title slide

1
Classic c-Fos Paradigms
1. Circadian Rhythms (light-induced c-Fos in SCN
-gt phase shift) 2. Seizures (kainic acid induced
c-Fos in hippocampus -gt excitotoxic cell
death) 3. Stimulants and Addiction (cocaine/amphet
amine induced c-Fos in striatum, accumbens)
2
(No Transcript)
3
History of c-Fos
1930s -- Watchpainters in NJ have an alarmingly
high rate of bone cancer (osteosarcoma) 1960s --
Drs Finkel, Biskis, and Jinkins identify a virus
activated by radiation that induces
osteosarcomas. 1980s -- Drs. Curran and Morgan
clone the viral oncogene, v-Fos, and the cellular
analog, c-Fos c-Fos turns out to be a
proto-oncogene, transcription factor, and
immediate-early gene.
4
c-Fos transgenic Mice
Overexpression of c-Fos leads to bone
tumors Deletion of c-Fos leads to bone
deformities
Fos KO
Ctrl
5
c-Fos as Marker of Neural Activation
c-Fos is expressed in bone during development,
but in other tissues during cell division or
after stimulation. Relatively low expression in
the brain except after stimulation.
6
c-Fos as transcription factor
7
Immunohistochemistry (aka immunocytochemistry)
peptide -gt rabbit, mouse -gt immune system -gt 1
antibodies rabbit -gt polyclonal, mouse used for
monoclonal (inject rabbit/mouse antibodes -gt
horse -gt 2 antibodies) perfuse animal, dissect
brain, cut 20-40 soak 1 antibodies with target
tissue 1 antibodies stick to target peptide 1)
detect 1 antibodies with fluorescent/radioactive
2 antibodies 2) soak target tissue with
biotinylated 2 antibodies 2 antibodies stick
to 1 antibodies incubate with
avidin/biotin/enzyme complex -gt macmolecular
complex with enzyme react enzyme with
chromogenic substract look at colored reaction
product
Amplification by increasing of tags,
enzyme-gtproduct
8
Immunohistochemistry (aka immunocytochemistry)
DAB H20 02
DAB H202
9
Immuno controls

• 1. Leave out 1 or 2 antibodies
• makes sure staining specific to antibodies
• 2. Preabsorb 1 antibody with target peptide
• makes sure 1 sticks to target-- but doesnt
  tell you
• if 1 sticks to something else (unknown,
  undesired?)
• 3. Confirm with Western Blot
• immuno gives you spatial resolution in section
  or cell
• (Western gives you molecular size resolution,
  to make sure its right protein)
• 4. Confirm with in situ hybridization/RT-PCR/North
  ern blot
• immuno gives you protein epitope, but can cross
  react with other proteins
• RNA detection methods use a sequence specific
  probe that is probably less cross-reactive -- but
  gives you mRNA (cell body) not protein
  (dendrites, axons, or whereever)

10
c-Fos example no treatment
11
c-Fos example magnetic field exposure.
12
How do we know c-Fos correlates with neural
activity?
Inject CCK i.p. Supraoptic Nucleus secretes
oxytocin into blood stream. Record APs from SON,
and sample oxytocin from blood. c-Fos mRNA
appears with increased firing rate.
13
c-Fos requires receptor activation, not APs
Carbachol injection stimulates SON, and induces
c-Fos. Electrical stimulation of SON axons does
not induce c-Fos. So depolarization alone is not
sufficient receptor activation is required.
14
Classic c-Fos Paradigms
1. Circadian Rhythms (light-induced c-Fos in SCN
-gt phase shift) 2. Seizures (kainic acid induced
c-Fos in hippocampus -gt excitotoxic cell
death) 3. Stimulants and Addiction (cocaine/amphet
amine induced c-Fos in striatum, accumbens)
15
Light-induced c-Fos in Suprachiasmatic Nucleus
16
Kainic acid induced seizures and c-Fos induction
Systemic KA -gt glutamate receptors widespread
depolarization -gt seizures. Huge influx of
calcium induces c-Fos, and ultimately causes cell
death.
Control
Kainic Acid (seizure2h)
17
Advantages of c-Fos as a marker of Neuronal
activity
1. Gives cellular resolution (vs. 2DG, MRI). 2.
Can map whole brain (vs. electrodes). 3.
Relatively long persistance of marker (vs.
synaptic activity). 4. Can quantify response by
counting c-Fos-positive cells. 5. Double-labeling
can reveal the phenotype of activated cell. 6.
Presence of c-Fos implies activation of specific
signaling pathways.
18
Double-labeling to identify c-Fos-positive cells
19
c-Fos promoter reveals sensitivity to signaling
pathways
CAMK, PKA
MAPK
FOS-JUN
20
Caveats to c-Fos as a marker
1. Terrible temporal resolution of min-hrs (vs.
msec with electrode). 2. Will only see cells that
express c-Fos when activated (so absence of c-Fos
is not equal to absence of activation). 3.
Inhibitory cells also express c-Fos. 4.
Relatively insensitive, so often need to
calibrate response. 5. Immuno detection is
all-or-nothing, so difficult to quantify the
response of an individual cell.
21
Properties of c-Fos
Responsive to cAMP and Ca via CREB and MAP
kinase Induced rapidly (mRNA within 10 minutes,
protein within 30 minutes) mRNA degrades very
rapidly (half-life of 15 min) Protein stabilized
by phosphorylation at C-terminus by MAPK or
Rsk90 Forms dimer with Jun family members to make
AP-1 proteins Binds to AP-1 sites to
enhance/repress target gene expression
22
Rapid degradation of c-fos mRNA via AU-rich
element
t1/2
c-Fos
15 min
45 min
15 min
23
Serine Phosphorylation Sites at C-terminus
24
c-Fos Protein stability enhanced by
phosphorylation
New protein labeled with brief pulse of
35S-methionine disappearance of radio-labeled
Fos band Fos degradation.
c-Fos
mutant
- mutant
mutant behaves as if always phosphorylated -
mutant cant be phosphorylated
25
AP-1 Family Members
c-Fos Fos-B Fra-1 Fra-2
c-Jun Jun-B Jun-D
A Fos protein dimerizes with a Jun protein to
form activator protein 1 complex (AP-1). Dimer
binds to AP-1 element of target genes. Some
combinations enhance expression (e.g. c-Fos
c-Jun). Other combinations repress expression
(e.g. c-Fos Jun-B). Some are constitutively
expressed (e.g. c-Jun) others are inducible (e.g.
c-Fos). Half-life varies (e.g. Fos-B is
long-lived).
26
AP-1 Family Members
Different members may contribute to different
functions/behaviors c-Fos -gt circadian rhythms
(KO has bad clock) delta FosB-gt drug addiction,
wheel running FosB -gt maternal behavior
27
FosB KO is a poor Mom
28
FosB KO is a poor Mom
29
Dimers cause bending of DNA at AP-1 Site
30
AP-1 regulates target gene expression
Well-established genes somatostatin enkephalin
Other target genes are not well-defined, but
either 1. house-keeping genes after acute
stimulation. 2. plasticity-related genes when
stimulation causes long-term change in neuronal
function. Need to demonstrate AP-1 binding,
block of AP-1 leads to change in gene expression
31
Remaining c-Fos issues

• Identifying other IEGs and target genes mediating
  long term changes.
• Correlating c-Fos with other measures of
  activity, e.g. fMRI in humans.

32
Conditioned Taste Aversion (CTA)
A form of associative learning in which an animal
learns to avoid or reject a novel food after the
taste of the food has been paired with a toxic
effect. when a sucrose solution is paired with a
mildly toxic injection of LiCl, rats subsequently
reject sucrose CTA learning has several unique
characteristics tolerates a long-delay
between taste and toxin requires only one
pairing of taste and toxin persists for months
to years CTA learning is mediated by robust
changes in neural networks requiring gene
expression and protein synthesis. The underlying
molecular mechanisms may reveal specific
adaptations to the physiological and temporal
niche of CTA.
33
CTA Learning A Change in Behavior
34
Intraoral Infusions of Sucrose Taste Stimulus
35
CTA Learning A Change in Neural Activity
c-Fos in brainstem, amygdala
36
c-Fos as Marker of Neuronal Activity
AP-1
target genes
37
CTA After Contingent Sucrose and LiCl
10
Sucrose LiCl
Sucrose LiCl
Sucrose LiCl
Sucrose
Alone
8
Weight Gain (g)
6
6 ml / 6 min
4
Intraoral
Infusion
2
0
Monday
Monday
Friday
Wednesday
1st Pairing
2nd Pairing
3rd Pairing
Final Test
38
Non-Contingent Sucrose and LiCl
10
Sucrose
Alone
8
6
4
2
0
Monday
Monday
Friday
Wednesday
Final Test
39
Non-Contingent Sucrose
iv
iNTS
40
Contingent (CTA) Sucrose
contingetn sucrose vs LiCl in the NTS
41
c-Fos induction in the iNTS after CTA Expression
Induced by CTAs with other tastes and
toxins. Correlates with learning, not
rejection. Correlates with the strength of the
CTA. Disappears with extinction. Diminishes with
forgetting. Is taste specific. Blocked by lesions
that block CTA expression.
see also Bernstein, Swank, Schafe, et al.
42
Overlapping Distribution of c-Fos induced by LiCl
or Cond. Sucrose
100
Extent of the Area Postrema
Cond. Sucrose
LiCl
80
60
c-Fos
Cells / Section
40
20
0
-240
240
480
720
960
1200
1440
1680
1920
2160
0
Caudal
Rostral
Distance from Obex (microns)
43
Are the same cells in the NTS activated by CTA
acquisition (LiCl) as by CTA expression (cond.
sucrose) ?
44
Protein Cond. Sucrose
mRNA LiCl
time
45
Contingent vs. Non-Contingent Pairings of
Intraoral Sucrose (5, 6 ml) and LiCl (76 mg/kg,
ip)
6
5
5 Sucrose Intake (g)
4
3


2
1




0
Pair 1
Pair 2
Pair 3
Test
46
c-Fos Protein (IHC) 3.3 h after Cond. Sucrose
47
c-Fos mRNA (ISH) 0.3 h after LiCl Injection
48
c-Fos Protein Induction by Conditioned
Sucrose c-Fos mRNA Induction by LiCl
c-Fos protein (IHC)
c-Fos mRNA (ISH)
296
100
15



80
12
Grains per Cell
Cells per Section
60
9
253
40
6
20
3
0
0
NC-NaCl
NC-LiCl
CS-NaCl
CS-LiCl
CS-NaCl
CS-LiCl
49
c-Fos Protein (Immunohistochemistry)
50
c-Fos mRNA (in situ hybridization)
51
Increased c-Fos mRNA in c-Fos protein cells after
LiCl Injection
Cond. Sucrose NaCl
Cond. Sucrose LiCl
c-Fos mRNA Grains/Cell
c-Fos mRNA Grains/Cell
52
Cond. Sucrose LiCl
dark, c-Fos Protein positive cells (CS)
53
Cond. Sucrose LiCl
c-Fos mRNA positive cells (LiCl)
54
Cond. Sucrose LiCl
c-Fos Protein and mRNA positive cells (CS and
LiCl)
55
Cond. Sucrose LiCl
56
c-Fos/c-Fos Double Labeling Conclusions
Approximately 40 of the cells activated by the
taste of sucrose during CTA expression can
be co-activated by a subsequent injection of
LiCl. Therefore, the taste response and the
toxin response can overlap at the neuronal level
in CTA learning. What other genes are expressed
in these cells? Sparsely scattered cells Multiple
genes