1) Corticotropin-releasing factor (CRF) expressing neurons in the amygdala, bed nucleus of the stria terminalis (BNST), and paraventricular nucleus (PVN) are implicated in anxiety and fear. 2) A transgenic mouse was developed that expresses Cre recombinase in CRF neurons, allowing manipulation of these neurons. 3) Preliminary analysis of 5 male CRF-Cre mice at 3 and 5 weeks found stable expression of Cre in the BNST, central amygdala, and PVN, supporting continued expression into adulthood. More research is needed with more mice across ages.

1. Amygdala:
CRF Cre Rosa
LacZ
Amygdala:
CRF expression
(Allen Brain Atlas)
References
©2014 Allen Institute for Brain Science. Allen Mouse Brain Atlas [Internet].
Available from: http://mouse.brain-map.org/
Gafford G, Jasnow AM, Ressler KJ (2014) Grin1 receptor deletion within CRF
neurons enhances fear memory. PloS one 9:e111009.
Gafford GM, Guo JD, Flandreau EI, Hazra R, Rainnie DG, Ressler KJ (2012) Cell-
type specific deletion of GABA(A)alpha1 in corticotropin-releasing factor-
containing neurons enhances anxiety and disrupts fear extinction. Proceedings of
the National Academy of Sciences of the United States of America 109:16330-
16335.
Martin EI, Ressler KJ, Jasnow AM, Dabrowska J, Hazra R, Rainnie DG, Nemeroff
CB, Owens MJ (2010) A novel transgenic mouse for gene-targeting within cells
that express corticotropin-releasing factor. Biol Psychiatry 67:1212-1216.
Discussion
Cre recombinase and CRF expression
• Cre recombinase expression remained stable
within the 3-5 week range we tested.
• Studying a greater age range of CRF-Cre mice
would offer further insight into developmental
changes in Cre expression.
Limitations:
• Small sample size (only five male mice)
• Difficulty in staining and imaging may have
hidden differences in Cre recombinase
expression.
• Signal loss due to difficulty mounting, staining,
or malfunctioning imaging hardware.
Future Directions:
• Analyses of a greater number of transgenic
mice in a greater age range are needed to
determine the true stability of Cre recombinase
transgene expression within the BNST, PVN,
and amygdala
Conclusions
• Our preliminary findings indicate that Cre
recombinase is stably expressed within the BNST,
CeA and PVN at 3 and 5 weeks.
• These preliminary results support the idea that the
cessation of Cre recombinase transgene and CRF
expression continues into adulthood in these mice.
• More CRF-CRE knockout mice will need to be
studied to determine whether Cre expression occurs
throughout adulthood.
Expression of the Cre recombinase transgene and
corticotropin-releasing factor in developing transgenic mice
Jon Giuliano1, Georgette Gafford, PhD2, and Kerry Ressler, MD, PhD2
Emory College of Arts and Sciences1, Yerkes National Primate Research Center2
Methods
Subjects
Five male CRF-Cre mice (Mus musculus) were euthanized at 3
weeks of 5 weeks of age using isofluorane. Their brains were
removed and flash frozen. Using a Cryostat, 35um brain sections
were cut and placed on slides.
Treatments
Day 1: Two cartridges (10 slides total) were prepared. For each
cartridge (5 slides) slides were rinsed in three 5-minute sessions with
1X PBS. Next, blocking buffer (16 mL 1X phosphate buffered saline
(PBS)), 64uL Triton X-100, 48 uL NGS, 160 mg BSA) was prepared,
and slides were blocked for 1 hour at room temperature. Next,
primary solution (16 mL 1X PBS, 64uL Triton X-100, 48 uL NGS, 160
mg BSA, 16 uL mouse monoclonal anti-Cre or anti CRF (Sigma)
primary antibody was prepared, and slides were bathed for 48 hours
at 4 degrees Celsius.
Day 2, Immunofluorescence: Slides were rinsed in three 5-minute
sessions with 1X PBS. Next, slides were incubated in a 1:1000
florescent 2º antibody: PBST solution for 2 hours at room
temperature. Slides were then rinsed in three 5-minute sessions with
1X PBS, then coverslipped and imaged.
Day 2, Immunohistochemistry: Slides were rinsed in three 5-minute
sessions with 1X PBS and then placed in a 1:1000 florescent 2º Ab
PBST solution for 2 hours at room temperature. Slides were rinsed in
1X PBS. Next, ABC solution (5 mL 1X PBS, 0.25% Triton X-100, 1:500
A and B) was prepared, and slides were incubated in the solution for
an hour. Slides were then rinsed 1X PBS. Finally, DAB solution was
prepared, and slides were incubated for 5 minutes, coverslipped and
imaged. Acknowledgements:0SIRE0Research0Partner0Program,02014
A.
AC AC
BLA
BLA
PVN PVN
BNST BNST
CeA CeA
PVN
Cre-
CeA
PVN
CeA
Cre+
A.) Rosa LacZ staining from
CRFp3.0CreLacZ mouse (Martin et al.,
2010) corresponds to localization of B.)
CeA CRF expression (Image credit: Allen
Institute for Brain Science). C.) LacZ
staining in Rosa LacZ CRF Cre mice
corresponds to CRF expression. D.) In
situ hybridization of CRF expression in
Cre- and Cre+ mice (Gafford et al., 2014)
0
20
40
60
80 Cre)
Cre+
Seconds
Plus maze – Time in open arm
Anxiety measure
*
0
20
40
60
80
1 2 3 4 5
%*Freezing*Behavior
*
Fear Conditioning– Freezing
Fear measure
E. F.
Cre%
Cre+
CRF Neurons are Localized to the Bed Nucleus of the Stria Terminalis (BNST),
Paraventricular Nucleus of the Thalamus (PVN), and Central Amygdala (CeA)
Manipulation of CRF Neurons can modulate anxiety and fear
E.) Significant
differences in anxiety
as measured in the
plus maze task
(Gafford et al., 2012)
F.) Fear (Gafford et al.,
2014) as measured in
fear conditioning occur
with manipulation of
different
subpopulations of CRF
neurons.
Preliminary data suggests Cre expression extends into adulthood
G). CRF expression (Allen Brain
Atlas) is comparable to Cre
expression in BNST of CRF Cre
mice. H.) Hoescht stain shows
nuclei of all cells. I.) Cre staining
of CRF neurons. J.) CRF
expression in PVN (Allen Brain
Atlas) is comparable to Cre
expression in PVN of CRF Cre
mice (K.) Hoescht stain shows
nuclei of all cells and (L.) Cre
staining of CRF neurons.
Preliminary findings in our samples
suggest Cre staining is stably
expressed across 3 and 5 weeks.
Allen0Brain0Atlas
BNST
BNST020X0Hoescht0staining BNST020X0Cre0staining
B.
C. D.
Introduction
In the United States alone, anxiety disorders
affect 40 million adults while PTSD affects 7.7
million adults. To address these common disorders,
we must understand neural factors associated with
anxiety, PTSD, and general fear acquisition.
Corticotropin-releasing factor (CRF) peptide,
released from a number of cell types, affects the
endocrine, autonomic, and behavioral stress
response; as such, it remains a factor of interest in
the study of anxiety disorders.
Cre recombinase is a 35-kDa enzyme which
recognizes 34-bp sequences known as “LoxP” sites
and excises DNA flanked by these LoxP sites
(“floxed”) via intrachromosomal recombination.
Martin and colleagues (Martin et al., 2010)
established a transgenic mouse in which CRF-
producing cells also express Cre (CRF Cre mouse).
We visualized the Cre recombinase transgene and
CRF expression in five male CRF-Cre mice of
varying age, using immunohistochemistry and
immunofluorescence in areas of CRF expression
(the bed nucleus of the stria terminalis (BNST),
central amygdala (CeA), and the paraventricular
nucleus of the hypothalamus (PVN) at 21 and 35
days postnatal.
Allen0Brain0Atlas
G. H. I.
J. K. L.
PVN020X0Hoescht0staining
PVN
PVN020X0Cre0staining