2.1 vector
2 days before training. Consistent with our previous finding, we
observed that the overall size of the arc+ memory trace (numberof overall LA neurons that were arc+) was stable across experiments, regardless of vector microinjected or memory strength(F3,30 = 2.06, p > 0.05). However, the distribution of arc+ neurons(in infected versus noninfected neurons) differed significantly
between vector groups.
In mice microinjected with either CREB or dnKCNQ2 vector,
infected neurons were three times more likely to be arc+ thantheir noninfected neighbors following a memory test. This suggests that these neurons with CREB or dnKCNQ2 were preferentially recruited to the memory trace, paralleling our previous
findings with CREB vector (Han et al., 2007, 2009). The opposite
pattern emerged from mice microinjected with Kir2.1 vector; infected neurons were over five times less likely to be arc+ than
noninfected neurons following a memory test (Figure 2E; Vector(GFP, CREB, dnKCNQ2, Kir2.1) X Neuron Infection Status (infected, noninfected) ANOVA, F3,30 = 18.78, p < 0.001, in micewith GFP vector, there was no difference between the probabilityof arc+ nuclei in infected versus noninfected neurons). Importantly, mice microinjected with Kir2.1 vector showed normal
memory (Figure 2D), suggesting that the majority of noninfected
neurons were sufficient to support normal memory. The relative
exclusion of neurons with Kir2.1 vector from the arc+ memory
trace (and normal memory) is strikingly similar to our previous
728 Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc.

finding in which we similarly microinjected a dominant-negative
CREB vector (HSV-mCREB). We previously observed that neurons with mCREB were also less likely to be allocated to thememory trace, but that mice microinjected with mCREB vector
showed normal memory (Han et al., 2007). It is of interest tonote that similar to Kir2.1, mCREB expression decreases
neuronal excitability (Dong et al., 2006; Han et al., 2006). Thesefindings support the notion that neurons are chosen for the memory trace based on their relative excitability.
Interestingly, following a memory test, noninfected neurons
were less likely to be arc+ in mice microinjected with CREB or
dnKCNQ2 vector than in mice microinjected with GFP vector(one-way ANOVA on the probability of noninfected neurons being arc+ in the three Vector groups [GFP, CREB, dnKCNQ2]
showed a significant difference, F2,27 = 11.87, p < 0.05; the likelihood of noninfected cells being arc+ was lower in mice microinjected with CREB or dnKCNQ2 vector than in mice microinjectedwith GFP vector [Newman-Keuls]). Together, these data areconsistent with the interpretation that neurons compete against
one another for inclusion in a memory trace, and that neurons
with relatively higher levels of excitability are more likely to win
this competition. In addition, these winning neurons may also
actively inhibit ‘‘loser’’ neurons, to maintain the stability of theoverall size of the memory trace. Indeed, our experimental
data were correctly forecast in an elegant modeling study (Kimet al., 2013), which showed that neuronal competition for memory allocation involves both relative neuronal excitability and a disynaptic inhibition process.
Using Genetically Encoded Mediators of Neural
Excitability to Transiently Increase Excitability in a Small
Portion of LA Neurons before, but Not Immediately after,
Training Enhances Fear Memory Formation
In the above experiments, we used HSV to express different
transgenes that affect excitability. Due to the nature of transgene
expression using the present HSV vector, mice were trained andtested with high levels of transgene expression. As such, theseexperiments do not address precisely when an increase in neuralexcitability is important in the observed memory enhancement.
To gain finer temporal control over neuronal excitability, weused two different systems to genetically manipulate neuralexcitability over a relatively brief time. First, we used HSV to express the DREADD (designer receptors exclusively activated bydesigner drug) hM3Dq. hM3Dq is an evolved Gq-coupledmuscarinic receptor, which has no constitutive activity anddoes not bind to the endogenous ligand (ACh) (Armbruster
et al., 2007; Nichols and Roth, 2009). However, binding of thesynthetic ligand, clozapine-N-oxide (CNO, an otherwise pharmacologically inert compound), to hM3Dq receptors increases
neuronal excitability (Armbruster et al., 2007; Nichols and Roth,2009). We first verified this in cultured hippocampal neurons.
Specifically, we found that neurons transfected with hM3Dq
show increased excitability only in the presence of CNO, andthat CNO without the hM3Dq receptor did not alter excitability(Figure 5A; two-way ANOVA on membrane potential as a measure of excitability, with within-group factor CNO Application[before versus after CNO application] and between-group factor
Transfection [transfected with hM3Dq versus untransfected]

NeuronIncreasing Excitability in LA Enhances Memory

Figure 5. Using hM3Dq DREADD to Increase Neuronal Excitability(A) Primary cultured hippocampal neurons transfected with hM3Dq (but not neighboring untransfected neurons) show depolarization only after (and not before)
CNO administration. hM3Dq construct, n = 5 neurons; untransfected, n = 5 neurons.
(B) LA neurons infected with hM3Dq vector show increased activity (cFos expression) following systemic injection of CNO. Mice microinjected with hM3Dq or
GFP vector were not trained but received systemic administration of CNO or VEH. cFos immunohistochemistry was examined 90 min later, and the number of
infected neurons positive for cFos was quantified. hM3Dq vector + CNO administration n = 6 mice, hM3Dq+VEH n = 5 mice, GFP+CNO n = 4 mice, GFP+VEH n =4 mice. Data presented are mean ± SEM. n.s., not statistically different.

revealed a significant interaction, F1,8 = 8.78, p < 0.05, as well as
significant main effect of Transfection, F1,8 = 8.56, p < 0.05, and
CNO Application, F1,8 = 6.44, p < 0.05; post hoc Newman-Keuls
tests performed on the significant interaction revealed that neurons transfected with hM3Dq were significantly more excitable
after CNO administration, while all other groups did not differ).
In addition, in mice microinjected with hM3Dq vector into theLA, we observed an increase in expression of an activity marker
in infected neurons only following systemically administered
CNO. Specifically, neurons with hM3Dq vector had higher levelsof cFos expression, a marker of neuronal activation (Morgan andCurran, 1991), only following administration of CNO, while CNO
on its own produced no effect in neurons with the control GFP
vector (Figure 5B; significant Vector X CNO Injection interaction,
F1,15 = 5.11, p < 0.05; posthoc Newman-Keuls tests confirmedthat infected neurons showed a higher probability of being
cFos+ in the hM3Dq+CNO group than in all other groups, which
did not differ from each other).
To transiently increase excitability in a small (10%), random
portion of LA principal neurons, specifically in the minutes before(and during) fear training, we microinjected hM3Dq (or GFP) vector as above (2 days before training), and systemically administered CNO (or vehicle [VEH]) before weak fear training. Mice
were tested 24 hr later, drug free (in the absence of CNO). In
this way, neuronal excitability was increased only in the minutes
before training. During the memory test, we observed an increase in freezing only in mice microinjected with hM3Dq vector
and administered CNO before training. We observed no memoryenhancement by administration of CNO alone (in mice with GFP
vector) or expression of hM3Dq alone (in mice administered
VEH). Therefore, increasing neuronal excitability in a random,small subset of neurons before training was sufficient to enhancememory formation (Figure 6A; Vector X CNO Injection, F1,29 =8.57, p < 0.05; mice microinjected with hM3Dq vector andadministered CNO before training froze more than all othergroups [Newman-Keuls]). Importantly, administering CNO
immediately after training failed to enhance memory in mice microinjected with hM3Dq vector (Figure 6B; no significant effect ofVector, CNO Injection, or Vector X CNO Injection interaction,

F1,28 = 0.22, p > 0.05). This finding suggests that increased
neuronal excitability at the time of memory encoding, rather
than during consolidation, determines the strength of memory
formation.
To examine whether this small portion of LA neurons that had
increased excitability only during the minutes before training waspreferentially allocated to the memory trace, we used catFISH forarc RNA following a memory test (as above). Before this memory
test, no CNO was administered. Specifically, we microinjected
mice with hM3Dq vector, administered CNO or VEH prior totraining, and 5 min following a drug-free memory test, examined
the overlap of arc (to identify neurons in the memory trace) andGFP (to identify infected neurons). In mice administered CNO
before training, hM3Dq-expressing neurons were 3.8 times
more likely to be included in the memory trace than their noninfected neighbors, whereas hM3Dq-expressing and noninfectedneighboring neurons were equally likely to be arc+ in mice administered VEH before training (Figure 6C; CNO Injection X Neuron
Infection Status, F1,18 = 14.52, p < 0.05, in mice microinjectedwith hM3Dq vector and administered CNO, infected neurons
were more likely to be arc+ than noninfected neurons; however,infected and noninfected neurons were equally likely to be arc+ inmice microinjected with hM3Dq vector but administered VEH[Newman-Keuls]). This result parallels the findings from mice microinjected with CREB or dnKCNQ2 vector, and suggests thatneurons with increased excitability in the minutes before (andduring) training are preferentially allocated to the memory trace.
Synthetic Activation of Neurons Allocated to a Memory
Trace Is Sufficient to Serve as a Retrieval Cue
To determine whether these neurons with increased excitability
at the time of training became critical components of the memory
trace, we next asked whether artificial activation of just theseneurons was sufficient to serve as a memory retrieval cue. In
our previous experiments, we probed conditioned fear memory
by placing mice in a novel context and presenting an external
retrieval cue (presentation of the tone CS that was paired withshock during training). In this experiment, however, we microinjected mice with hM3Dq (or GFP) vector and administered CNO
Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc. 729

NeuronIncreasing Excitability in LA Enhances Memory

Figure 6. Increasing Excitability Using hM3Dq DREADD in a Small Portion of LA Neurons Immediately before, but Not Immediately after,
Training Enhances Fear Memory Formation; These Neurons Are Preferentially Allocated to Memory Trace; Artificial Reactivation of These
Neurons Alone Serves as a Sufficient Memory Retrieval Cue(A and B) Transiently increasing excitability in a small portion of LA neurons before, but not after, training enhances fear memory formation. Microinjecting hM3Dq
vector 2 days before weak auditory fear conditioning and systemically administering CNO before, but not immediately after, (B) training enhances fear memory
formation. CNO before training; GFP vector + CNO administration n = 7 mice, GFP+VEH n = 8 mice, hM3Dq+CNO n = 10 mice, hM3Dq+VEH n = 8 mice. CNO after
training; n = 8 mice for each group.
(C) In mice microinjected with hM3Dq vector, infected (GFP+) were more likely than noninfected neighbors (GFP ) to be allocated to arc+ memory trace if CNO
was administered before training. No difference between distribution of arc+ in infected and noninfected neurons in mice microinjected with hM3Dq vector but
administered VEH. CNO administered only before training (not before test). hM3Dq vector + CNO administration n = 15 sections from 8 mice, hM3Dq+VEH n = 5
sections from 5 mice.
(D) Artificially activating neurons that had increased excitability before strong training (hM3Dq+CNO) is sufficient to serve as a memory retrieval cue in the absenceof an external retrieval cue. Mice with hM3Dq vector administered CNO before training and again before a test in a novel context show higher levels of freezing
than control groups (left). However, all groups froze at equivalent levels when an appropriate external cue was replayed (middle, the tone previously paired withshock). GFP vector + CNO administration n = 8 mice, GFP+VEH n = 8 mice, hM3Dq+CNO n = 8 mice, hM3Dq+VEH n = 8 mice. This enhancement in freezing
following CNO administration is specific to fear memory; we observed no increase in freezing in these mice when similarly microinjected with hM3Dq vector,
administered CNO, and placed in a novel context, but not trained (right, before tone-shock pairing). GFP vector + CNO n = 16 mice, hM3Dq+CNO n = 16 mice.
Data presented are mean ± SEM. n.s., not statistically different.

to all groups before strong training. Rather than assessing memory by exposing mice to the tone (CS), we probed memory byplacing mice in a novel context following administration of
CNO (to reactivate the neurons that we hypothesize are part ofthe memory trace) and measured freezing. Activation of thesehM3Dq-expressing neurons alone (without external tone
retrieval cue) was sufficient to induce freezing (Figure 6D, left;
Vector X CNO Injection at Test, F1,28 = 9.80, p < 0.05, micewith hM3Dq vector administered CNO before test froze morethan all other groups [Newman-Keuls]). All groups froze at equivalently high levels when an external cue (the tone previously
730 Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc.

paired with shock) was replayed during a subsequent memory
test (Figure 6D, middle; Vector X CNO Injection at Test, F1,28 =0.37, p > 0.05), indicating that all groups were equally capableof strongly expressing the memory if presented with a retrievalcue that closely matched the training cue (Koutstaal et al.,2001). Importantly, increased freezing was not observed if
mice with hM3Dq vector were administered CNO and placedin a novel context before the training session, indicating that simply activating a small population of LA neurons alone does not
induce freezing (Figure 6D, right; no significant effect of Vector[hM3Dq versus GFP] [all mice administered CNO beforeNeuronIncreasing Excitability in LA Enhances Memory

Figure 7. Increasing Excitability in a Small
Portion of LA Neurons Using Optogenetics
Immediately before Training Enhances
Memory Formation(A) Primary hippocampal neurons transfected with
ChR2 (but not neighboring untransfected neurons)
show depolarization during, but not before, illumination with a blue light. ChR2 construct, n = 9
neurons; untransfected, n = 4 neurons.
(B) Optogenetically activating a small portion of LA
neurons immediately before training enhanced
memory formation. GFP vector + no light n = 6
mice, GFP vector + light n = 6 mice, ChR2 vector +no light n = 9 mice, ChR2 vector + light n = 11 mice.
Data presented are mean ± SEM. n.s., not statistically different.

training], F1,30 = 0.01, p > 0.05). These data support the interpretation that nonspecifically increasing activity in a random, small
population of LA neurons (that are not part of the fear memory
trace) does not induce freezing. Instead, these results indicatethat artificially reactivating a key component of the fear memory
trace (those neurons with increased excitability at the time oftraining) is sufficient to induce memory recall. The finding thatfreezing was lower following artificial reactivation of a memory
trace than following exposure to the CS that predicted shock isin keeping with the encoding specificity principle, which suggests that memory retrieval is most effective when information
available at encoding is also present at retrieval (Tulving andThomson, 1973).

before training was sufficient to enhance memory formation (Figure 7B; Vector [ChR2 versus GFP] X Light condition immediately
before training (light versus no light) ANOVA showed a significant
interaction, F1,28 = 6.83, p < 0.05; only mice microinjected with
ChR2 and trained immediately after light on showed enhanced
memory; there was no difference in freezing levels between
mice with GFP vector in either the light or no-light condition or
mice with ChR2 vector in the no-light condition [NewmanKeuls]). Together, these data support the conclusion that
increased neural excitability at the time of training mediates
memory formation and neuronal allocation to a memory trace.

Using Optogenetic Mediators of Neural Excitability toTransiently Increase Excitability in a Small Portion of LA
Neurons Immediately before Training Enhances Fear
Memory Formation
To gain further temporal precision over neuronal excitability, weused HSV to express channelrhodopsin-2 (ChR2), a light-activated cation channel, in a small portion of LA neurons. When
activated by blue light, ChR2 induces depolarization (Deisseroth,
2011; Sparta et al., 2013; Tye and Deisseroth, 2012). We
confirmed this effect in cultured hippocampal neurons (Figure 7A; two-way ANOVA on membrane potential as a measureof excitability with within-group factor Light [before versus during] and between-group factor Transfection [transfected with
ChR2 versus untransfected] revealed a significant interaction,
F1,11 = 9.47, p < 0.05; posthoc Newman-Keuls tests revealed
there was no difference between untransfected hippocampal
culture depolarization before and during blue light; however,neurons transfected with ChR2 showed significantly more depolarization during light).
We microinjected HSV expressing ChR2 or control GFP into
the LA of mice as above. Immediately before training (in whicha tone CS was paired with a 0.5 mA shock), we illuminated theLA with blue (473 nm) light pulses (continuous train at 20 Hz lightpulses, with a 5 ms pulse for 30 s immediately before tone-shock
pairing). Additional groups were similarly microinjected with
ChR2 or GFP vector but were trained with the light off. All mice
were given a tone test (without light) 24 hr after training. Optogenetically activating a small portion of LA neurons immediately

Together, these experiments indicate that increasing excitability(without directly manipulating CREB function) of a small, random
population (10%) of LA pyramidal/principal neurons immediately before training was sufficient both to bias neuronal memory
allocation and enhance memory formation. Moreover, the memory-enhancing effect of increasing CREB function was prevented by decreasing excitability specifically in these neurons.
Artificial reactivation of neurons that were highly excitable during
training subsequently served as a memory retrieval cue in theabsence of external retrieval cues, indicating that activation ofthis small population of neurons was sufficient for memory
expression. Therefore, these results indicate that memory allocation is at least partially based on relative neuronal excitability
at the time of training.
Our finding that increasing excitability in LA neurons mediates
neuronal fear memory allocation is also consistent with previous
findings examining avoidance learning in the piriform cortex(Choi et al., 2011). Choi and colleagues used lentivirus to express
ChR2 in a random population of roughly 10% of excitatory neurons in the piriform cortex. Optogenetic activation of this small
population of neurons was paired with a shock in an avoidance
paradigm. In a memory test, activation of these neurons alone
was sufficient to induce avoidance, suggesting that these neurons were allocated to the avoidance memory trace, and that
subsequent activation of these neurons alone could serve as aretrieval cue for this memory.
Our finding that neurons are recruited to a memory trace
based on neuronal excitability was predicted by a recent

DISCUSSION

Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc. 731

NeuronIncreasing Excitability in LA Enhances Memorybiophysical modeling study. Kim and colleagues (Kim et al.,2013) used a 1,000-LA-cell conductance-based model, which
mirrored the known relative composition of principal neurons
and interneurons in the LA. To test the prediction that neurons
were allocated to a memory trace based on their relative excitability, principal neurons were randomly divided into three
categories that differed only with respect to their intrinsic excitability (low, medium, or high excitability produced by differential
expression of K+ currents) and not to their initial type or numberof inputs. The network was trained (‘‘auditory fear conditioned’’)and the proportion of principal neurons assigned to the low, medium, or high excitability categories which showed plasticity to
the tone CS (neurons with increased CS responsiveness after
training, as a proxy of neurons recruited into the memory trace)compared. Similar to our experimental results, this model founda much larger proportion of plastic cells (analogous to our arc+
memory trace neurons) among the principal cells assigned tothe high excitability category (analogous to our neurons infectedwith either CREB or dnKCNQ2 vector) than would be expectedby chance. Specifically, while only 1% of model LA neurons
with low excitability became part of the memory trace, over
40% of the more excitable neurons (medium and high excitability) were allocated to the memory trace.
Here we observed that CREB and dnKCNQ2 expression not
only increased the likelihood that the infected neurons wereallocated to the memory trace, but these manipulations also
decreased the likelihood that noninfected neighboring neurons
were allocated to the memory trace. Consistent with this, weobserved that the overall size of the LA memory trace (neurons
arc+ after a memory test) did not vary with any of our manipulations or level of freezing during the test. Indeed, we previously
showed that a similar size of memory trace supports both strong
and weak fear memories (Han et al., 2007). This finding indicatesthat the strength of memory is not coded by number of neurons
in the LA fear memory trace. In addition, this observation implies
that although competition takes place between neurons withhigh versus low intrinsic excitability, there is an additional formof competition that may also play a critical role in neuronal allocation to a memory trace. Kim et al. suggest that the substrate ofthis additional form of competition is disynaptic inhibition. Thismodeling study showed that the outcome of neuronal competition is also mediated by the distribution of excitatory connections
between principal cells and the amount of disynatpic inhibition
they generate in other projection cells. That is, collections of principal LA neurons band together to form a Hebbian ensemble,
which suppresses plasticity in other principal cells via the recruitment of inhibitory interneurons.
Intrinsic excitability is regulated by several mechanisms (Aizenman et al., 2003; Daoudal and Debanne, 2003), including
learning. Across several species, learning transiently increases
intrinsic excitability of some neurons (Alkon, 1974, 1984; Alkon
et al., 1985; Moyer et al., 1996; Oh et al., 2003; Thompson
et al., 1996) by downregulating K+ currents which mediate the
AHP (Saar et al., 2002). In the present experiments, we artificiallymodified intrinsic excitability by overexpressing CREB, manipulating K+ channel function, or using chemicogenetics or optogenetics. However, endogenous changes in intrinsic excitability arelinked to learning in a variety of species (from Hermissenda
732 Neuron 83, 722–735, August 6, 2014 ª2014 Elsevier Inc.

[Alkon, 1974] to rodents [Thompson et al., 1996]), suggestingthat the current manipulations, while artificial in nature, tap intoand mimic an endogenous underlying fundamental memory process. Moreover, this process of neuronal allocation based onrelative excitability may also play a role in ‘‘preplay,’’ a recently
described phenomenon in which the emergence of hippocampal
place cell firing is predicted by neuronal activity patterns that
occur in the minutes before actual exposure to a novel spatial
context (Dragoi and Tonegawa, 2011, 2013).
EXPERIMENTAL PROCEDURES
Mice
Adult (<12 weeks of age) male and female F1 hybrid (C57BL/6NTac X 129S6/
SvEvTac) mice were used for all experiments, except where noted. All procedures were conducted in accordance with policies of the Hospital for Sick Children Animal Care and Use Committee and conformed to both the CanadianCouncil on Animal Care (CCAC) and NIH Guidelines on the Care and Use ofLaboratory Animals.
HSV Vectors
Wild-type full-length CREB (kindly provided by Dr. Satoshi Kida, Tokyo University of Agriculture, Tokyo, Japan), dnKCNQ2 (hKCNQ2-G279S, kindly provided
by Dr. Dirk Isbrandt, DFG Heisenberg Team Experimentelle Neuropädiatrie),
Kir2.1 (kindly provided by Dr. Eric Nestler, Mt. Sinai, NY), hM3Dq (kindly provided by Dr. Bryan Roth, University of North Carolina), and ChR2 (fused with
YFP, kindly provided by Dr. Karl Deisseroth, Stanford) cDNAs were subcloned
into an HSV vector backbone that coexpresses GFP as a fluorescent reporter(HSV-p1005 [Russo et al., 2009]). Transgene expression using this viral system
typically peaks at 3 days, and dissipates within 7–12 days, following microinjection (Barrot et al., 2002; Cole et al., 2012; Josselyn et al., 2001; Vetere
et al., 2011). The average titer of the virus stocks was 4.0 3 107 infectious
units/ml. See Supplemental Experimental Procedures for details.
Surgery
Mice were pretreated (atropine sulfate 0.1 mg/kg, i.p.), anesthetized (chloral hydrate, 400 mg/kg, i.p.), and viral vectors were infused (1.5 ml/side, 0.1 ml/min)into the LA (AP = 1.45, ML = ± 3.45, V = 5.0 mm from bregma), CeA (AP =1.35, ML = ± 3.3, V = 5.3 mm from bregma), or BA (AP = 1.4, ML = ±3.45, V = 5.5 mm) (Paxinos and Franklin, 2001). For the optogenetic experiment, mice were similarly microinjected with HSV encoding ChR2, and bilateral
optical fibers were implanted slightly above each LA (Sparta et al., 2012; Stuber
et al., 2011).
Electrophysiology
Hippocampal Cultured Neurons
Low-density cultures of dissociated mouse hippocampal neurons were prepared as previously described (Acton et al., 2012). After 10–15 days in culture,neurons were transfected with DNA constructs (GFP, CREB, dnKCNQ2,
Kir2.1, CREB+Kir2.1). Electrophysiological recordings were performed on
both transfected and untransfected (control) neurons from the same culture
dish; transfected neurons were identified by GFP fluorescence. Neuronal
excitability was determined in current-clamp mode by injecting current in
10 pA steps from 50 pA to 150 pA.
To verify that hM3Dq expression increased neuronal excitability following
CNO, and that ChR2 expression increased excitability upon blue light application, we estimated the change in the baseline membrane potential for neurons
transfected with these constructs (as well as untransfected controls) both
before and after CNO/light, respectively.
Acute Ex Vivo LA Slices
Mice (4 weeks of age) were microinjected with viral vectors (GFP, CREB,
dnKCNQ2) into the LA, as above. After 1 day, brains were removed and placed
into chilled modified aCSF. Coronal slices (350 mM) containing the LA wereprepared. Recordings were obtained from infected neurons (identified by
GFP fluorescence in the LA) and their uninfected neighbors.

NeuronIncreasing Excitability in LA Enhances Memory

Stereological Cell Counting
To determine the number of neurons infected by our viral microinjections, weused unbiased stereological principles and systematic sampling techniques toestimate the percentage of the total number of LA neurons infected by our viral
manipulations. See Supplemental Experimental Procedures for details.

SUPPLEMENTAL INFORMATION

cFos Immunostaining
To further verify that the DREADD hM3Dq increases neuronal activity in vivo
only following CNO administration, we examined cFos levels (as a marker ofneuronal activity) in infected cells. Mice were microinjected with hM3Dq or
GFP vector, and 2 days later these homecage mice were administered VEH
or CNO systemically. After 90 min, mice were perfused transcardially with
4% PFA. Brains were sliced coronally (50 mm) and incubated with antibodies
directed against cFos (Rabbit, K0306; 1:1,000, Calbiochem) and GFP(Chicken, 1:1,000, Millipore). The number of infected neurons that were also
positive for cFos was assessed by two experimenters blind to the treatment
condition. See Supplemental Experimental Procedures for details.

AUTHOR CONTRIBUTIONS

Auditory Fear Training and Testing
Auditory Fear Conditioning
Fear conditioning training consisted of placing mice in a conditioning chamberand 2 min later presenting a tone (2,800 Hz, 85 dB, 30 s) that coterminated witha shock (2 s, 0.3 mA = weak training; 2 s, 0.6 mA = strong training; 0.5 mA for
optogenetic experiment; Figure 7B). For the optogenetic experiment, the implanted optrodes were tethered to a laser source (473 nm; Laserglow) througha split optic fiber (Precision Fiber Products) during conditioning. Light stimulation, given for 30 s immediately preceding the tone, consisted of a 20 Hz trainof 5 ms pulses, and the average output light power was calibrated to 1 mW at
each fiber end.
Auditory Fear Testing
Testing for auditory fear conditioning occurred 24 hr after conditioning (except
where explicitly stated). Mice were placed in a novel chamber, and 2 min later
the tone CS was presented (for 1 min). The amount of freezing (defined as animmobilized, crouched position, with an absence of any movement except
respiration [Blanchard and Blanchard, 1969; Bolles and Fanselow, 1982]) during tone was assessed.
Statistical Analysis
Amount of freezing spent during the tone was compared across groups by
ANOVA. Where appropriate, significant effects were further analyzed using
Newman-Keuls or Fisher’s Least Square Difference (LSD).

ACKNOWLEDGMENTS

Assessing Anxiety-like Behavior
Open Field
Mice were placed in an open field for 10 min, and total distance and amount oftime spent in the periphery were examined.
Elevated plus Maze
Mice were placed in the center of the maze, and behavior was monitored for
5 min. We analyzed time in open versus closed arms, crossings in open versus
walled (closed) arms, and total distance traveled. A decrease in open arm time
and/or open arm crossings is taken to reflect an anxiety-like phenotype.

Aizenman, C.D., Huang, E.J., and Linden, D.J. (2003). Morphological correlates of intrinsic electrical excitability in neurons of the deep cerebellar nuclei.
J. Neurophysiol. 89, 1738–1747.
Alkon, D.L. (1974). Associative training of Hermissenda. J. Gen. Physiol. 64,
70–84.

catFISH or arc Fluorescent In Situ Hybridization for the ActivityDependent Gene arc
We identified neurons specifically activated by the fear memory test (and presumably part of the fear memory trace), and we used the cellular localization ofarc mRNA as a marker of neuronal activity, a technique that takes advantage ofthe unique transcriptional time course of the activity-dependent gene arc (Guzowski et al., 1999, 2001), as previously described (Han et al., 2007, 2009).

Armbruster, B.N., Li, X., Pausch, M.H., Herlitze, S., and Roth, B.L. (2007).
Evolving the lock to fit the key to create a family of G protein-coupled receptors
potently activated by an inert ligand. Proc. Natl. Acad. Sci. USA 104, 5163–
5168.

CNO
CNO (Toronto Research Chemicals [TRC]) was made in a stock solution of
10 mg/ml in DMSO and then diluted in saline to desired concentration. CNO
was injected at a dose of 2.0 mg/kg i.p., 15 min and 3 hr before training. For
the after-training (Figure 6B) and before-testing (Figure 6D) experiments, we
injected the same dose of drug.
Description of the experimental procedures is included in the Supplemental
Experimental Procedures.

Supplemental Information includes one figure and Supplemental Experimental
Procedures and can be found with this article online at http://dx.doi.org/10.
1016/j.neuron.2014.07.017.

S.A.J. and P.W.F. designed, directed, and coordinated the study along with
S.A.K. In addition, A.P.Y. and H.L.H. conducted behavioral experiments andperformed the surgeries with help from M.M.T., and V.M. designed and performed the cell culture and immunohistochemistry experiments. C.Y. performed the in situ hybridization. A.J.R. designed the viral constructs andgenerated viral vectors. B.R., J.P., V.M., and M.A.W. designed and performed
the electrophysiological experiments in the M.A.W. lab. A.P.Y. performed statistical analysis. S.A.J. and P.W.F. wrote the manuscript, with help from all
authors.

We thank Mika Yamamoto, Russell Braybon, and Antonietta (Toni) De Cristofaro for excellent technical assistance and members of the S.A.J./P.W.F. labfor their helpful suggestions throughout this project. This work was supported
by grants from the Canadian Institutes of Health Research (CIHR; MOP74650), Brain and Behavior Foundation (NARSAD), EJLB Foundation, and
the Alzheimer’s Society of Canada to S.A.J., and CIHR (MOP- 86762) to
P.W.F., and an NSERC Discovery Grant to M.A.W. In addition, A.P.Y.,
A.J.R., and C.Y. received support from Restracomp Fellowships (Hospitalfor Sick Children), A.P.Y. from the Alzheimer’s Society of Canada, C.Y. froma CIHR training grant (Sleep and Biological Rhythms Toronto), and B.R. was
supported by a Banting Postdoctoral Fellowship (CIHR).
Accepted: July 2, 2014
Published: July 24, 2014
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