Download Li Hao - USD Biology

Hao Li
Normal
condition
Inhibitory effects of
GABA on CRH neurons
Decreasing activity of
the HPA axis
THDOC
(StressStress
derived
neurosteroid) condition
Excitatory effects of
GABA on CRH neurons
Increasing activity of
the HPA axis
δ subunit-containing GABAA receptors
Animals
CRH-GFP mice which express GFP specifically in CRH neurons by crossing GFP
mice and CRH-Cre mice (?)
Gabrd(GABA receptor subunit delta) knocked out mice (control)
If two loxP sites are in the
DNA like the figure
shows , the target gene
will be excised.
If two DNA contain single
loxP sites, they will be
fused.
Detection
• How the Cre recombinase is expressed in CHR
neurons
• How many GFPs are linked with CRH
• How the CRH is expressed in PVN
X-gal staining assay kit
CRH-Cre mice X Rosa26 reporter mice
fluorogold
lacZ reactivity(black) fluorogold labeling( purple)
GFP antibody
CRH-GFP mice
All the GFP and
Cre recombinase
are in CRH neurons
Methods
Whole-cell patch clamp
Perforated patch clamp
Results
GABAA receptors δ subunit expression in the PVN
GABAA receptors δ subunit largely exist in CRH neurons
Neurosteroid regulation of CRH neurons
IPSCs
Average
decay
time of
IPSCs
SR95531:
GABAAR
inhibitor
Tonic
currents
Neurosteroid has
effects on GABAAR
δ subunit.
Spontaneous firing rate
Binned firing rate (?)
Neurosteroid makes CRH neurons harder
to fire.
Neurosteroid actions on CRH neurons regulate corticosterone levels
Under non-stress condition,
neurosteroid decreased the
corticosterone level (HPA
axis activity).
Under stress condition, by
effects on δ subunit,
neurosteroid increased the
corticosterone level (HPA
axis activity) and blockade of
the neurosteroid seemed to
turn off the HPA axis activity.
Neurosteroids increase the activity of CRH neurons following stress
Perforated patch, current
champ I=0
Spontaneous firing rates of
CRH-GFP neurons
Neurosteroid increased
firing rates under stress and
decreased under normal
condition.
Animals were THDOC-treatment.
Most CRH neurons depolarized
following stress.
When treated with the GABAAR
inhibitor, neurons switched from
excitatory to inhibitory.
Decreased phosphorylation of KCC2 residue Ser940 in the PVN following stress
Biotinylated KCC2 =
expression of KCC2 on cell
surface
The expression of KCC2 both in
total and on surface and
phosphorylation of KCC2
Ser940 decreased following
stress.
Neurosteroidogenesis mediates stress-induced anxiety
Blockade of neurosteroid by
finasteride largely decreased
animals’ anxiety behaviors.
Discussion
谢谢 ！
Thank you!