Slide: Why study memory?
There are a number of clinically important cases where memory is lost or compromised
including senile dementia and Alzheimer’s Disease.
• Memory enhancers-Helicon and Memory Inc
• Memory decline in aging
• Memory disorders:
Alzheimer’s Disease
Anxiety disorders: Phobias, PTSD
Slide: Definitions
Short-Term Memory: Memory lasting minutes to hours, does not depend on de novo
transcription.
Long-Term Memory: Memory lasting hours to months, depends on de novo transcription.
Several forms of memory including spatial, contextual, and object recognition are formed
in, and initially stored in the hippocampus.
Memories can be stored in the hippocampus for approximately one month after which
they are stored in the cortex.
Several biochemical changes occur in the hippocampus during memory formation
including activation of Erk 1/2, cAMP increases and stimulation of CRE-mediated
transcription.
Slide: Hippocampus-dependent memory
There are several forms of memory that are destroyed when the hippocampus is
ablated. Operationally, we define them as hippocampus-dependent memory. This
includes:
Contextual fear conditioning
Passive avoidance
Spatial (Morris water maze)
Novel object recognition
Slide: Erk MAP kinase, CRE-transcription and protein synthesis: The CNS has the
remarkable capacity to process and store information. Consequently, there is intense
interest in mechanisms underlying memory as well as the relationship between memory
and specific forms of synaptic plasticity. Although a number of signaling pathways may
contribute, there is considerable evidence that cross-talk between the Ca2+, Erk/MAP
kinase, and cAMP regulatory pathways may play a pivotal role in memory formation.
The MAP kinase pathway is thought to play a crucial role in memory because it mediates
activity-dependent stimulation of CRE-mediated transcription as well as translation, two
events required for memory consolidation.
Slide: The cAMP/Calcium Response Element, CRE
1. Responds to cAMP and calcium. Can integrate signals from multiple kinase pathways.
Pairing of calcium and cAMP signals gives synergistic stimulation of CRE-mediated
transcription.
2. Palindromic Sequence, TGACGTCA which binds to transcription factors including
CREB, CREM and ATF-1.
3. Phosphorylation of CREB at Ser-133 is necessary but not sufficient for CRE-mediated
transcription.
4. Kinases that can phosphorylate CREB at Ser-133 include CaM-kinase IV, PKA, MSK1, Rsk2.
Slide: There are four lines of evidence that implicate CRE-mediated transcription
in LTM.
1. Disruption of the CREB gene inhibits memory formation…These studies were not
entirely convincing because disruption of the gene for CREB causes compensating
increases in other transcription factors. Furthermore, disruption of the CREB gene by
different labs did not yield a consistent phenotype.
2. Administration of CRE oligonucleotide decoys to the hippocampus blocks memory.
3. CREB is phosphorylated at Ser-133 during training for contextual memory.
4. CRE-mediated transcription is stimulated during memory formation.
Slide: Signaling pathway for calcium/cAMP activation of CRE-mediated
transcription
Hippocampus-dependent, LTM is initiated by activation of NMDA receptors and a
postsynaptic Ca2+ increase. This Ca2+ signal activates several pathways including
MAP kinases and adenylyl cyclases.
In brain, calcium and cAMP are coupled through calmodulin which mediates
Ca2+ stimulation of type I and type 8 adenylyl cyclases.
Today, I will present evidence supporting the hypothesis that activation of MAP
Kinase and its nuclear translocation during memory formation depends on a cAMP
signal… generated through these adenylyl cyclases.
Slide: Circadian Oscillation of the pathway
It is also hypothesized that persistence of hippocampus-dependent memory depends on
circadian oscillation of this pathway in the hippocampus…an oscillation that is driven by
changes in calcium-sensitive adenylyl cyclase activity.
These are the issues that I will discuss in these two lectures.
Slide: CRE-lac Z mouse
One of the tools used to address these issue is a CRE-reporter mouse strain, which
allows one to monitor CRE-mediated transcription during behavioral studies.
This mouse has a reporter construct comprised of multiple CRE sequences, a minimal
promoter, and the LacZ gene.
You can use antibodies against beta-galactosidase to monitor CRE-mediated
transcription.
Slide: Contextual Fear Paradigm
This mouse was used to show that training for several forms of hippocampus-dependent
memory including contextual memory stimulates CRE-mediated transcription in the area
CA1 in the hippocampus.
In this paradigm, a mouse is trained for context by placing it in a training box and
shocking 2 min later. It learns to associate the context with the shock and develops a
strong memory for the training context. This is manifested as increased freezing upon
return to context.
Unpaired controls are mice placed in context for several hours and then shocked…or
those that are shocked immediately after being placed in context.
Slide: Contextual Fear and CRE-mediated transcription
The unpaired controls do not develop memory for context and nor do they show traininginduced increases in CRE-mediated transcription.
Mice that develop memory for context show enhanced CRE-mediated transcription in
area CA1 of the hippocampus. In this same paper, it was shown that contextual training
stimulates CREB phosphorylation on Ser-133.
Approximately 10% of the neurons showed a positive signal for CRE-mediated
transcription.
Slide: Sterotaxic cannulation apparatus
Is CRE-mediated transcription required for contextual memory. To answer this
question mice were bilaterally cannulated to deliver CRE-oligonucleotide decoy to area
CA1 of the hippocampus. This picture shows an anesthesized mouse in a sterotaxic
frame used to insert cannula into specific areas of the mouse brain.
Slide: CRE oligo blocks contextual fear memory
Mice were bilaterally infused in area CA1 with either a CRE decoy oligonucleotide or a
control scrambled oligonucleotide of identical composition. The CRE decoy
competitively binds to any transcription factor, including CREB, that normally binds to the
CRE sequence…thereby blocking CRE-mediated transcription.
a. In sections taken from animals infused with fluorescent-labeled decoy, the
diffusion and cellular uptake was restricted to the CA1 region of the hippocampus.
b. Infusion of the oligonucleotides did not affect the sensitivity of mice to shock
during training
c. Infusion of CRE-decoy completely blocked memory for context, while the
scrambled decoy was without effect.
This is evidence that contextual memory requires activation of CRE-mediated
transcription…
Slide: MAP kinase activation with contextual training
The major pathway for Ca2+ stimulation of CRE-mediated transcription in hippocampal
neurons is the MAPK pathway.
Indeed, when a mouse is trained for contextual memory, you see transient activation of
MAPK in the hippocampus…that can be monitored using a phosphopeptide-specific
antibody that recognizes phosph-Erk1 and Erk2.
Slide: MAP kinase activation in area CA1 of the hippocampus
The major increase in MAPK activity following training is in area CA1 of the
hippocampus.
In this enlargement of the CA1 you can see many more neurons staining positive for pErk in the trained animals compared to unpaired controls
Slide: 10 percent of neurons activated
Surprisingly, as many as 10% of CA1 neurons show training-induced MAPK activation
which averages 9 to 10 fold higher than in surrounding neurons
However, only one or two dendrite branches were labeled in each of these neurons
Slide: Activation PKA and MAPK in same neurons
The hypotheses under consideration assumes that PKA and MAPK are coactivated in
the same neurons during acquisition of contextual memory. This was confirmed by
monitoring trained-induced PKA activity and pERK in trained mice. PKA activity was
measured using an antibody that recognizes PKA phosphorylated substrates. Here is an
example of two neurons showing coactivation of PKA and MAPK during training. When
we look at large number of neurons that show a trained-induced increase in Erk
activation, there is a strong correlation with PKA activation
Slide: Nuclear MAP kinase activity
Confocal imaging was used to measure nuclear pERK and PKA activity in neurons from
trained mice. Here is an example of a neuron in which there is robust increases in
nuclear p-Erk and PKA activity, and another here where there is not.
Slide: Quantification of Nuclear MAP kinase activity
Data obtained from a large number of trained and untrained mice indicate a seven-fold
increase in nuclear p-Erk following training. There was also a significant increase in
PKA activity in the nucleus.
Slide: Activation of CREB kinases during training
There are several Erk-activated, CREB kinases in hippocampal neurons …including
MSK1 and RSK-2. When mice are trained for context, you see significant increases in
the activity of both of these CREB kinases in the hippocampus…illustrated here for
MSK1. Furthermore, there is a strong correlation between neurons showing coactivation
of MSK-1 and MAPK. This suggests that MSK-1 phosphorylates CREB during formation
of contextual memory.
Slide: MEK inhibitor blocks MAPK activation
This training-induced increase in MAPK activity can be blocked by bilateral
administration of PD 98059 to area CA1 in the hippocampus of cannulated mice.
This drug blocks MEK activity, which is an upstream activator of MAPK. This data shows
P-Erk from 4 trained mice…two received vehicle and two received the MEK inhibitor.
Slide: MEK inhibitor blocks contextual memory formation
The bilateral administration of the MEK inhibitor to the hippocampus completely blocks
contextual memory.
Unilateral administration is without effect.
Slide: MEK inhibitor blocks CREB phosphorylation
Bilateral infusion of the MEK inhibitor also completely inhibits training-stimulated
increases in CREB phosphorylation at Ser-133, a phosphorylation that is required for
stimulation of CRE-mediated transcription.
Slide: MEK inhibitor blocks CRE-mediated transcription
Most importantly, the training-stimulated increases in CRE-mediated transcription are
also blocked by the MEK inhibitor.
This data indicates that the training-stimulated increases in the CREB/CRE
transcriptional pathway depend on activation of MAP kinase.
Slide: AC1 x AC8 knockout reference
To evaluate the role of Ca2+-stimulated adenylyl cyclases for MAPK activation during
training a double knockout strain lacking both AC1 and AC8 was examined.
These transgenic mice exhibit short-term memory but cannot consolidate to form
long-term memories.
Slide: Calcium stimulated AC activities, AC1-/-, AC8-/- and Double knockout
Double knockouts lacking both AC1 and AC8 show no calcium activated adenylyl
cyclase activity in membranes isolated form whole brain, hippocampus or the
cerebellum.
Slide: passive avoidance paradigm
These mice were examined for several forms of hippocampus-dependent memory
including passive avoidance memory.
In this paradigm, a mouse is placed in a two chambered cage. One side is lighted and
the other is dark. Mice prefer the dark and quickly cross over into the dark side. When
they cross over they receive a mild footshock. They develop a strong memory for the
experience and if they are tested at a later date, mice that remember the experience
have a greatly increased cross over latency for the dark.
Slide: Calcium-Stimulated Adenylyl Cyclase is Required for Passive Avoidance
Memory
Although single knockouts in AC1 or AC8 show normal memory for passive avoidance
24 hr or 8 days after training ( note the increased cross-over latency after training). The
double knockouts show little or no memory for passive avoidance memory….
Slide: DKO Mice Show Normal Learning and Short-Term Memory
Nevertheless, these transgenic mice show short-term memory for passive avoidance
when examined 5 to 10 minutes after training…
they learn, they have short-term memory but no long term memory.
Slide: Forskolin adminIstration to the hippocmpus
To determine if this memory defect is due to the loss of cAMP increases, we cannulated
DKO mice to deliver forskolin to area CA1 of the hippocampus.
Forskolin is a general activator of the other adenylyl cyclases present.
Verification of the cannula placement and forskolin penetration to area CA1 was done
by infusing bodipy-forskolin, a fluorescent-labeled forskolin. Bodipy-forskolin penetrated
to area CA1 only on the cannulated side of the brain.
Slide: LTM is Restored to DKO Mice by Infusion of Forskolin into Area CA1 of the
Hippocampus
Administration of forskolin 15 min before training completely reverses the memory defect
seen with DKO mice, they show LTM for passive avodiance.
We conclude that Ca2+-stimulated adenylyl cyclase activity is essential for some forms of
LTM, and that either AC1 or AC8 can produce the critical cAMP signal for memory
formation.
These mice are also deficient in other forms for hippocampus-dependent memory
including spatial memory.
Slide: L-LTP is compromised in DKO mice
L-LTP is a form of synaptic plasticity that depends on transcription. It is an
electrophysiological model for LTM. When this synapse in the hippocampus is tetanically
stimulated repeatedly, one gets a prolonged increase in synaptic efficiency that lasts
many hours. L-LTP is lost in DKO mice.
Slide: Impaired Training-Induced ERK1/2 Activation
in Mice Lacking CaM-Stimulated Adenylyl Cyclases
When DKO mice were trained for context, there was no training-induced increase in
pERk. However, the basal levels of MAPK are elevated in the double knockouts,
perhaps reflecting a compensating increase in MAPK expression.
Nevertheless, there was no trained-induced activation of MAPK in the cell body or
dendrites of neurons from the adenylyl cyclase deficient mice.
Slide: No increase in nuclear MAPK activity
Nor was there an increase in nuclear p-ERK in the mice lacking these adenylyl
cyclases.
Slide: Nuclear /Cytoplasm ratio does not increase with DKO mice
When wild-type mice are trained for context, they show about a six-fold increase in the
ratio of nuclear to cytoplasmic pERK.
This doesn’t occur with the adenylyl cyclase deficient mice.
Slide: Signaling model for CaM-stimulated adenylyl cyclases and CREB-mediated
transcription.
From these data and related experiments, we conclude that stimulation of the CaMstimulated adenylyl cyclases during training provides a cAMP signal required for the
activation and nuclear translocaion of Erk MAP kinase. Activation of MAPK by cAMP
could be mediated through EPAC1 or through PKA and Rap1.
Slide: AC1+ mice
From these experiments, it seemed that one might be able to enhance memory
genetically by increasing cAMP in the hippocampus. So we made transgenic mice that
overexpress AC1 under the control of the alpha-CaM Kinase II promoter. This restricts
increased expression of AC1 to the forebrain
Ca2+-stimulated adenylyl cyclase activity in hippocampal membranes was 3 fold in this
transgenic mouse strain.
Slide: AC1+ mice have increased LTP
These AC1-overexpressing mice have enhanced CA1 LTP, a measure of synaptic
plasticity. A single train of HFS which normally produces short-term LTP in wild type
mice, generated L-LTP in the AC1+ mice.
Slide: AC1+ mice show increased memory for novel objects
In this test, a mouse is introduced to two new objects, A and B, for 5 minutes. At a later
time, the same mouse is put in the cage with one of the original objects, B, and a new
object, C. Mice are naturally curious and will prefer a new object over an old one that
they have seen before. So…if they remember the original object B, they will show
preference for the new object C.
In a single training session, wild type mice have a memory for novel object that persists
for an hour but is gone after 24 hrs.
Mice over-expressing AC1 have memory for object recognition that lasts at least 24
hour. With a wild type mouse you have to train it four times in order for it to develop 24
hr memory for object recognition. …
If the mice are trained 2x for 10 minutes AC1+ mice but not wild type mice have 5 day
memory.
This is a remarkable improvement in memory, and one of the few transgenic mouse
strains with enhanced memory.
Slide: AC1+ Mice Have Superior Remote Memory
When wild type mice are trained for context by shocking one in the training context, they
maintain contextual memory for at least 3 weeks. After 22 weeks is almost completely
gone. In contrast the AC1+ mice maintain significant memory for context, even after 22
weeks. So increasing the activity of this enzyme in the hippocampus results in more
persistent LTM
Slide: The enhanced memory is due to increases in training-induced
MAPK/CREB transcriptional pathway.
Basal MAPK activity and the training-induced increases in MAPK were greatly increased
with mice over-expressing AC1.
Furthermore, training-induced stimulation of CREB phosphorylation is also increased in
the transgenic mouse.
These experiments identify AC1 as a useful drug target site to enhance
memory….particularly because this enzyme is neurospecific…it is the only neurospecific
signaling component in the cAMP signal transduction system.
A good strategy would be to design drugs that only activate AC1 when it is stimulated by
calcium. In that way, one would increase cAMP at specific synapses in response to
activity-dependent calcium increases at specific synapses representing a particular
memory trace and not have general saturating cAMP levels across the brain.
Slide: AC1+ extinction
There is one concern that I have about using drugs to increase calcium-stimulated
adenylyl cyclase in the hippocampus. There may be undesirable side effects. For
example, when a wild type mouse is trained for context and then brought back
repeatedly into context without a shock, it undergoes memory extinction. It learns that
the context is no longer associated with a shock and shows decreased freezing with
each exposure.
The extinction curve for AC1+ mice is greatly retarded. Either the memory is much
stronger and it cannot forget as easily or the mouse is deficient in reversal learning.
Either way, it is a warning signal that there are always unexpected consequences when
you play with mother nature.
Slide: How do we maintain memories over extended time?
Training-induced increases in transcription protein synthesis appear to be only transient.
How are memories maintained for long periods of time when the half-lives of most
proteins are on the order of hours to days?
Slide: The memory consolidation pathway undergoes circadian oscillation
Although activation of this pathway is necessary for contextual memory, it seems
unlikely that a single round of transcription can maintain hippocampus-dependent LTM
because they average half-life of synaptic proteins is not long enough.
This dilemma stimulated Kristin Mahan, a Pharmacology graduate student, to monitor
MAPK activity in the hippocampus over an extended period of time. Her working
hypothesis was that this pathway is reactivated periodically and that this reactivation is
required for the persistence of memory.
Slide: pERK Oscillates in the Hippocampus
When mice are maintained on a normal 12 hr L/12 hr D cycle, they exhibit a pronounced
diurnal oscillation in p-Erk in the hippocampus with maximal activity during the day, the
inactive phase of their circadian rhythm. Remember, mice are nocturnal and sleep
during the day and are activie during the dark.
Slide: MEK Activity in the Hippocampus Oscillates
The activity of the upstream regulator of MAP kinase, MEK, also shows a diurnal
oscillation with a maximum corresponding to the peak of MAPK.
Slide: Phospho-CREB in the Hippocampus Oscillates
In addition, phosphorylation of CREB at Ser-133, which is required for CREB stimulation
of CRE-mediated transcription, also shows significant oscillation.
These data indicate that the MEK/Erk MAPK /CREB transcriptional pathway in the
hippocampus is higher during the day, during the period of inactivity and sleep for mice.
Since we knew that this was the major pathway for memory consolidation, it was
impossible to ignore these data…especially because the peak of activity was during the
rest phase of the mouse circadian cycle. There is plenty of data suggesting that memory
consolidation may occur during sleep.
Slide: p-Erk oscillates under free-running conditions
Most diurnal oscillations are also circadian under free-running conditions (dark/dark
conditions).
This actogram represents the activity of mice over 24 hr periods for many days. The dark
areas represent motion the white areas, inactivity. Mice retain this activity cycle, even
under 24 hr dark conditions…we call this free-running because there is no light to entrain
the circadian cycle. This intrinsic circadian rhythm is dictated by the SCN in the
hypothalamus.
Therefore, MAPK activity was measured under free-running conditions. Animals
were entrained to a L/D cycle for 10 days and then placed in complete darkness (D/D)
for five days before being analyzed for hippocampal MAPK activity.
Oscillations in MAPK activity were maintained in the hippocampus under Freerunning conditions… suggesting that the fluctuation in MAPK activity is circadian.
Slide: cAMP oscillates in the hippocmapus
You may remember that in CNS neurons cAMP activates MAPK. Therefore, cAMP was
measured throughout the L/D cycle. Hippocampal cAMP was significantly higher during
the day than at night. This suggested the interesting possibility that cAMP may be driving
the circadian oscillation of MAPK in the hippocampus.
Slide: Calcium-Activated Adenylyl Cyclase Activity in the Hippocampus is Higher
During the Day than Night
To determine if this variation in cAMP is due to CaM-stimulated adenylyl cyclases ,
Ca2+-activated adenylyl cyclase activity in the hippocampus were measured during the
day and at night. This activity was significantly higher in the daytime compared to the
night…
Slide: Oscillations in pERK and cAMP are Absent in Mice Lacking CalciumActivated Adenylyl Cyclases 1 and 8
To determine if the oscillations in cAMP and MAPK activity depend on AC1 and AC8.
Mice lacking these two enzymes were analyzed for MAPK activity and cAMP during the
day and night. In contrast to wild type mice, the MAPK activity in the hippocampus of
DKO mice did not change during the circadian cycle… Nor did cAMP.
It is interesting that that disruption of Ca-stimulated adenylyl cyclases ablates the
oscillation in MAP kinase and completely blocks memory consolidation…suggesting that
the MAPK oscillation may be required to sustain contextual memory.
Slide: Ras.GTP shows a circadian oscillation
Activated Ras shows the same oscillation as MEK and Erk activities.
So cAMP must be activating upstream of Ras, perhaps through a cAMP-GEF such as
EPAC-1
Slide: L/L conditions impairs circadian rhythm
Another way to disrupt MAPK oscillations in the hippocampus is by putting animals
under L/L conditions.
Exposure of animals to 24 hr L/L conditions for several days severely disrupted the
circadian rhythm of the animals.
Slide: Exposure to Light/Light Conditions
Disrupts Oscillations in Hippocampal pERK
Exposure to constant light was sufficient to disrupt pErk oscillations in the
hippocampus………indicating pErk oscillations are under tight circadian control.
Slide: Exposure to Light/Light Conditions
Impairs Memory Maintenance
To test the effect of L/L conditions on contextual memory, the experiment illustrated here
was carried out. After training for context, half of the mice were exposed to light-light
conditions for a week.
The animals exposed to LL after training showed much poorer memory for context.
From these data you can begin to get a feeling about the importance of the circadian
rhythm and sleep for retention of memory.
Slide: Inhibition of MAPK at ZT0 and ZT4 but Not at
ZT12 and ZT16 Impairs Memory persistence
a. Another way to disrupt the oscillation of MAPK is to introduce MEK inhibitors at
the peak of the circadian oscillation of MAPK. MAPK activity in the hippocampus
can be inhibited by infusion of MEK inhibitors, in this case UO126.
d. When the MEk inhibitor was infused at the peak of MAPK activity contextual
memory was impaired.
g. When the MEK inhibitor was infused at the trough of MEK activity there was no
effect on the persistence of contextual memory.
These data indicate two important facts:
1. Two days after a memory is acquired and consolidated, you need MAPK
activity to maintain the memory.
2. You need MAPK activity during the rest phase for the animal to maintain
contextual memory.
Slide: Model Slide
It is interesting that disruption of the MAP kinase oscillation in the hippocampus
genetically or by changing the lighting conditions both impaired the persistence of
memory, without affecting acquisition.
Although this is correlative evidence, it strongly suggests that the circadian oscillation of
Ca2+-sensitive adenylyl cyclase activity drives a MAPK oscillation which contributes to
the persistence of LTM.
So here is the idea…During the day you acquire various memories, some of which are
especially significant and will be converted to LTM during the sleep. Activated synapses
that represent the memory trace for specific memories are biochemically tagged during
acquisition for consolidation during the rest phase or sleep. During the night the cAMP
/MAPK/CREB consolidation pathway is activated on a circadian cycle to strengthen
those memories at tagged snapses. Repeated activation over the circadian cycle may
serve to maintain the memories.
Slide: Electrolytic lesion of the SCN
A key unanswered question is whether the circadian oscillation of this signaling pathway
is intrinsic to the hippocampus or is driven by the master circadian clock in the
suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice
by electrolytic lesion and examined hippocampus-dependent memory as well as
adenylyl cyclase and MAPK activities when the SCN is destroyed bilaterally.
Electrolytic lesioning was done by bilateral insertion of electrodes to the SCN that were
insulated except at the tip. Delivery of current destroyed the SCN and circadian rhythm.
The Sham-lesioned mice retained their circadian rhythm.
Slide: The SCN is Required for the Persistence of Memory
In this experiment, mice were trained for contextual fear memory and two days later
underwent electrolytic lesioning or sham lesion. After a two week period they were
tested for contextual memory.
The Sham-lesion controls showed contextual memory comparable to non-surgery
controls.
SCN electrolytic lesioning greatly impaired contextual. Memory. This data indicates that
the SCN is required for the persistence of contextual memory. Why??
Slide: The SCN is Required for the oscillation in adenylyl cyclase activity
Ablating the SCN blocked the circadian oscillation of Calcium Sensitive Adenylyl cyclase
activity in the hippocampus.
Slide: The SCN is Required for Oscillation of MAPK Activity
Similarly, the circadian oscillation of MAPK activity in the hippocampus is also
dependent on the SCN. We don’t know the mechanism by which the SCN regulates the
circadian oscillation of signaling events in the hippocampus…
Slide: Return to model slide
In conclusion, we find it interesting that disruption of the MAP kinase oscilaltion in the
hippocampus genetically, by changing the lighting conditions, by use of MEK inhibitors,
or lesioning of the SCN impaired the persistence of memory.
Although this is correlative evidence, as is everything that we do in this field, it strongly
suggests that the circadian oscillation of Ca2+-sensitive adenylyl cylacase activity drives
an oscllation in MAPK activity MAPK that contributes to the persistence of LTM.
Slide: Monitoring Sleep in Mice
Another relevant question is whether or not the cAMP/MAPK/CREB pathway is activated
during sleep during the circadian cycle--So we carried out experiments to monitor these
activities during sleep using EEG and EMG recordings.
EMG, a measure of muscular activity, readily distinguishes animals that are awake from
those sleeping.
REM sleep which is characterized by fast, low amplitude EEG traces, can be
distinguished from NREM sleep which is characterized by slow, high-amplitude EEG
traces.
Slide Sleep profile of mice
It is notable that mice spend about 6% of their total sleep time in REM sleep, much less
time than in NREM sleep.
Mice exhibit polyphasic sleep architecture during the daytime spend some time awake,
or in REM or NREM sleep.
Sleep: MAPK activity is high during REM sleep
Mice were monitored continuously during the day time, rapidly sacrificed when they were
awake, in REM or NREM sleep and analyzed for MAPK activity.
P-Erk levels in the hippocampus were significantly higher in REM sleep compared to
waking, but not elevated in NREM sleep.
Slide: Elevated pCREB immunoreactivity in the CA1 and DG during REM sleep
Similarly, p-CREB levels were also elevated in REM sleep relative to waking but not in
NREM sleep.
Slide: cAMP/PKA signaling increases during REM sleep.
Moreover, cAMP levels in the hippocampus were higher in REM sleep than in waking
but not in NREM sleep.
This is reflected in increased PKA phosphorylation of PKA substrates in REM sleep
compared to waking.
Slide: Adult neurogenesis in the brain
Another feature of memory is its dependence on adult neurogenesis…. the
formation of new neurons in the adult brain. Adult neurogenesis occurs in two regions of
the adult brain: the subgranular zone of the Dentate Gyrus, and the Sub-Ventricular
Zone along the lateral ventricles where newborn cells migrate along RMS to the olfaction
bulb.
Slide: Factors affecting adult neurogenesis in the brain
Stimulation of adult neurogenesis by:
-Environmental enrichment
-Exercise--running
-Anti-depressants
-Novel odors
-Pheromones
-Mating
inhibition of adult neurogenesis by:
-Stress
-Alcohol
-Drugs
-Aging
-Circadian disruption
Slide: ERK5 Is Only Expressed In The Neurogenic Regions In Adult Mouse Brain
Slide: Hypothesis
1. ERK5 May Play A Novel And Critical Role In Regulating Adult Neurogenesis In Both
SVZ/Olfactory Bulb And SGZ/Hippocampus.
2.Adult neurogenesis may play as role in memory
Slide: Design of a transgenic mouse to conditionally knockout Erk5 only in
neurons in the neurogenic regions of the adult brain.
To evaluate the effect of loss of ERK5 on adult neurogenesis in vivo, Josh Pan, an MCB
student in the Xia lab, generated inducible and conditional ERK5 KO mice by crossing
the ERK5 floxed mice with the nestin-cre-ER tm driver line. This strain, has a nestin-creERtM, so ERK5 is only KO in nestin-expressing neural stem cells when you give the
mice tomoxifen. So if you administer tomoxifen to adult mice, then erk5 is deleted in
adult neural stem cells expressing nestin; leaving the developing nervous system
undisturbed.
As shown on the left, when the mouse is given tamoxifen, you block expression of Erk5
in the SGZ.
Slide: Conditional Deletion Of ERK5 In Adult Neurogenic Regions Reduces
Hippocampal Neurogenesis By Delaying Neuronal Differentiation
Tamoxifen treatment decreased the number of total ERK5+ cells in the SGZ by 75%.
This led to reduced the number of adult-born, mature neurons that are NeuN and BrdU
double-positive. NeuN is a marker expressed in mature neurons.
Concomitantly, there was an increase in the number of BrdU+ cells co-labeled with DCX
or Calretinin, markers for immature granular neurons, suggesting that erk5 deletion
delays neuronal differentiation of adult born neurons.
Slide: ERK5 icKO Mice Are Normal In Spatial Memory In The Morris Water Maze
Test
1.ERK5 icKO mice learned this task as well as wt mice during the training sessions.
2.There was also no difference in the probe test.
(training: 4 sessions each day, 30 min apart, 40 sec swimming, rest on platform for 15
sec)
Slide: ERK5 icKO Mice Show Impaired Acquisition Of Newer Spatial Information
We then subjected animals to reversal training, in which the hidden platform was moved
to the opposite location, in quadrant #3.
This is a more challenging task, because animals have to forget the previous location
and learn to find the hidden platform in a new location.
Interestingly, ERK5 icKO mice swam longer distances than control mice to find the
new platform during training.
Furthermore, during the subsequent reversal probe test, control mice spent much more
time in the new target quadrant (#3) than in the old one (#1). However, ERK5 icKO mice
spent the same amount of time searching both the old and new locations.
(The deficits of ERK5 icKO mice in the reversal hidden platform and fear memory
extinction tests could be explained by two different mechanisms. ERK5 icKO mice could
be impaired in memory extinction per se and could not learn the more challenging tasks
of dissociating the hidden platform from the original location or dissociating the context
from the shock. Alternatively, one could argue that ERK5 icKO and inhibition of adult
neurogenesis made the initial memory more stable and long lasting, thus more difficult to
erase or suppress. However, the fact that ERK5 icKO mice don’t have any remote fear
memory argues against a more stable or stronger memory in the KO mice)
Slide: Representative Track Plots During Probe Tests
These are examples of computer-generated tracing of their swimming patterns during
the initial probe test and reversal probe test.
You can see clearly the differences between wt and ERK5 icKO mice during the reversal
probe test.
These data suggest that ERK5 icKO mice exhibit impaired acquisition of newer spatial
information and did not learn the new location of the hidden platform as well as control
mice.
Slide; ERK5 icKO Mice Have No Remote Memory In The Passive Avoidance Test
(Hippocampus-dependent fear conditioning)
(The testing apparatus has a dark and light chamber, separated by a trapping door. You
place the animal in the light side; because of their natural curiosity and preference for
dark over light , they will cross-over to the dark side. During training session, when the
animal enters the dark side, the trapping door was closed and a mild foot shock was
delivered. When you bring the animal back to the light chamber later, if they remember
the foot shock, they will stay out here in the light chamber and not enter the dark
chamber. )
The time lapse before they enter the dark chamber is recorded and defined as crossover
latency.
ERK5 icKO mice showed similar cross-over latency as WT mice when tested 24 hr later,
suggesting that they learned and can retrieve the memory.
Interestingly, ERK5 icKO mice showed no remote memory measured 21 days after
training.
(A 0.7 mA foot shock (for 2 s) was delivered immediately after a mouse enters the
darkened half of the chamber.)
Slide: Conditional caMEK5 expression in adult neurogenic regions extends long
term spatial memory
Can you enhance long-term memory by increasing adult neurogenesis in the
hippocampus? In this experiment, adult neurogenesis was enhanced using a transgenic
mouse in which you can conditionally increase the expression of the upstream activator
of MEK5 by tamoxifen. In this measurement of spatial memory using the Morris water
test, the wild type mice retained spatial memory measured by the probe test for 223
days while mice with enhanced neurogenesis retained spatial memory for 51 days.
Conclusion:
If you want to maintain long-term memories, increase adult neurogenesis by
exercise, traveling, and sex.