Scientists discover brain “glue” that helps memories last a lifetime
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Summary:
Scientists discover brain “glue” that helps memories last a lifetime.
A groundbreaking study has revealed the critical role of the molecule KIBRA in forming long-term memories, acting as a "glue" that helps stabilize and strengthen synapses. Researchers found that KIBRA binds with an enzyme called PKMzeta, which plays a key role in memory retention. This discovery could pave the way for new treatments for memory-related conditions like Alzheimer's and other cognitive disorders.
Memory formation has always been a mystery, particularly how memories remain stable despite the constant turnover of molecules in our brain. Neurons store information by strengthening certain synapses, but these synapses are constantly changing, making it hard to understand how long-term memories endure. This study, published in Science Advances, offers a solution by showing how KIBRA works with PKMzeta to maintain the stability of these synapses over time.
The researchers focused on KIBRA, a protein that has been linked to memory performance in humans. They discovered that KIBRA interacts with PKMzeta at synapses involved in memory formation. Once these synapses are activated, KIBRA attaches to them, and PKMzeta binds to the KIBRA tag, ensuring the synapses remain strong. This process helps the memory persist even as the molecules involved are constantly replaced.
One of the most fascinating aspects of this discovery is that breaking the bond between KIBRA and PKMzeta can erase memories. The researchers also explained how their findings clarify why enhancing PKMzeta in the brain can improve weak or faded memories, by acting only on the synapses tagged by KIBRA. This persistent synaptic tagging mechanism provides a new understanding of memory storage and offers insight into neurological and psychiatric disorders.
The study also connects to a hypothesis proposed by Francis Crick in 1984, which likened memory storage to the maintenance of Theseus’s Ship, where new components replace old ones while maintaining the integrity of the structure. This analogy now makes sense, thanks to the discovery of KIBRA and PKMzeta and their role in memory formation.
KIBRA anchoring the action of PKMζ maintains the persistence of memory
Panayiotis Tsokas https://orcid.org/0000-0002-1679-8980, Changchi Hsieh https://orcid.org/0000-0003-0488-1565, Rafael E. Flores-Obando https://orcid.org/0000-0002-0736-9527, Matteo Bernabo, Andrew Tcherepanov https://orcid.org/0009-0001-3424-2812, A. Iván Hernández, Christian Thomas https://orcid.org/0000-0002-6642-7774, Peter J. Bergold, James E. Cottrell,
and Todd C. Sacktor https://orcid.org/0000-0002-8625-8701Authors
Info & Affiliations
Abstract
How
can short-lived molecules selectively maintain the
potentiation of activated synapses to sustain long-term
memory? Here, we find kidney and brain expressed adaptor
protein (KIBRA), a postsynaptic scaffolding protein
genetically linked to human memory performance, complexes with
protein kinase Mzeta (PKMζ), anchoring the kinase’s
potentiating action to maintain late-phase long-term
potentiation (late-LTP) at activated synapses. Two
structurally distinct antagonists of KIBRA-PKMζ dimerization
disrupt established late-LTP and long-term spatial memory, yet
neither measurably affects basal synaptic transmission.
Neither antagonist affects PKMζ-independent LTP or memory that
are maintained by compensating PKCs in ζ-knockout mice; thus,
both agents require PKMζ for their effect. KIBRA-PKMζ
complexes maintain 1-month-old memory despite PKMζ turnover.
Therefore, it is not PKMζ alone, nor KIBRA alone, but the
continual interaction between the two that maintains late-LTP
and long-term memory.
INTRODUCTION
How
molecules lasting only hours to days can maintain memory
that persists weeks to years is a long-standing
fundamental question in neuroscience (1, 2). In 1984,
Crick (1) proposed
that the continual interaction between synaptic proteins
maintains the strengthening of synapses in the face of
molecular turnover. Such a general mechanism can be
constrained by the requirement of most theories of memory
that information is stored by the persistent enhancement
of only the activated synapses of a neuron (3). Long-term
potentiation (LTP) is widely considered a putative
physiological substrate of memory because strong afferent
synaptic stimulation persistently potentiates only
activated synaptic pathways; unstimulated pathways remain
unchanged (4). Thus, the
molecular interaction that maintains synaptic enhancement
might also continually target the action of potentiating
molecules to activated synapses. Progress toward
elucidating this mechanism has been slow, however, because
the molecules potentiating synaptic transmission during
late-LTP and memory maintenance have not been clearly
established.
One
molecule that persistently potentiates synaptic
transmission is the autonomously active protein kinase C
(PKC) isoform, protein kinase Mzeta (PKMζ) (5–10). PKMζ
consists of the independent catalytic domain of the
atypical isoform PKCζ. Unlike other PKCs, PKMζ lacks an
autoinhibitory regulatory domain and is constitutively and
thus persistently active without second messenger
stimulation (11). Instead,
the amount of PKMζ determines its activity (5, 11). PKMζ is
selectively expressed in neurons from a dedicated PKMζ
mRNA, which is transported to dendrites and, under basal
conditions, is translationally repressed (12–15). Strong
afferent synaptic stimulation derepresses the PKMζ mRNA (12). This
derepression up-regulates new PKMζ synthesis and increases
the amount of the kinase in neurons (12, 16–19), including
in dendritic spines and postsynaptic densities (20). In
late-LTP maintenance recorded in hippocampal slices, the
increased steady-state amount of PKMζ persists for hours
in CA1 pyramidal cells (16, 17, 19). In
spatial long-term memory maintenance, the increases last
for weeks in selective hippocampal neuronal circuits that
were transcriptionally active during initial memory
formation (19). Spatial
memory formation also persistently increases PKMζ in
extrahippocampal regions involved in spatial information
processing such as retrosplenial cortex but not in
thalamus (19). Likewise,
skilled motor learning resulting in long-term procedural
memory increases PKMζ for over a month in sensorimotor
cortex (21). In
contrast to all other PKCs, the persistent increases in
PKMζ in LTP and long-term spatial memory maintenance
correlate with the extent of persistent synaptic
potentiation (11, 16, 17) and memory
retention (18).
Evidence
for PKMζ’s causal role in maintenance initially came from
ZIP, an inhibitor of the kinase’s catalytic site that,
unlike inhibitors of any other signaling molecule,
disrupted established late-LTP and long-term memory
without affecting basal synaptic transmission (5, 7). However,
knockout mice lacking PKMζ (Prkcz−/− mice;
PKMζ-null mice) still express LTP and memory that is
reversed by ZIP (22, 23). These
mutant mice, however, compensate for the loss of PKMζ by
the persistent activation of PKCs that show short-term
increases in wild-type mice, including another atypical
isoform, PKCι/λ, that is also sensitive to ZIP (17). Blocking
PKMζ synthesis with short hairpin RNA (shRNA) or antisense
oligodeoxynucleotides that selectively suppress the
translation of PKMζ mRNA, but not PKCι/λ mRNA, prevents
late-LTP and long-term memory formation in wild-type
animals (17, 24, 25). Thus,
PKMζ synthesis is necessary for wild-type late-LTP and
long-term memory, and PKMζ action is sufficient to
potentiate synaptic transmission (5, 6, 17, 25).
LTP
induction that increases PKMζ synthesis within a neuron (19) results in
potentiation exclusively at activated synapses during LTP
maintenance (17). Likewise,
memory training increases PKMζ (19) as well as
synaptic strength (26) in
selective dendritic compartments of memory-activated
hippocampal neurons for at least a month despite evidence
of the kinase’s rapid turnover (27, 28). Moreover,
viral overexpression of PKMζ within neocortical neurons
does not degrade memory, as predicted by saturating
potentiation of all synapses (29, 30); instead,
it enhances previously established long-term memory,
presumably by strengthening a subpopulation of synapses
activated during learning (31). PKMζ,
however, lacks the regulatory domain by which other PKCs
translocate to membrane (11).
Therefore, how PKMζ action persistently targets activated
synapses remains unclear but could be through interaction
with another molecule.
A
clue to such a partner of PKMζ might come from human
memory (32). Human
episodic memory is a highly polygenic trait involving
genes linked in networks (33), and one
important network node encodes a member of the WW and C2
domain–containing protein (WWC) family, the PKMζ-binding,
postsynaptic scaffolding protein, kidney and brain
expressed adaptor protein (KIBRA, also known as WWC1) (28, 32, 34, 35). Human
genome-wide studies reveal that alleles of the KIBRA gene
associate with variation in normal memory performance (32, 33). KIBRA’s
function in memory might be evolutionarily conserved
because overexpression of a dominant-negative KIBRA in
cultured neurons of the molluscan model system Aplysia disrupts
a cellular analog of classical conditioning (36, 37), and
knockdown of KIBRA gene
expression in mice blocks LTP and memory formation (28, 38–40).
Coexpressing KIBRA with PKMζ in cultured cells stabilizes
the kinase and increases its steady-state level by
decreasing PKMζ’s elimination through the
ubiquitin-proteosome pathway (28, 36, 37). However,
an interaction between endogenous KIBRA and PKMζ in
neurons has not been observed. We therefore began by
asking whether synaptic stimulation facilitates persistent
KIBRA-PKMζ interactions in late-LTP maintenance.
RESULTS
KIBRA-PKMζ complexes persistently increase in LTP maintenance
We
used in situ proximity ligation assay (PLA) to detect
molecular complexes of KIBRA and PKMζ in late-LTP
maintenance (Fig.
1 and fig. S1A). In PLA, pairs of
antibodies are linked to oligonucleotides, and if the
molecules recognized by the antibodies are within 40 nm,
the oligonucleotides generate a DNA that is amplified
and detected by fluorescent probes (Fig.
1A) (41). To
establish late-LTP, we prepared mouse hippocampal slices
and briefly tetanized Schaffer collateral/commissural
fibers in CA3 stratum (st.) radiatum to potentiate field
excitatory postsynaptic potential (fEPSP) responses
recorded in CA1 st. radiatum for 3 hours (Fig.
1B) (42). As
control, we recorded low-frequency test responses in an
adjacent slice from the same hippocampus for the
equivalent time. The strong stimulation facilitates
persistent increases in KIBRA-PKMζ complexes in st.
radiatum (Fig.
1B), which accumulate in ~1-μm puncta along CA1
pyramidal cell dendrites (Fig.
1C). In contrast, the complexes do not increase in
st. lacunosum-moleculare that receive unstimulated
synaptic projections, despite high levels of complexes
under basal conditions in this dendritic region. The
relatively few complexes in CA1 pyramidal cell bodies of
st. pyramidale also do not increase.
In
parallel with PLA, we examined KIBRA and PKMζ
colocalization a second way by measuring the proteins
individually by immunocytochemistry (Fig.
2 and fig. S1B). Stimulation
persistently increases the amounts and colocalization of
KIBRA and PKMζ in st. radiatum (Fig.
2A). The colocalized proteins accumulate in ~1-μm
puncta along CA1 pyramidal cell dendrites (Fig.
2B). Comparing immunocytochemistry and PLA in CA1
pyramidal cell bodies, however, reveals high levels of
KIBRA and PKMζ yet low levels of KIBRA-PKMζ complexes;
therefore, somatic KIBRA and PKMζ do not appear to
interact (Figs.
1B and 2A).
Thus, strong afferent synaptic activity acts locally
within neurons to persistently increase KIBRA-PKMζ
interaction.
Blocking the KIBRA-binding site in PKMζ reverses late-LTP maintenance
As
most signaling events triggered by strong afferent
synaptic activity last for only seconds to minutes (11, 43–45), the
persistence of KIBRA-PKMζ complexes for hours in
late-LTP suggests that sustained KIBRA-PKMζ interaction
might maintain late-LTP in wild-type mice. To test this
“KIBRA-PKMζ maintenance” hypothesis, we used the
small-molecule PKMζ-inhibitor, ζ-stat
(1-naphthol-3,6,8-trisulfonic acid), which has been
proposed to selectively block the allosteric
KIBRA-binding site in the ζ-catalytic domain (Fig.
3A) (37). To
assess this mechanism of action, we isolated KIBRA-PKMζ
heterodimerization/multimerization using the split-Venus
bimolecular fluorescence complementation reporter assay
(BiFC) (Fig.
3B) (28). In this
assay, KIBRA and PKMζ fused with complementary fragments
of the fluorescent reporter Venus are transfected into
human embryonic kidney (HEK) 293T cells, and the
interaction of KIBRA and PKMζ produces a fluorescent
signal by bringing the two fragments of split-Venus into
close apposition (Fig.
3B, left, and fig. S2) (28). BiFC
reveals that ζ-stat inhibits KIBRA-PKMζ interaction
[median inhibitory concentration (IC50)
= ~1 μM; Fig.
3B, middle]. In contrast, the ζ-stat at 10 μM has
no measurable effect on the interaction of KIBRA with
the other atypical isoform, PKCι/λ, which produces BiFC
with KIBRA ~10-fold less than PKMζ (Fig.
3B, right). We further validated ζ-stat’s
mechanism of action by generating a mutant
PKMζ[PKCι/λ-P291Q;F297S] with the ζ-stat–binding site in
PKMζ changed to the analogous amino acids in PKCι/λ.
Interaction of KIBRA with PKMζ[PKCι/λ-P291Q;F297S] is
similar to PKCι/λ, and, like PKCι/λ, ζ-stat does not
inhibit complexes of mutated PKMζ with KIBRA. ζ-stat
also has no effect on KIBRA’s interaction with any
conventional or novel PKC, the two other classes of PKC
isoforms (fig. S3, A and B). Ca2+/calmodulin-dependent
protein kinase II alpha (CaMKIIα), another kinase that
potentiates synaptic transmission and is important for
early-LTP (44, 46), does
not measurably interact with KIBRA (fig. S3C).
Now,
we can test the central prediction of the KIBRA-PKMζ
maintenance hypothesis that decoupling PKMζ from KIBRA
reverses potentiation of activated synapses. We
simultaneously recorded two independent synaptic
pathways in st. radiatum within a hippocampal slice. We
stimulated one pathway by high-frequency tetanization to
induce late-LTP, and in the second pathway, we recorded
low-frequency test fEPSP responses for the equivalent
time. After establishing late-LTP for 3 hours, we
applied 10 μM ζ-stat to the bath and recorded responses
in both pathways for 4 hours (Fig.
4A and fig. S4A). ζ-stat reverses
late-LTP maintenance in the stimulated synaptic pathway
without measurably affecting synaptic transmission in
the control pathway. Thus, ζ-stat disrupts the
maintenance of enhanced synaptic transmission
selectively at activated synapses, as predicted by the
KIBRA-PKMζ maintenance hypothesis.
We
tested if the effect of ζ-stat requires PKMζ by
examining PKMζ-null mice that recruit compensatory
PKMζ-independent mechanisms of late-LTP (17). In
notable contrast to wild-type mice, ζ-stat has no effect
on late-LTP maintenance in mice lacking PKMζ (Fig.
4B and fig. S4A).
If
KIBRA-PKMζ coupling in wild-type mice maintains LTP,
then, when ζ-stat is washed out, the reversal of
potentiation should persist. If another mechanism
maintains LTP and KIBRA-PKMζ coupling is a transient,
downstream signaling pathway that expresses synaptic
potentiation, then, when the drug is washed out,
potentiation should return. To distinguish between the
KIBRA-PKMζ maintenance hypothesis and this alternative
KIBRA-PKMζ downstream-expression hypothesis, after
establishing wild-type late-LTP, we applied ζ-stat for 3
hours and then washed the drug out for an additional 4
hours. The reversal of potentiation persisted (Fig.
5A and fig. S4B). To examine if the
washout was effective, in separate experiments, we
applied the drug for the same 3-hour duration,
suppressing late-LTP in one synaptic pathway, and then
initiated the washout (Fig.
5B and fig. S4C). After only 1 hour
of washout, we stimulated a second synaptic pathway and
produced late-LTP, indicating effective drug washout.
These results are predicted by the KIBRA-PKMζ
maintenance hypothesis and not by the KIBRA-PKMζ
downstream-expression hypothesis.
Blocking the KIBRA-binding site in PKMζ disrupts established long-term memory
We
next tested the predictions of the KIBRA-PKMζ
maintenance hypothesis in active place avoidance, a
hippocampus-dependent spatial long-term memory task (Fig.
6). We rapidly conditioned wild-type mice to
actively avoid a shock zone by three 30-min training
sessions with 2-hour intertrain intervals and then, 1
day later, injected ζ-stat or vehicle bilaterally in
hippocampus. Two days after the injection, we tested
long-term memory retention with the shock off. Mice that
had received vehicle remember to avoid, whereas mice
that had received ζ-stat show persistent loss of
retention for the shock zone location. In contrast,
PKMζ-null mice receiving ζ-stat avoided the shock zone
as well as those that had received vehicle, thus
controlling for off-target effects of the drug in memory
maintenance.
We
examined if ζ-stat disrupts maintenance as opposed to
other aspects of memory (fig. S5A). Repeating the
experiments in wild-type mice, we measured the
persistent loss of long-term memory retention without
shock and then immediately retrained the mice to avoid
another shock zone defined by the cues of a novel
context. The mice that had previously been injected with
ζ-stat do not remember the location of the first shock
zone that was established before the injection, but they
learn to avoid and remember a new second location as
well as the mice that had received only vehicle. The
mice that had earlier received ζ-stat have learning
curves in the second context indistinguishable from
vehicle-treated mice, demonstrating that ζ-stat does not
suppress expression of the avoidance behavior (fig.
S5B). Thus, ζ-stat persistently disrupts previously
acquired information but, once eliminated, does not
impair formation, maintenance, or expression of newly
acquired spatial information.
We
also examined the consequences of KIBRA-PKMζ decoupling
on auditory-cued fear/threat memory maintenance in the
basolateral amygdala (BLA) of wild-type and PKMζ-null
mice (Fig.
7). One day after conditioning, both wild-type and
PKMζ-null mice show increased freezing to tone,
confirming previous findings in PKMζ-null mice (23). Then, 1
day after the first memory retention testing, the mice
were injected with ζ-stat or vehicle bilaterally in BLA
and retested the following day. The results reveal that
ζ-stat impairs memory retention in wild-type mice and
not in PKMζ-null mice. We note that the ζ-stat treatment
in wild-type mice results in freezing that is
indistinguishable from freezing before presentation of
the conditioned stimulus [for analysis of variance
(ANOVA), see Fig.
7 legend; post hoc analysis shows P =
0.34].
As
PKMζ-null mice show a form of PKMζ-independent memory
maintenance, we asked if wild-type mice might as well.
Previous results with the first-generation
PKMζ-inhibitor ZIP revealed that, whereas the agent
disrupts cued fear/threat memory, ZIP in hippocampus
does not affect contextual fear/threat memory (47, 48). We
found that, like ZIP, ζ-stat has no effect on contextual
fear/threat memory in hippocampus, in either wild-type
mice or PKMζ-null mice (fig. S6). Therefore, wild-type
mice store information by both PKMζ-dependent mechanisms
and, like PKMζ-null mice, PKMζ-independent mechanisms.
Peptide mimicking the PKMζ-anchoring site in KIBRA reverses late-LTP maintenance and disrupts long-term memory
To
further test that blocking KIBRA-PKMζ interaction is an
effective way to reverse LTP and memory, we inhibited
KIBRA-PKMζ dimerization using a cell-permeable,
myristoylated peptide that mimics KIBRA’s PKMζ-anchoring
sequence [myr-N-FVRNSLERRSVRMKRPS-C, KIBRA-based zeta
antagonist peptide (K-ZAP); Fig.
8, A and B] (28). K-ZAP
applied 3 hours after tetanization reverses late-LTP
maintenance in wild-type mice, with no measurable effect
on baseline synaptic transmission (Fig.
8C and fig. S7). Like ζ-stat, K-ZAP
has no effect on late-LTP maintenance in PKMζ-null mice.
Intrahippocampal injection of K-ZAP 1 day after active
place avoidance conditioning disrupts established
spatial long-term memory retention assayed 2 days after
injection in wild-type mice (Fig.
9A). K-ZAP has no effect on memory retention in
PKMζ-null mice.
Persistent KIBRA-PKMζ coupling maintains remote memory
We examined
if KIBRA-PKMζ interaction maintains memory despite PKMζ
turnover. PKMζ turns over within a few hours in cultured
hippocampal neurons (27) and
within days in hippocampus in vivo, as indicated by the
loss of PKMζ protein after shRNA knockdown of PKMζ mRNA
in wild-type animals (24, 25) or
inducible deletion of the PKMζ gene in Prkczfl/fl mice
(fig. S8). Although PKMζ molecules are replaced, active
place avoidance conditioning of wild-type mice produces
a stable persistent increase in the steady-state level
of PKMζ in hippocampus that lasts for over 4 weeks, and
the conditioned behavior remains hippocampus-dependent
in the face of systems consolidation (7, 19). We
therefore trained mice on active place avoidance and, 4
weeks later, injected either K-ZAP or vehicle
bilaterally in hippocampus (Fig.
9B). Retention testing 2 days after injection
reveals K-ZAP disrupts remote spatial memory.
DISCUSSION
Previous
efforts to understand how molecules store long-term memory
focused on the individual actions of single molecules (9), such as
the persistently active kinases CaMKII and PKMζ that
potentiate synaptic transmission (10, 46), or the
prion-like properties of translation factors that might
perpetuate protein synthesis (49). Other
investigators have searched for synaptic or extrasynaptic
proteins that are especially long-lived (50, 51). Here,
building upon a suggestion of Crick (1) that
persistent molecular interactions sustain memory, we
tested the hypothesis that the continual coupling between
an autonomously active kinase and a postsynaptic
scaffolding protein sustains late-LTP and long-term memory
(Fig.
10). Our results reveal that coupling of PKMζ and
KIBRA is necessary for long-term memory maintenance,
providing a mechanism that also addresses the general
question of how an increase in/activation of kinase
signaling specifically targets action only at activated
synapses. Thus, it is not PKMζ alone, nor KIBRA alone, but
the interaction between the two that maintains LTP and
memory (Figs.
4 to 9).
Synaptic
stimulation that induces LTP facilitates the formation of
KIBRA-PKMζ complexes that persist at least 3 hours in
late-LTP maintenance (Fig.
1), and functional KIBRA-PKMζ coupling maintains
late-LTP in hippocampal slices and memory lasting weeks in
vivo (Figs.
4 to 9).
This persistent coupling of KIBRA and PKMζ contrasts with
the activities of other molecules that last for only
seconds to minutes after strong synaptic stimulation,
including CaMKII, cyclic adenosine monophosphate–dependent
protein kinase, and most PKC isoforms (11, 43, 44), as well
as local modulators of actin dynamics (45) and
activators of gene expression that increase the synthesis
of proteins cell-wide (42, 52). These
signaling molecules also act transiently for only minutes
to hours during initial cellular memory consolidation (44, 45, 52), as
compared to the persistent action of KIBRA-PKMζ coupling
necessary for memory maintenance lasting weeks (Fig.
9B). Blocking PKMζ with antisense
oligodeoxynucleotides or shRNA shows no effect on learning
or initial short-term forms of memory while preventing
long-term memory (17, 25).
Therefore, coupling PKMζ action to KIBRA is likely not
essential for initial, transient forms of memory.
KIBRA-PKMζ coupling, however, could play a role in the
initiation of late-LTP and long-term memory, and this
important question requires investigating the coupling
during the formation of these fundamental processes.
We
propose that KIBRA acts as a synaptic tag aligning PKMζ at
activated synapses (Fig.
10). KIBRA interacts with postsynaptic
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptors (AMPARs), functioning as a scaffolding protein
to regulate trafficking of the receptors (38). Blocking
PKMζ from binding the KIBRA tag during and after
tetanization prevents the formation of late-LTP, allowing
for early-LTP (Fig.
5B). Therefore, a simple hypothesis for the
initiation of late-LTP is that the increased number of
postsynaptic AMPARs in early-LTP sequesters KIBRA at
activated synapses, and the scaffolding protein then acts
as a tag at these synapses to anchor PKMζ (Fig.
10). Whether KIBRA accumulates at activated synapses
first and PKMζ then binds the scaffolding protein or the
proteins translocate to activated synapses as dimers (as
shown in Fig.
10) requires further analysis of the translocation
and complex formation of KIBRA and PKMζ in LTP induction.
In LTP maintenance, both KIBRA and PKMζ are present in CA1
pyramidal cell somata, but they do not seem to be in a
complex (Figs.
1B and 2A).
This suggests that some other molecule, perhaps another
PKC competing with PKMζ for KIBRA (fig. S3, A and B), is
inhibiting their interaction in cell bodies.
Alternatively, the soma might lack an element present at
activated synapses that facilitates the alignment of KIBRA
and PKMζ. One possible facilitating molecule is protein
interacting with C kinase 1 (PICK1), which forms complexes
with both KIBRA and PKMζ (38, 53). PICK1,
KIBRA, and PKMζ all regulate GluA2 subunit-dependent AMPAR
trafficking (38, 53–55). KIBRA
also interacts with the actin-associated postsynaptic
proteins, synaptopodin, angiomotin, and dendrin (35), as well
as with other PKCs that are transiently activated in LTP
(fig. S3, A and B) (11). In
addition, KIBRA forms homodimers and also heterodimers
with other members of the WWC family (56) and
inducible genetic knockdown of KIBRA/WWC1 that suppresses
LTP and memory formation down-regulates WWC2 as well,
suggesting cooperative effects among WWC family members (40). Thus,
KIBRA-PKMζ coupling might be a core interaction within a
larger network of molecules that initiates “persistent
synaptic tagging.”
Once
established, the continual alignment of KIBRA and PKMζ
maintains late-LTP and long-term memory (Fig.
10). Antagonists that block the KIBRA-binding site
in PKMζ or mimic the PKMζ-binding site in KIBRA reverse
late-LTP at activated synapses when applied 3 hours after
tetanization and have no measurable effect on unactivated,
resting synaptic pathways in the same slice preparation (Figs.
4A and 8C)
or on baseline synaptic transmission (Fig.
5B). These data support the specificity of the
potentiating effects of KIBRA-PKMζ coupling in activated
synapses. The exclusive action of KIBRA-PKMζ inhibitors on
late-LTP maintenance contrasts with CaMKII inhibitors,
which either block LTP induction but not maintenance (57–59) or affect
LTP within an hour of tetanization (60). In
spatial memory, PKMζ increases selectively in
subpopulations of neurons active during memory formation
(marked by Arc promoter
activation) and in subsets of synaptic spines of these
active neurons (19). Further
work is required to show coupling of the PKMζ to KIBRA
specifically in activated synapses during memory storage
by techniques such as enhanced green fluorescent protein
reconstitution across synaptic partners (dual-eGRASP) (61).
Both
antagonists of KIBRA-PKMζ coupling erase established
late-LTP and long-term memory (Figs.
4A, 5A, 6, 7, 8C,
and 9,
and fig. S5); however, neither antagonist affects LTP or
memory in PKMζ-null mice (Figs.
4B, 6, 7, 8C,
and 9A).
The PKMζ-independent maintenance of these mutant mice and
also wild-type mice (fig. S6) could be through the
prolonged actions of other PKCs (17), CaMKII (60), or other
molecular mechanisms (49, 50, 62–65). The
transient increase in PKCι/λ during early-LTP in wild-type
mice becomes persistent during late-LTP in PKMζ-null mice
(17). Whether
KIBRA anchors and stabilizes PKCι/λ at activated synapses
after genetic deletion of PKMζ remains to be explored but
could provide a parsimonious account for compensation in
PKMζ-null mutant mice. An initial, weak PKCι/λ interaction
with KIBRA (Fig.
3B), which is displaced by the strong interaction of
newly synthesized PKMζ in wild-type mice, might persist in
the absence of PKMζ. A PKCι/λ inhibitor disrupts late-LTP
and long-term memory maintenance exclusively in PKMζ-null
mice and not wild-type mice (17). These
pharmacogenetic experiments with ζ and ι/λ antagonists
support (i) the KIBRA-PKMζ maintenance hypothesis that
late-LTP and long-term memory in wild-type mice share a
common PKMζ-dependent molecular mechanism of information
storage (7) and (ii)
mutant mice lacking PKMζ recruit compensatory
PKMζ-independent maintenance mechanisms (17).
The
persistent anchoring of PKMζ by KIBRA at activated
synapses that maintains physiological long-term memory
could help explain how overexpression of PKMζ and KIBRA
can enhance memory. PKMζ overexpression after memory
formation enhances weak long-term memories that have faded
over time (31). This
result was unexpected because overexpressed PKMζ might
enhance synapses indiscriminately and, as a consequence,
degrade information stored by differences in synaptic
weights in neuronal circuits (3, 29, 30). Our
findings, however, suggest the possibility that the
overexpressed PKMζ might target KIBRA at appropriate
activated synapses to replace the endogenous PKMζ that,
like synaptic potentiation (26), declines
as memories fade (18). Likewise,
in a mouse model of the human tauopathy associated with
Alzheimer’s disease, the endogenous KIBRA is deficient in
anchoring PKMζ, and overexpression of a fragment of KIBRA
that traffics to synapses and binds PKMζ restores the
ability to form long-term memory (66). Our
results suggest that providing the fragment of KIBRA
reconstitutes the physiological link between activated
synapses and PKMζ’s potentiating action, thus overcoming
the defective PKMζ anchoring in the tauopathy of
Alzheimer’s disease.
The
persistent coupling of KIBRA to PKMζ’s potentiating action
at activated synapses maintains memory longer than the
predicted life spans of individual KIBRA (28, 39, 40) and PKMζ
molecules (24, 25) (Fig.
9B and fig. S8). Thus, as in Crick’s
hypothesis, the molecules of KIBRA and PKMζ must be
replaced with new molecules, and these newly synthesized
proteins must be targeted to appropriate sites at
activated synapses. The degrading KIBRA and PKMζ are
likely exchanged by new molecules produced by local
dendritic synthesis from PKMζ mRNA (15) and
perhaps KIBRA mRNA as well (67). A
positive feedback loop by which PKMζ up-regulates
dendritic synthesis, including that from its own mRNA, has
been reported (68, 69). KIBRA
anchoring PKMζ’s action may further localize this
dendritic synthesis to activated synapses.
The
KIBRA-PKMζ complexes in activated synapses might then
align the newly synthesized KIBRA and PKMζ with the
synapses’ AMPAR trafficking machinery in a
self-perpetuating molecular mechanism. The autonomous
activity of PKMζ sustains synaptic potentiation by
increasing the number of postsynaptic AMPARs (5, 6). This
action is through persistently decreasing the endocytosis
of recycling GluA2 subunit-containing AMPARs (53, 55). These
AMPAR subunits interact with KIBRA (38).
Therefore, we speculate that, in a process distinct from
the initial setting of the tag that establishes the
commitment to late-LTP (42, 70), the
continual accumulation of KIBRA at potentiated synapses
serves as a persistent synaptic tag in late-LTP
maintenance for newly synthesized PKMζ (8). The
antagonists that block formation of KIBRA-PKMζ complexes (Figs.
3 and 8,
A and B) reverse established late-LTP within a few
hours of their application (Figs.
4A, 5A,
and 8C).
Therefore, the exchange of complexes within activated
synapses may be rapid. However, we do not know if the
antagonists might also disrupt established complexes. This
is because the reconstitution of split-Venus used in the
BiFC assay to test the inhibitors is likely irreversible (71). The
question of the rate of complex turnover in activated
synapses can be investigated by the development of
technologies such as live-imaging dual-eGRASP that could
identify these synapses in vivo (61).
Nonetheless, both KIBRA (28, 39, 40) and PKMζ (24, 25) (fig. S8)
appear from knockdown experiments to turn over in neurons
within hours to days, while KIBRA-PKMζ interactions
sustain memory for at least 4 weeks (Fig.
9B). Thus, as Theseus’ Ship was sustained for
generations by continually replacing worn planks with new
timbers (72), long-term
memory can be maintained by continual exchange of
potentiating molecules at activated synapses, a concept we
call persistent synaptic tagging.
MATERIALS AND METHODS
Experimental design
Reagents
Unless
otherwise stated, reagents were from MilliporeSigma.
ζ-stat (NSC 37044) was obtained from Drug Synthesis
and Chemistry Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute. Upon arrival, the drug was
dissolved in phosphate-buffered saline (PBS) (pH 7.4),
aliquoted at 10 mM stock solution, and stored at
−20°C. K-ZAP (myr-N-FVRNSLERRSVRMKRPS-C) was
custom-synthesized by AnaSpec (Fremont, CA) and stored
in PBS (pH 7.4) at −20°C.
Animals and cell lines
This study was
performed in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of
Health. All animals were handled according to approved
Institutional Animal Care and Use Committee (IACUC)
protocols [no. 11-10274, 15-10467 of the State
University of New York (SUNY) Downstate Health
Sciences University, or 2000-4512 of McGill
University]. The protocols were approved by the IACUC
of SUNY Downstate Health Sciences University (animal
welfare assurance number: D16-00167) and McGill
University (animal welfare assurance number:
F16-00005). All efforts were made to minimize animal
suffering and to reduce the number of animals used.
C57/B6 mice and PKMζ-null mice on a C57/B6 background
at SUNY Downstate Health Sciences University were
genotyped as previously described (17).
PKMζ-null mice at McGill University were a generous
gift from W. Sossin, Montreal Neurological Institute,
McGill University. Prkczfl/fl mice
were a generous gift from S. Ghosh, Yale University.
Male mice were examined in this study, and KIBRA-PKMζ
interaction in LTP and memory maintenance in both
sexes will be compared in a future study. HEK293T cell
line was obtained from the ATCC (American Type Culture
Collection).
Hippocampal slice recording and stimulation
Acute mouse
hippocampal slices (450 μm) were prepared as
previously described (17, 73).
Hippocampi from 2- to 6-month-old male C57/B6 or
PKMζ-null mice as previously described (17) were
dissected, bathed in ice-cold dissection buffer, and
sliced with a McIlwain tissue slicer in a cold room
(4°C). The dissection buffer contained 125 mM NaCl,
2.5 mM KCl, 1.25 mM NaH2PO4,
26 mM NaHCO3,
11 mM glucose, 10 mM MgCl2,
and 0.5 mM CaCl2 and
was bubbled with 95% O2/5%
CO2 to
maintain pH at 7.4. After dissection, the slices were
immediately transferred into an Oslo-type interface
recording chamber (31.5° ± 1°C) (73). The
recording superfusate consisted of 118 mM NaCl, 3.5 mM
KCl, 2.5 mM CaCl2,
1.3 mM MgSO4,
1.25 mM NaH2PO4,
24 mM NaHCO3,
and 15 mM glucose, bubbled with 95% O2/5%
CO2,
with a flow rate of 0.5 ml/min. In a subset of
experiments, a custom-made recirculation system using
piezoelectric pumps was used for recycling the
superfusate (Bartels Mikrotechnik GmbH, Dortmund,
Germany) (73).
fEPSPs were
recorded with a glass extracellular recording
electrode (2 to 5 megohms) placed in the CA1 st.
radiatum, and concentric bipolar stimulating
electrodes (CBBRE75 and 30200; FHC, Bowdoin, ME) were
placed on either side within CA3 or CA1. Test
stimulation rate was once every 30 s, alternating
every 15 s between stimulating electrodes. Based on a
preestablished exclusion criterion, a slice was not
used if fEPSP spike threshold was <2 mV on initial
input-output analysis. Pathway independence was
confirmed by the absence of paired-pulse facilitation
between the two pathways. A single stimulating
electrode was used for PLA/immunocytochemistry with a
test stimulation rate of once every 30 s. The
high-frequency stimulation, optimized to produce a
relatively rapid onset of protein synthesis-dependent
late-LTP (74),
consisted of two 100-Hz/1-s tetanic trains, at 25% of
spike threshold, spaced 20 s apart. The maximum slope
of the rise of the fEPSP was analyzed on a PC using
the WinLTP data acquisition program (75).
Proximity ligation assay
Methods used
were as described in the Sigma Duolink PLA Probemaker
Guide (MilliporeSigma, St. Louis, MO). Because PLA is
highly sensitive, standard immunocytochemical blocking
methods used to detect independent fluorescent signals
from two primary antisera of the same species (fig.
S1B) may not be sufficient; therefore, anti-PKMζ–PLUS
and anti-KIBRA–MINUS probes were generated by directly
conjugating the individual rabbit primary antibodies
[C2 (12, 19) and
ab216508, Abcam, Waltham, MA] with PLA
oligonucleotides (PLUS and MINUS, respectively).
Briefly, following purification with Microcon
Centrifugal Filters (Millipore, Burlington, MA), the
carrier- and preservative-free anti-PKMζ and
anti-KIBRA primary antibodies (1 mg/ml in PBS) were
incubated with lyophilized nucleotides (PLUS or MINUS,
respectively) at 20°C overnight. The reaction was
terminated by incubation with the Stop Reagent for 30
min at 20°C, and an equal total volume of Storage
Solution was added.
Hippocampal
slices were fixed by immersion in ice-cold 4%
paraformaldehyde in 0.1 M phosphate buffer (PB) (pH
7.4) immediately after recording and postfixed for 48
hours. Slices were then washed with PBS (pH 7.4) and
cut into 20 μM sections using a Leica VT 1200S
vibratome. Free-floating sections were permeabilized
in 96-well plates with PBS-TX100 for 6X 10 min at 20°C
and blocked with Duolink Blocking Solution
(Sigma-Aldrich, DUO82007-8 ml) for 1 hour at 37°C in a
preheated humidity chamber. The sections were then
incubated overnight at 4°C with anti-PKMζ–PLUS probe
(1:400) and anti-KIBRA–MINUS probe (1:400) mixed in
Duolink Probe Diluent. After 6X 10-min washes with
Duolink In Situ Buffer A, the sections were incubated
in a preheated humidity chamber for 30 min at 37°C
with Ligation Buffer (5X Duolink Ligation buffer
diluted 1:5 in high-purity water) containing the
Ligase enzyme. Following 6X 10-min washes with Duolink
In Situ Buffer A, the sections were incubated in a
preheated humidity chamber for 100 min at 37°C with
Amplification Buffer (5X Duolink Amplification buffer
diluted 1:5 in high-purity water) containing
Polymerase enzyme (Duolink In Situ Detection Reagents
Red; Sigma-Aldrich, DUO92008). The sections were then
washed for 6X 10 min with Duolink In Situ Buffer B at
20°C, followed by a 1-min wash in 0.01X Wash Buffer B.
The sections were mounted with Sigma-Aldrich Duolink
In Situ Mounting Medium containing
4′,6-diamidino-2-phenylindole (DAPI) (DUO82040). A
single confocal plane consisting of individual tiles
was captured using the Tiles tool of a Zeiss LSM800
AxioObserver Z1/7 confocal microscope with a
Plan-Apochromatic 20X/0.8 M27 lens. For each
fluorophore, all parameters (pinhole, excitation
wavelength, emission power, and detector gain) were
held constant for all imaging sessions. To correct for
tiling artifacts that result from uneven illumination,
an estimated shading profile was calculated for each
channel with the BaSiC tool for illumination
correction, using an ImageJ/Fiji Plugin (https://github.com/marrlab/BaSiC)
(76).
Shading correction was applied in Zen 2.6 software
using the “Shading Correction” function. In a subset
of experiments when conjugating MINUS probe was
unavailable from the manufacturer, the
anti-KIBRA–MINUS probe was substituted with a
biotinylated (Abcam Lightning-Link ab201795)
anti-KIBRA ab216508 primary (1:400 in Duolink Antibody
Diluent; incubated overnight at 4°C). After 6X 10-min
washing (Duolink In Situ Buffer A), a goat anti-biotin
secondary (1:200 in Antibody Diluent; Sigma-Aldrich,
B3640-1MG) was applied for 2 hours at 20°C. After
another 6X 10-min washing, the sections were incubated
with Duolink PLA donkey anti-goat (DAG)–minus (1:5;
DUO92003) in Antibody Diluent at 37°C for 1 hour. Both
methods yielded similar results.
Immunocytochemistry
Hippocampal
slices were fixed by immersion in ice-cold 4%
paraformaldehyde in 0.1 M PB (pH 7.4) immediately
after recording and postfixed for 48 hours. Slices
were then washed with PBS (pH 7.4) and cut into 20 μM
sections using a Leica VT 1200S vibratome.
Free-floating sections were permeabilized in 96-well
plates with PBS containing 0.3% Triton X-100
(PBS-TX100) for 6X 10 min at 20°C and blocked with 10%
normal donkey serum in PBS-TX100 for 2.5 hours at
20°C. The sections were then incubated overnight at
4°C with primary rabbit anti-KIBRA antibody (ab216508,
Abcam, Waltham, MA) at 1:100 in PBS-TX100. After
washing 6X 10-min in PBS-TX100 at 20°C, the sections
were incubated with Alexa Fluor 488–conjugated donkey
anti-rabbit (1:200 in PBS-TX100; Jackson
ImmunoResearch, West Grove, PA) for 2 hours at 20°C.
After 6X 10-min washes in PBS-TX100 at 20°C, the
slices were blocked for 2 hours at 20°C with 5% normal
rabbit serum in PBS-TX100, followed by 6X 10-min
washes in PBS-TX100 at 20°C. The slices were then
further blocked with 10% AffiniPure Fab Fragment
donkey anti-rabbit IgG H+L (Jackson ImmunoResearch)
PBS-TX100 for 2 hours at 20°C. This additional
blocking step was followed by 6X 10-min washes in
PBS-TX100 at 20°C. The sections were then incubated
overnight at 4°C with primary antibody rabbit
anti-PKCζ/PKMζ C2 [1:4000 (19);
generated as previously described (12)] in
PBS-TX100. After 6X 10-min washes in PBS-TX100 at
20°C, the sections were incubated with Alexa Fluor
647–conjugated donkey anti-rabbit (1:200 in PBS-TX100;
Jackson ImmunoResearch) for 2 hours at 20°C. After
washing 6X 10-min in PBS-TX100, the sections were
incubated with streptavidin-conjugated Alexa Fluor 647
(1:250 in PBS-TX100; Jackson ImmunoResearch) for 2
hours at 20°C. After 6X 10-min washes at 20°C with
PBS-TX100 and 10 min with PBS, the sections were
mounted with DAPI Fluoromount-G (Southern Biotech).
This procedure produces no bleedthrough between KIBRA
and PKMζ fluorescent signals (fig. S1B). A single
confocal plane consisting of individual tiles was
captured using the Tiles tool of a Zeiss LSM800
AxioObserver Z1/7 confocal microscope with a
Plan-Apochromatic 20X/0.8 M27 lens. For each
fluorophore, all parameters (pinhole, excitation
wavelength, emission power, and detector gain) were
held constant for all imaging sessions. To correct for
tiling artifacts that result from uneven illumination,
an estimated shading profile was calculated for each
channel with the BaSiC tool for illumination
correction, using an ImageJ/Fiji Plugin (https://github.com/marrlab/BaSiC)
(76).
Shading correction was applied in Zen 2.6 software
using the Shading Correction function.
Three-dimensional protein modeling
Molecular
graphics performed with UCSF ChimeraX (version 1.6.1),
developed by the Resource for Biocomputing,
Visualization, and Informatics at the University of
California, San Francisco, with support from National
Institutes of Health R01.GM129325 and the Office of
Cyber Infrastructure and Computational Biology,
National Institute of Allergy and Infectious Diseases
(77, 78). The
simulated protein models of KIBRA and PKMζ for
illustration are developed by ModBase (79) and T.
Ko at the University of Pennsylvania, respectively.
BiFC assay
Transfection constructs
KIBRA-PKMζ BiFC
was performed as previously described (28),
using pVen1-FLAG-KIBRA and pVen2-HA-PKMζ, in which
PKMζ was cloned between Eco RI and Bam HI sites. The
constructs pVen1 and pVen2 encode the N terminus
(amino acids 1 to 154) and C terminus (amino acids
155 to 238) of the Venus protein, respectively, and
the Venus fragments were on the N terminus of the
fusion proteins. pVen2-HA-PKMζ[PKCι/λ-P291Q;F297S]
was generated by site-directed mutagenesis using
forward (cctggagAagcAAatccggatcccccggtCcctgtccgtc)
and reverse
(gacggacaggGaccgggggatccggatTTgctTctccagg) primers
and the QuickChange II XL Site-Directed Mutagenesis
Kit (Agilent Technologies), following manufacturer’s
instructions. Additional pVen2-HA-PKC isoforms and
pVen2-HA-CaMKIIα in the pVen2-HA vector were
generated by cloning the N terminus of the coding
sequence of each kinase (table S1) to obtain an
in-frame fusion at the C terminus of the pVen2-HA.
Human versions of KIBRA and kinases were used, and
all polymerase chain reaction amplified sequences
and constructs were verified by DNA sequencing.
Cell culture and transfection
HEK293T cells
were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% heat-inactivated fetal bovine
serum, penicillin (100 U/ml ), streptomycin (100
mg/ml), 2 mM glutamine, and 10 mM Hepes in a T75
tissue culture flask with canted neck and ventilated
cap. Twenty-four hours prior to transfection, 1 × 105 HEK293T
cells in 1 ml of media were plated on poly-d-lysine–treated
coverslips in 24-well plates. One hour prior to
transfection, 0.5 ml of the media was removed from
each well, and ζ-stat or K-ZAP was added to the
media that had been removed. The remaining media in
each well was then discardeXd and substituted with
the 0.5-ml preconditioned media containing ζ-stat or
K-ZAP at designated concentrations. For
transfection, a total of 50 ng of DNA was delivered
to each well using Lipofectamine 3000 (Invitrogen)
and OptiMEM 1X reduced serum medium (Invitrogen) at
a plasmid-to-plasmid ratio of 1:1. The amount of
transfected DNA was optimized to produce relatively
low concentrations of proteins during
overexpression. The cells were cotransfected with
pVen1-FLAG-KIBRA and pVen2-HA-PKMζ, as previously
described (28), or
pVen1-FLAG-KIBRA and
pVen2-HA-PKMζ[PKCι/λ-P291Q;F297S]/PKC
isoforms/CaMKIIα. As controls, cells were
cotransfected with pVen1-FLAG-KIBRA and pVen2-HA or
pVen1-FLAG and pVen2-HA-PKMζ. After transfection,
the cells were incubated for 24 hours, fixed with 4%
paraformaldehyde, and kept on 0.02% sodium azide in
1X PBS at 4°C until immunostaining.
Immunostaining and confocal microscopy
To
detect FLAG and HA tags in cells transfected with
BiFC plasmids, the cells were blocked and
permeabilized using blocking buffer (2% bovine serum
albumin, 0.05% Tween 20, and 0.1% Triton X-100 in 1X
PBS) for 15 min at 20°C. The primary antibodies,
mouse anti-FLAG (1:100, Sigma-Aldrich) and rabbit
anti-HA (1:100, Sigma-Aldrich), were diluted in
blocking buffer. The blocking solution was then
removed, 200 μl of the diluted primary antibody was
added to each well, and the samples were incubated
overnight at 4°C. The primary antibody was then
removed, followed by 3X 5-min washes with 0.05%
Tween 20 in 1X PBS. The cells were then incubated
for 1 hour in the dark at 20°C with secondary
antibodies, Alexa Fluor 647–conjugated goat
anti-mouse (1:250, Invitrogen) and Alexa Fluor
594–conjugated goat anti-rabbit (1:250, Invitrogen)
diluted in blocking buffer. The secondary antibody
solution was removed, and the same washing steps as
with the primary antibody solution were performed.
The cells were mounted on glass slides with DAPI
Fluoromount-G (Southern Biotech). Z-stack images of
random microscopic fields were acquired with a 1-μm
z-step on a confocal microscope Zeiss LSM800
AxioObserver Z1/7 using a Plan-Apochromatic 63X/1.4
NA (numerical aperture) oil objective and exported
as maximum intensity projection. All imaging
parameters (pinhole, excitation wavelength, emission
power, and detector gain) were constant for all
experimental conditions. To determine the number of
interacting BiFC puncta per cell, the images were
converted to grayscale and the ImageJ 1.52n (80)
freehand or wand selection tools were used to create
an outline of each cell, which was added to the
region of interest manager. To ensure that HEK293T
cells were transfected with both pVen1-FLAG-KIBRA
and pVen2-HA-PKMζ (or pVen1-FLAG-KIBRA and
pVen2-HA-PKMζ[PKCι/λ-P291Q;F297S]/PKC
isoforms/CaMKIIα), only cells that were positive for
FLAG and HA tags signals were included in the
analysis. The “find maxima” algorithm from ImageJ
was used to count the number of local maxima per
cell with a noise tolerance of 10, as previously
described (81).
Individual data points for each experimental
condition (control and treatment) consist of the
means of >50 cells obtained from two independent
cultures.
Behavior—Active place avoidance
Intrahippocampal injection
For spatial
long-term memory experiments, we adapted the
approach used by Garcia-Osta et al. (82).
Mice were ~12 weeks old at surgery. Briefly, to
implant the injection cannula hardware, mice were
anesthetized by an intraperitoneal injection of a
mixture of dexmedetomidine (5 mg/kg body weight) and
ketamine (28 mg/kg body weight) and mounted in a
Kopf stereotaxic frame (Tujunga, CA). The tips of
guide cannula (Plastics One, Roanoke, VA; part
number: C235GS-5-2.0) were targeted above the
injection target in the dorsal hippocampus
[anterior/posterior (AP), −1.94 mm;
medial/lateral(ML), ±1.00 mm; dorsal/ventral (DV),
−0.90 mm]. The other injection hardware (part
numbers: C235DCs-5 and 303 DC/1; cannula dummy and
cannula cap, respectively) was assembled, and
antisedan (0.65 mg/kg body weight,
intraperitoneally) was administered to reverse the
sedation at the end of surgery.
Three to 4
weeks after surgery, the animals received active
place avoidance training. Before testing the effect
of the drug injection on place avoidance, the
animals received a bilateral injection of vehicle
[PBS (pH 7.4), 0.5 μl per side] and were left in the
home cage to habituate to the procedure. Depending
on the experimental design, injection of vehicle or
drug, either ζ-stat or K-ZAP (5 nmol in 0.5 μl of
PBS per side), is 1 day or 4 weeks after the
training session. During the injection, the animals
were restrained, the cannula cap and dummy were
removed, and the injection needle (Plastics One,
Roanoke, VA; part number: C235IS-5) was inserted
into the guide cannula so that it protruded from the
end of the guide by 0.5 mm. The other end of the
needle was connected to a 10-μl Hamilton syringe via
Tygon tubing. The drug or vehicle was infused for 1
min, and after the infusion, the needle was left in
place for 5 min before removal. The animals were
then returned to their home cages until the memory
retention tests that were conducted 2 days later.
Conditioning
Active place
avoidance was conducted with a commercial
computer-controlled system (Bio-Signal Group, Acton,
MA). The mouse was placed on a 40-cm diameter
circular arena rotating at 1 rpm. The specialized
software, Tracker (Bio-Signal Group, Acton, MA), was
used to detect the animal’s position 30 times per
second by video tracking from an overhead camera. A
clear wall made from PET-G (polyethylene
terephthalate glycol–modified) was placed on the
arena to prevent the animal from jumping off the
elevated arena surface. A 5-pole shock grid was
placed on the rotating arena, and the shock was
scrambled across the 5 poles when the mouse entered
the shock zone. All experiments used the
“Room+Arena-” task variant that challenges the mouse
on the rotating arena to avoid a shock zone that was
a stationary 60° sector (7).
Every 33 ms, the software determined the mouse’s
position, whether it was in the shock zone and
whether to deliver shock. After the animal enters
the shock zone for 500 ms, a constant current
footshock (60 Hz, 500 ms) was delivered and repeated
with the interval of 1500 ms until the mouse left
the shock zone. The shock intensity was 0.2 or 0.3
mA, which was the minimum amplitude to elicit flinch
or escape responses. The animal was forced to
actively avoid the designated shock zone because the
arena rotation periodically transported it into the
shock area.
The tracked
animal positions with timestamps were analyzed
offline (TrackAnalysis, Bio-Signal Group, Acton, MA)
to extract several end-point measures. The time to
first enter the shock zone estimates ability to
avoid shock and was taken as an index of
between-session memory. The number of entrances
within one trial was taken as another index to
examine the animal learning curve throughout all
training trials. A pretraining habituation period on
the apparatus equivalent in time to a training
session, but without shock, was provided.
The
training schedule was as follows: 2 hours after a
30-min pretraining habituation, the animals received
three 30-min training trials, with an intertrial
interval of 2 hours. Bilateral intrahippocampal
injection of ζ-stat or vehicle (Fig.
6) or K-ZAP or vehicle (Fig.
9) was 24 hours (Figs.
6 and 9A)
or 4 weeks (Fig.
9B) after the training session. Retention
testing was a 30-min trial without shock 2 days
after injection. In fig. S5, the animals first
received the conditioning in context A, injection,
and a memory retention test as described above.
Immediately after retention testing, the animals
were conditioned in context B with a different set
of spatial cues, and memory for active place
avoidance in context B was tested 3 days later.
Preestablished exclusion criterion was if, after
sacrificing the animals and performing histology for
cannula placement, cannulae were found to be
incorrectly targeted. The data from two animals were
excluded from behavioral analysis of the effects of
ζ-stat on PKMζ-null mice because histology revealed
misplaced cannulae.
Behavior—Auditory-cued fear/threat conditioning
Animals
Mice were 8 to
10 weeks old at the time of cannulation and 9 to 11
weeks old at the beginning of behavioral
experiments. Mice were housed with cage mates in
plastic cages and provided with food and water ad
libitum. Mice were maintained on a 12-hour:12-hour
light:dark cycle (lights on at 7:00 a.m.) and
behavioral experiments began at 9:00 a.m.
Surgery
Mice were
injected intraperitoneally (1 ml/100 g body weight)
with an anesthetic cocktail containing ketamine (10
mg/ml) and xylazine (2 mg/ml). Mice were provided
with analgesic treatment prior to surgery
(carprofen; 5 mg/ml). Guide cannulae (Plastics One,
Roanoke, VA) were implanted bilaterally in the BLA
(from bregma: AP, −1.7 mm; ML, ±3.0 mm; DV, −4.4 mm)
and secured to the skull with three jeweler’s screws
and dental cement. Antisedan (0.66 ml/100 g body
weight of 0.5 mg drug per ml solution) was given via
intraperitoneal injection after surgery to reverse
the anesthesia.
Conditioning
For 7 days
following surgery, mice were handled by freely
exploring the experimenter’s palm for 2 to 5 min.
Mice were habituated, trained, and tested in the
same conditioning box (Coulbourn Habitest, Coulbourn
Instruments) with differing floors and walls to
produce two different contexts (context A and
context B). For each day of the behavioral
experiment, mice were brought to the experiment room
at 9:00 a.m. and allowed to acclimatize for 30 min.
Mice were then habituated to the testing context
(context A with smooth floor and flat, blank walls)
for 20 min each day for two consecutive days. The
next day, mice were trained in a second context
(context B with a grid floor, patterned walls, and a
curved wall). Training consisted of 2 min of
exploration of context B followed by a tone (2800
Hz, 85 dB, and 30 s) coterminating with a footshock
(0.7 mA, 1 s). Mice received two tone-shock pairings
separated by 1 min and remained in context B for an
additional 1 min before returning to their home
cage. Mice were tested 24 hours after training in
context A (test 1). During testing, mice were placed
in the conditioning box and, after 2 min, were
exposed to a 30-s tone (2800 Hz, 85 dB). The next
day, mice received bilateral infusions and were
tested a second time 24 hours after infusion in
context A (test 2). Freezing behavior (cessation of
all movement except breathing) during the tone on
test 1 and test 2 was scored by an experimenter
blind to the conditions. Scores are reported as the
percent of time spent freezing during the tone.
Preestablished exclusion criteria were (i) if the
mouse froze less than 25% of the time after the
conditioned stimulus was presented at the first
test, which is the standard cutoff used in the Nader
lab to distinguish mice that had learned or not
learned the conditioned stimulus-unconditioned
stimulus association; and (ii) if after sacrificing
the animals and performing histology for cannula
placement, cannulae were found to be incorrectly
targeted.
Drug infusions
Mice
were bilaterally infused with ζ-stat obtained from
Drug Synthesis and Chemistry Branch, National Cancer
Institute or vehicle [PBS (pH 7.4)]. Mice were
infused with 6 nmol of ζ-stat in 0.3 μl at a rate of
0.2 μl/min into each BLA. Drugs were infused with
28-gauge microinjectors (Plastics One, Roanoke, VA)
connected to Hamilton syringes (26 gauge, model
1701N) by way of polyethylene tubing (Braintree
Scientific Inc., Braintree, MA). After infusion,
injectors remained in place for 1 min to ensure that
drug diffused sufficiently away from the injector
tip.
Behavior—Contextual fear/threat conditioning
Animals
Mice were 8 to
10 weeks old at the time of cannulation and 9 to 11
weeks at the beginning of behavioral experiments.
Mice were housed with cage mates in plastic cages
and provided with food and water ad libitum. Mice
were maintained on a 12 hours light/dark cycle
(lights on at 7:00 a.m.) and behavioral experiments
began at 9:00 a.m.
Surgery
Mice were
injected intraperitoneally (1 ml/100 g body weight)
with an anesthetic cocktail containing ketamine (10
mg/ml) and xylazine (2 mg/ml). Mice were provided
with analgesic treatment prior to surgery
(carprofen; 5 mg/ml). Guide cannulae (Plastics One,
Roanoke, VA) were implanted bilaterally in the
dorsal hippocampus (from bregma: AP, −2.1 mm; ML,
±1.8 mm; DV, −1.2 mm) and secured to the skull with
three jeweler’s screws and dental cement. Antisedan
(0.66 ml/100 g body weight of 0.5 mg drug per ml
solution) was given via intraperitoneal injection
after surgery to reverse the anesthesia.
Conditioning
For 7 days
following surgery, mice were handled by freely
exploring the experimenter’s palm for 2 to 5 min.
Mice were trained and tested in the same
conditioning box (Coulbourn Habitest, Coulbourn
Instruments), which consisted of a grid floor and
blank walls. For each day of the behavioral
experiment, mice were brought to the experiment room
at 9:00 a.m. and allowed to acclimatize for 30 min.
On the first day (training), mice explored the
context for 2 min. We then delivered two 0.7-mA
(1-s) shocks spaced 1 min apart. Mice remained in
the box for an additional minute before returning to
their home cage. The next day, mice received
bilateral infusions and were tested 24 hours after
infusion. During testing, mice were placed in the
same context for 4 min with no shock. Freezing
behavior (cessation of all movement except
breathing) during the 4-min session was recorded by
an experimenter blind to the conditions. Scores are
reported as the percent of time spent freezing
during the 4-min session. Preestablished exclusion
criterion was if, after sacrificing the animals and
performing histology for cannula placement, cannulae
were found to be incorrectly targeted.
Drug infusions
Mice
were bilaterally infused with vehicle [PBS (pH 7.4)]
or ζ-stat (10 nmol in 0.5 μl per hemisphere at a
rate of 0.2 μl/min per side). Drugs were infused
with 28-gauge microinjectors (Plastics One, Roanoke,
VA) that protruded 0.3 mm from the guide cannulae
connected to Hamilton syringes (26 gauge, model
1701N) by way of polyethylene tubing (Braintree
Scientific Inc., Braintree, MA). After infusion,
injectors remained in place for 1 min to ensure drug
diffusion away from the injector tip.
Immunoblotting
Immunoblots
for fig. S8 were performed as previously described (19).
Statistical analysis
Replicates
are biological because there is only one measurement for
each sample. Drug/vehicle comparisons were performed
blindly. Sample sizes vary for the different
experimental approaches (biochemistry, slice physiology,
and behavior) and were established by power analyses
based on effect size estimates from published works or
preliminary experiments. The observed effect sizes for
binary comparisons are reported as Cohen’s d and
as η2p for
ANOVA effects. Multifactor comparisons were performed
using ANOVA with repeated measures, as appropriate.
Two-population Student’s t tests
were performed to compare protein immunofluorescence
intensity between control and potentiated hippocampal
slices. For LTP experiments, the responses were first
normalized to the mean of the 30-min period prior to
tetanization (or equivalent in control pathways), and
then the means over 5-min periods were used for
statistical comparisons (e.g., pretetanization and
beginning and end of drug application). Paired Student’s t tests
were used to compare the change in the potentiated
response at time points at the beginning and end of drug
application. The degrees of freedom for the critical t values
of the t tests
and the F values
of the ANOVAs are reported as subscripts. Post hoc
multiple comparisons were performed by Newman-Keuls
tests as appropriate. Statistical significance was
accepted at P <
0.05 or appropriate Bonferroni correction for multiple
comparisons.
Acknowledgments
P.T.
is an Alexander S. Onassis Public Benefit Foundation
Scholar.
Funding: This
work was supported by the National Institutes of Health
[grant R37 MH057068 (T.C.S.)], National Institutes of
Health [grant R01 MH115304 (T.C.S. and A.A.F.)], National
Institutes of Health [grant R01 NS105472 (A.A.F.)],
National Institutes of Health [grant R01 MH132204
(A.A.F.)], National Institutes of Health [grant R01
NS108190 (P.J.B. and T.C.S.)], Natural Sciences and
Engineering Research Council of Canada Discovery [grant
203523 (K.N.)], and the Garry & Sarah S. Sklar Fund
(P.T.).
Author
contributions: Conceptualization:
T.C.S., A.A.F., K.N., H.Z.S., P.T., C.H., R.E.F.-O.,
A.I.H., M.B., J.E.C., J.K., and A.T. Methodology: P.T.,
C.H., R.E.F.-O., A.T., A.I.H., M.B., K.N., A.A.F., and
T.C.S. Investigation: P.T., C.H., R.E.F.-O., M.B., A.T.,
J.K., C.T., and T.C.S. Project administration: T.C.S.,
A.A.F., and P.T. Resources: P.T., C.H., A.T., J.E.C.,
J.K., K.N., A.A.F., and T.C.S. Data curation: C.H., P.T.,
R.E.F.-O., and A.T. Validation: P.T., C.H., R.E.F.-O.,
M.B., A.T., J.K., A.A.F., and T.C.S. Supervision: T.C.S.,
A.A.F., P.T., K.N., A.I.H., and J.E.C. Software: A.A.F.
Visualization: C.H., P.T., A.I.H., and T.C.S. Formal
analysis: C.H., P.T., R.E.F.-O., A.A.F., and T.C.S.
Writing—original draft: T.C.S. Writing—reviewing and
editing: A.A.F., T.C.S., K.N., P.J.B., H.Z.S., P.T., C.H.,
R.E.F.-O., M.B., A.T., A.I.H., C.T., J.E.C., and J.K.
Funding acquisition: T.C.S., A.A.F., P.J.B., K.N., and
J.E.C.
Competing
interests: T.C.S. is an inventor on a
patent application (serial number 63/597,518) submitted by
The Research Foundation for The State University of New
York on 11/9/2023, pertaining to results presented in the
paper. The other authors declare that they have no
competing interests.
Data
and materials availability: All data
needed to evaluate the conclusions in the paper are
present in the paper and/or the Supplementary Materials.
Requests for plasmids, antisera, and other materials used
in this study may be made to T.C.S.
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