RotationProposal_ShorrockM_D1

18 March 2013 MCB220L – 4th
rotation proposal Meghann Shorrock
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Spacing effect on formation and maintenance of maternal behavior
SUMMARY
The ability to store long term memories (LTM) for days to decades is vital for adaptive living. In
mammals, acquisition and maintenance of maternal behavior is especially critical for the
evolutionary success due to the enormous effect it has on offspring mental fitness. Yet, the
mechanism underlying maternal behavior maintenance remains poorly understood and the
probable role of LTM has never been addressed. The Stolzenberg lab offers some of the first
evidence linking LTM with maternal behavior. Using mice, we demonstrated that experience
dependent maternal behavior is subject to the same temporal patterning which promotes
accurate and long term retention of memories. Specifically, that learning is most effective when
spaced widely over time (spaced training), rather than when presented with little to no rest
interval (massed training). We hypothesize that maternal behavior is generated and maintained
via spacing effect mechanisms.
BACKGROUND
In humans and other mammals, experience with infants has a substantial effect on the quality
of subsequent maternal care which in turn effects infant development. Increased mothering
has been shown to decrease infant stress and secure infant attachment in nearly all mammals
from human to rodent (Chen et al, 2012; LaPlant et al., 2010; Champagne 2008; Champagne et
al., 2008). Often, mothering quality and quantity is increased through mother-infant
interactions, suggesting an experience dependent component for maternal care. This
experience dependent aspect has been observed in rats (Lee et al, 2000), although a strong
endocrine prerequisite (usually from giving birth) is first required to inhibit the rat’s inherent
aversion to pubs (Lee et al., 2000; Stolzenberg et al., 2011). Laboratory mice, however, do not
have a hormonal roadblock to overcome. Similar to observed human behavior, they become
highly responsive to pubs after repeat exposure even if they have never given birth
(Stolzenberg et al, 2012). Understanding how this experience with infants is stored and
maintained to alter maternal care long term could help explain what goes wrong when mothers
fail to bond with their infants, as in postpartum depression. However, molecular mechanisms of
maternal care have been largely overlooked in favor of behavioral approaches. Recent work
from the Stolzenberg lab has begun to address this knowledge gap.
Using mice, Stolzenberg et al. (2012) demonstrated that experience-induced changes in
behavior were mediated by chromatin modifications, which in turn alters gene expression to
promote maternal care. Following administration of sodium butyrate, a histone deacetylase
inhibitor, there was a significant increase in potentiated maternal responsiveness as well as the
expression of several genes also known to play a critical role in long term potentiation (LTP) and
long term memory formation, included cyclic-AMP response element binding protein (CREB)
and oxytocin. Furthermore, investigation into the increased rapidity of maternal response
following subsequent infant interactions revealed the surprising result that retention of
maternal experience was subject to spaced learning paradigm. Specifically, that learning is most
effective when spaced widely over time (spaced training), rather than when presented with
little to no rest interval (massed training). Mice presented with massed learning did not

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maintain long term memory of maternal care and were unresponsive when reintroduced to
pubs 24h post interaction (Stolzenberg et al., 2012). Spaced learning illicit a specific cascade of
molecular events to promote LTM that is observed in organisms ranging from nematode to
human (Maximilian et al, 2012; Li et al., 2013; Genoux et al., 2011; 2002; Naqib et al. 2012).
Several of these factors had already been implicated in the previous studies, further
strengthening the probable role of LTM molecular mechanisms in maternal experience. Two of
the best candidates are CREB and the atypical protein kinase M zeta (PKMζ).
A key player in spaced but not massed training is CREB. It is crucial for LTM formation (Dash et
al, 1990; Yin et al., 1994) and required for normal nurturing behavior in mice (Jin et al., 2005).
CREB is unique in that it spans bothe regulation for chromatin remodeling through histone
acetylation transferase (Korzus et al., 2004) as well as transcription factors which lead to
downstream upregulation of factors promoting synaptic plasticity, dendritic translation, and
recruitment of LTP molecules to the synapse (Koshibu et al.,2009; Maximilian et al., 2012;
Genoux et al., 2011; 2002). One of the possible downstream function of CREB is thought to
function by interacting with a predicted CRE domain of PKMζ.
PKMζ is vital for LTM maintenance and is unique in its potency (Mei et al, 2011; Miques et al.,
2010; Shao et al, 2012; Westmark et al, 2010; Yao et al, 2008; 2013). Inactivating PKMζ
anywhere from 30 min to 30 days post learning abolishes established memories, indicating its
critical role in maintaining memories (Yae et al 2008, 20013). The constitutively active PKMζ
protein is translated locally at the synapse and functions to stabilize GluR2 containing AMPA
receptors (AMPAR) at the post synaptic density (PTD) through the NSF/GluR2 trafficking
pathway. AMPA receptors are the major excitatory receptor of the central nervous system and
are have been shown to be critical in a variety of brain functions including the synaptic plasticity
responsible long term potentiation (LTP) and long term memory (Santos et al., 2009; Yao et al.,
2008; Migues et al., 2010; Ehrlich et al., 2004; Hu et al., 2007; Kalashnikova et al., 2010).
Translational repressors like PIN1 can block PKMζ activation and inhibit formation of LTM. Like
CREB, PKMζ is found throughout the CNS including hippocampus (Hernandez et al, 2003),
dorsalmedial/lateral striatum (Pauli et al 2012), basolateral amygdala (Migues et al, 2011), and
spinal cord (Laferriere et al, 2011). Importantly, local trnalsation of PKMζ can be regulated by
oxytocin (Lin et al., 2012) which is a potent maternal care molecule (Stolzenberg et al., 2012;
2011; Patisaul et al., 2003; Shahrokh et al., 2010) identified in our initial investigation.
The ubiquitous distribution and parallel activity of CREB and PKMζ make them attractive
candidates to explain long term maintenance of maternal behavior. Therefore, we hypothesize
that maternal behavior is generated and maintained via spacing effect mechanisms of CREB and
PKMζ. The present study will use biochemical, genetic, and behavioral approaches to (1)
establish spaced-learning molecules are present in maternal behavior, and (2) demonstrate
spaced-learning mechanisms function in maternal behavior. Characterizing this link will help
establish maternal behavior as a the first innate and natural model for LTM, providing a
powerful alternative to artificial paradigms used in nearly all learning and memory studies to
date.

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SPECIFIC AIMS
To address this hypothesis the following specific aims will be completed,
Aim 1: Establish spaced learning molecules are present in maternal behavior
Aim 2: Establish spaced learning mechanisms function in maternal behavior
Aim 1: Establish spaced-learning molecules are present in maternal behavior
(1) Experimental design Experience induced maternal behavior will be performed as before
(Stolzenberg et al., 2011). Briefly, virgin female C57BL/6J mice will be exposed to pubs at a rate
of spaced-full (2h for 4 days), spaced-short (2h for 2 days), or massed (8h in one day). Pup naive
mice will be used as a negative control. Maternal behavior will be scored through observed
licking and grooming (LG) and crouching during the first hour of each pup exposure. Pup-
retrieval on the T-maze during training (days 1-4) and post training (days 5 and 30) will test
acquisition and storage of maternal motivation, respectively. Brain samples will be gathered 2h
following last pup exposure at days 1-5, and 30 days. Punches (2mm) from the MPOA, VTA,
hippocampus, BLA, and NA will be generated and transcript levels of CREB, PKMζ , eukaryotic
initiation factor (eIF2α), activating transcription factor (ATF4), protein phosphatase (PP1), and
glutamate receptor subunit 2 (GluR2) will be determined using qPCR. Additionally, protein
levels will be measured by western blot.
(1) Expected results and anticipated caveats This study will confirm previous findings in which
naive mice are unresponsive, massed training produces some short term behavior with little
retention, spaced-half training yields responsive and retained maternal behavior though not as
robust as spaced-full training. We expect to the molecular mechanisms underlying maternal
behavior to mimic those underlying traditional learning. This will be demonstrated by observing
the LTM transcriptional program in the behavioral response circuit (MPOA, VTA, hippocampus,
BLA, NA). Specifically, CREB and PKMζ will be elevated in spaced but not massed training
indicating enhanced learning. Levels of upstream and downstream targets will support the
predicted enhanced learning. Inhibitors of LTM (eIF2α, ATF4, PP1) will be downregulated while
the functional output of PKMζ (GluR2 containing AMPAR) will be upregulated. These
transcriptional patterns will be present after 2 days of spaced training and be maintained over
long period of time as confirmed by 30 days post-training. However gene expression in neurons
is regulatory complex with transcription and translation often being uncoupled. Therefore,
changes protein could be observed without a concurrent change in transcripts. This highlights
the importance of looking at both protein and transcript levels. This approach will allow for
determination of LTM molecular expression as well as discovery of novel expression regulators.
Indeed, in a system of such regulatory complexity it could be difficult to interpret expression
levels. A strength of this outlined approach is that the maternal circuit and molecular players
are well characterized, permitting an informational context in which to better interpret
expression patterns and validate through internal agreement.
(1) Future directions This experiment will identify where in the maternal behavior circuit CREB
and PKMζ mediate long term learning. However, their functional significance remains
unexplored. Future studies can systematically inactivate CREB and PKMζ during and after

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maternal training to determine their effect on maternal behavior storage. Additionally, use of
genetic backgrounds with disrupted learning could be used to further determine the extent to
which maternal experience and memory are linked. The use of constitutaional knock down
mutants would be an even more precise and useful approach. For example, CREB dependent
HAT activity can be induced to disrupt chromatin remodeling and impairs memory formation.
Therefore, the next step should be to funcationally characterize the identified maternal based
maintenance molecules.
Aim 2: Demonstrate spaced-learning mechanisms function in maternal behavior
(2) Experimental design Virgin female C57BL/6J mice will undergo spaced-full maternal training
and testing as outlined above. Following training at 24 h and 30 day, mice will be injected with
PKMζ inhibitor, ZIP, or negative control scrambled-ZIP. A rescue from PKMζ inhibition will be
performed by a pre-injection with a GluR2 inhibitor, GluR23Y, or a scrambled control. Effect of
treatments will be determined using T-maze pub-retrieval. Mice will undergo a second spaced-
full maternal training to establish treatments did not affect ability to learn.
(2) Expected results and anticipated caveats As with previous memory studies (Mei et al.,
2011; Migues et al., 2010; Yao et al., 2008), inhibition of PKMζ activity will result in erasure of
previously learned task of maternal behavior. Therefore, trained mice treated with ZIP will
behave similar to pup-naive mice. Pre-injection with GluR23Y will negate this effect and trained
mice will perform as expected. While treated mice will lose established memories, they will
retain the ability to make and maintain new behavioral memories.
(2) Future directions This key experiment will demonstrate PKMζ operates to maintain
maternal behavior and that maternal behavior is subject to the same molecular mechanisms as
traditional learning and memory. Yet the interactions between proposed molecular players that
are critical to memory formation and maintenance, such as PP1 and PIN1, have not been
addressed. Additionally, how AMPAR trafficking is involved in PKMζ mediated memory
maintenance and the chaperones involved in its stabilization or unknown. Future studies should
determine the functional significance of downstream PKMζ activity as well as its upstream
regulators such as CREB.
DISCUSSION
The futility of massed training to generate long lived memories has been observed across
animal phyla from mechanosensory in worms (Lie et al., 2013) to exam cramming in students.
Alternatively, spaced training provides the best molecular and cellular environment to permit
acquisition and long term storage of experience, with maternal experience being no exception.
Quality of maternal care has a huge impact on offspring success and can influence subsequent
descendents for generations (Champagne et al., 2008). Yet almost nothing is known of the
molecular mechanisms underpinning such an omnipotent force on early life development. This
study is well suited in that it has already generated a maternal model which adheres to the
space learning paradigm. Therefore, we can immediately start identifying candidate molecules,
such as PKMζ and CREB and oxytocin, and manipulating the system to determine function.

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