# Viral Vector Induction of CREB Expression in the Periaqueductal Gray Induces a Predator Stress-Like Pattern of Changes in pCREB Expression, Neuroplasticity, and Anxiety in Rodents

Authors: Robert Adamec; Olivier Berton; Waleed Abdul Razek
Journal: Neural Plasticity
 (2009)

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## Abstract

Predator stress is lastingly anxiogenic. Phosphorylation of CREB to pCREB (phosphorylated cyclic AMP response element binding protein) is increased after predator stress in fear circuitry, including in the right lateral column of the PAG (periaqueductal gray).  Predator stress also potentiates right but not left CeA-PAG (central amygdala-PAG) transmission up to 12 days after stress. The present study explored the functional significance of pCREB changes by increasing CREB expression in non-predator stressed rats through viral vectoring, and assessing the behavioral, electrophysiological and pCREB expression changes in comparison with handled and predator stressed controls.  Increasing CREB expression in right PAG was anxiogenic in the elevated plus maze, had no effect on risk assessment, and increased acoustic startle response while delaying startle habituation.  Potentiation of the right but not left CeA-PAG pathway was also observed. pCREB expression was slightly elevated in the right lateral column of the PAG, while the dorsal and ventral columns were not affected. The findings of this study suggest that by increasing CREB and pCREB in the right lateral PAG, it is possible to produce rats that exhibit behavioral, brain, and molecular changes that closely resemble those seen in predator stressed rats.


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## Body

## 1. Introduction

Study of the neurobiology of
long-lasting changes in affect occurring after stressful events is of interest,
an interest heightened by the fact that fearful events may precipitate
affective psychopathologies  [1, 2].  In
extreme cases, a single aversive experience may induce posttraumatic stress
disorder (PTSD) [3, 4].  Animal models are useful to enhance
understanding of the impact of stress on brain and behavior, permitting
simulation of a human condition in a controlled setting allowing study of
disorder development. Conditioned fear paradigms, behavior in unfamiliar
situations that are fear or anxiety provoking, and more recently, predator
stress, are all models used to understand the neurobiology of the impact of
fearful events on affect.Predator stress in our hands
involves the unprotected exposure of a rat to a cat [5].  Predator stress may model aspects of PTSD for
several reasons.  First, predator stress
has ecological validity due to the natural threat posed by the predatory nature
of the stressor.  Second, duration of
anxiety-like effects in rats after predator stress, as a ratio of life span, is
comparable to the DSM IV duration of psychopathology required for a diagnosis
of chronic PTSD in humans. Third, predator stress has neurobiological face
validity in that right amygdala and hippocampal circuitry are implicated in
behavioral changes produced by predator stress, and these areas are consistent
with brain areas thought to be involved in PTSD [6–9].  For example, brain imaging  implicates hyperexcitability of the right
amygdala in response to script-driven trauma reminders in the etiology of PTSD [10–14].  Fourth, parallel path analytic studies  using data from Vietnam
veterans suffering from
PTSD and predator stressed rodents find that in both humans and rodents,
features of the stressor predict the level of anxiety [6].  For example, in predator stressed animals,
the more cat bites received, the higher the level of anxiety measured a week
later.  Finally, similar lasting changes
in startle and habituation of startle are seen in both predator stressed rats
and humans with PTSD [6, 15–18].Predator stress is fear provoking
and stressful [19–22]. Moreover, cat exposure produces long-lasting
increases in rat anxiety-like behavior (ALB)
[5, 23],
with some behavioral changes lasting three weeks or longer [5, 6, 24].
Behavioral effects of predator stress have been evaluated in a number of
tests including hole board, elevated plus maze (EPM), unconditioned acoustic
startle, light/dark box, and social interaction.
Anxiogenic effects of predator stress are NMDA receptor-dependent.  Systemic administration of both competitive
and non-competitive NMDA receptor antagonists 30  minutes before, but not 30 minutes after, predator stress prevents lasting changes in ALB [16, 25]. Moreover, local NMDA receptor block in
the amygdala prevents predator stress-induced increases in ALB [26].In addition to the behavioral
changes, amygdala efferent and afferent neural transmission is altered after
predator stress.  Specifically, predator
stress causes a long-lasting potentiation in neural transmission from the right
amygdala (central nucleus-CeA) to the right lateral column of the
periaqueductal gray (PAG), and
from the hippocampus via the right ventral angular bundle (VAB) to the right
basolateral amygdala (BLA) [9, 23, 27]. Moreover,
potentiation in these pathways is NMDA receptor-dependent [7]. In addition, NMDA receptor antagonists produce
anxiolytic-like effects when microinjected into the dorsolateral PAG  [28, 29].  The PAG  is also implicated in rodent ALB [30], and is activated by predator stress [31]. Together, these data suggest NMDA receptor-dependent long-term potentiation (LTP)-like change in amygdala afferent and
efferent transmission following predator stress contribute to the lasting
anxiogenic effects of cat exposure [7, 9, 16]. In
support of this conclusion are the findings that amygdala afferent and efferent
LTP-like changes are highly predictive of severity of change in ALB following predator stress [9, 23, 27].Predator stress induced changes in ALB and amygdala neural transmission are
accompanied by changes in phosphorylated cAMP response element binding protein
(pCREB). Specifically, pCREB-like-immunoreactivity (lir) is elevated in the
basomedial (BM), BLA, CeA,  and lateral (La) amygdala after predator stress
compared to control rats [32].  This is consistent with the elevation of
pCREB-lir in the amygdala after forced swimming stress [33, 34],
fear-conditioning in mice [35], retrieval of a
cued-fear memory [36], and electric shock [37].  In
addition to the amygdala, predator stress increases pCREB-lir in the right
lateral column of the PAG (lPAG) [23].As mentioned,  NMDA receptor antagonism prior to predator
stress blocks increases in ALB and
potentiation of amygdala afferent and efferent neural transmission.  Since phosphorylation of CREB may be regulated by NMDA receptors [38, 39] and pCREB-lir is increased after predator
stress [23, 32], the question of whether NMDA
receptor antagonism can block predator stress induced enhancement of pCREB-lir
was recently tested. Blocking NMDA receptors with the competitive blocker, CPP,
30 minutes prior to predator stress, prevented stress induced increases in pCREB
expression in amygdala, and right lPAG  [40]. Of
importance, the same dosing regime also blocks predator stress  effects on affect and amygdala afferent and
efferent transmission [7, 16, 25, 26].Together these findings provide
compelling evidence that predator stress induced increase in pCREB  is an
important contributor to the changes in brain and behavior of predator stressed
rodents. The purpose of the present study was to directly manipulate CREB  and pCREB  expression to confirm this notion.Local changes in gene expression in
the brain can be achieved with viral vectoring as a method of delivering
recombinant genes directly into neurons [41].
There are a variety of viral vectors available but several characteristics of
the herpes simplex virus (HSV) make it an ideal candidate for this study. The
non-toxic replication defective  HSV
vector is capable of infecting most mammalian differentiated cell types, it
accepts very large inserts and has high efficiency in  infecting neurons, being naturally
neurotrophic [41, 42].  One of the earliest studies to utilize this
method and apply it to rodent anxiety tests found that HSV vectored expression
of CREB  in the BLA  increased behavioral measures of anxiety in
both the open field test and the EPM, and enhanced cued fear conditioning [43].The present study was designed to
test the functional significance of
pCREB  changes within the right lateral column of the PAG. To do this we genetically induced increased
expression of CREB  in the right
lPAG  with HSV vectors and determined the effects of these manipulations on
behavior and amygdala efferent transmission (CeA-lPAG). We transfected  the neurons of the right PAG  in an area where pCREB  levels and CeA-PAG transmission are elevated after predator
stress  (see Adamec et al. [8, 23]).


## 2. Methods

### 2.1. Ethical Approval

The procedures involving animals
reported in this paper were reviewed  by
the Institutional Animal Care Committee of Memorial University and found to be
in compliance with the guidelines of the Canadian Council on Animal Care. Every
effort was made to minimize pain and stress to the test subjects while using as
few animals as possible.


### 2.2. Animals

Subjects were male hooded Long Evans
rats (Charles River Canada). Rats were housed singly in
clear polycarbonate cages measuring 46 cm× 24 cm × 20 cm for one week prior to
any testing. During this week, rats were acclimatized to their cage, and
handled. Handling involved picking up the rat and gently holding it on the
forearm. Minimal pressure was used if the rat attempted to escape, and grip was
released as soon as the rat became still. Rats were handled in the same room as
their home cage for one minute each day during the week long adaptation period.
Rats were given food and water ad lib and were exposed to a 12-hour light/dark cycle with lights on at seven a.m. The rats weighed
approximately 200 g on arrival and between 230 and 280 g on the day of testing.


### 2.3. Groups

After lab adaptation and handling,
the 12 subjects were randomly assigned to one of three groups of four. One
group served as a handled control (Handled GFP) while another was predator
stressed (Predator Stressed GFP).  Both
these groups were injected in the right lateral PAG (described in what follows) with the HSV-GFP vector before further treatment. This vector
consisted of an HSV virus carrying a green fluorescent protein gene (GFP), a
reporter used to visualize vector placement and virus induced gene expression.
This injection also served to control for any effects that GFP per se might
have. The third group was also handled (Handled CREB)
and before further treatment received an injection in the right lateral PAG  with an HSV-CREB  vector.  This vector included genes for
both CREB  and GFP.  The GFP served as a reporter of gene
expression, and the CREB  gene
elevated CREB  levels in the target
area.It is recognized that a group size
of four is small for behavioral studies of this nature. The small numbers were
necessitated by the availability of the virus. The implications of the small
group size are addressed further at the end of Section.


### 2.4. Surgical Microinfusion of Viral Vectors

Virus injections were done in the
lateral column of the right PAG,
where pCREB  increases in predator stressed rats have been observed [23, 32, 40]. The injections involved lowering a sterile
25 gauge needle attached to a microliter syringe into the brain using a
sterotaxically mounted microliter syringe holder. The coordinates for the
microinfusion according to the atlas of Paxinos and Watson [44] were, 6.3 mm posterior to bregma, 0.5 mm lateral
from the midline, and 5.5 mm below the skull. The injection of 0.5 μL
(in a concentration of 4.0 × 107 infectious units/mL supplied from
University of Texas South West Medical School) was given at a rate of 0.5 μL
per five minutes with the needle left in place for five minutes post injection.
This dose and rate were derived from the experience with the vector of one of
us (Berton). Moreover, in pilot studies with HSV-GFP, a 0.5 μl
injection at this rate produced GFP expression localized to the right lateral
column of the PAG  over an AP plane
range of.7 mm at three days post injection, the time of maximal protein
expression induced by this vector [43, 45].Injections  were performed under chloral hydrate
anesthesia (400 mg/kg, IP)  using
aseptic technique. Preanaesthetic doses of atropine were given (1.2 mg/kg).  Local anesthesia of wound edges was achieved
with marcaine and epinephrine (2%) infusion and supplemented as needed.  Holes in the skull were closed with sterile
gel foam and sealed with sterile bone wax and scalp wounds sutured. Rats were
kept warm under a lamp post surgery until they began to walk and groom, at
which time they were returned to their home cage. Surgery took approximately
one hour for each subject.


### 2.5. Cat Exposure and Handling Procedures

Three days after
virus (HSV) injection, when viral expression is peaking [43, 45],
rats were either handled or predator stressed. On the day of testing, predator
stressed rats were exposed to the same adult cat as described elsewhere [5].  The cat
exposure lasted 10  minutes  and was videotaped to capture the activities of both the
cat and the rat.  The cat generally
observed the rat at a distance with the intermittent approach and sniffing. On
occasion, the cat would mildly attack the rat but no injuries were ever
observed. At the end of the test, the rat was placed back into its home cage
and left undisturbed.  Rats in the other two groups whose treatment
included only handling did not come into contact with the cat, cat odors or
rats that had previously been exposed to cats. On the day of testing, rats in
these groups were weighed and handled as previously mentioned for 1 min. After
this handling period the rats were returned to their home cage and left
undisturbed. Handled and predator stressed rats home cages were kept in
separate rooms.


### 2.6. Behavioral Testing and Behavioral Measures

Four
days after HSV injection and one day after treatment, ALB was measured in the hole board, EPM and startle tests. The hole board test took
place just before the EPM as an independent test of activity and exploratory
tendency [46].


#### 2.6.1. Hole Board and Elevated Plus Maze Testing and Measures

The hole board and EPM were constructed and
used as described elsewhere [5]. The behavior of
the rats in the hole board and EPM was videotaped remotely for later analysis.
Rats were first placed in the hold board for 5 minutes. At the end of this time
period they were transferred by gloved hand to the EPM for a further 5  minutes of
testing. At the end of this testing period the rats were returned to their home
cages.Several measures of activity and exploration
were taken while the rat was in the hole board. They included frequency of
rearing (activity), and head dips, a measure of exploratory tendency scored
when the rats placed its snout or head into a hole in the floor. Fecal boli
deposited were also counted.  A measure
of thigmotaxis was time spent near the wall of the hole board. This measure was
quantified as the rat having all four feet in the space between the holes for
head dipping and the wall. Time spent in the center of the hold board was also
recorded. A rat was considered to be in the middle when all four feet were in
the center space defined by a square drawn through the four holes in the floor
of the box.In the EPM, exploration and activity
were scored as the number of entries into the closed arms of the maze (closed
arm entries). An entry was only recorded when the rat had all four feet inside
one arm of the maze. Other measures of exploration included head dips, scored
when a rat  placed its snout or head over
the side of an open arm, and rearing as a measure of activity. These behaviors
were divided into three types: protected (rat had all four feet in closed arm
for rearing or hindquarters in the closed arm for head dips), center (rat has
all four feet in center of maze), and unprotected (rat has all four feet in an
open arm). Time spent grooming was also recorded using the same three
subdivisions.Measures of anxiety-like behavior
were also taken. Two measures assessed open arm exploration: ratio time and
ratio entry. Ratio time was the time spent in the open arms of the maze divided
by the total time spent in any arm of the maze. The smaller the ratio the less
open arm exploration indicating a more “anxious” rat. Ratio entry was the
number of entries into the open arms of the maze divided by the total entries
into any arm of the maze. Again, the smaller the ratio, the less the open arm
exploration experienced, the more “anxious” the rat.Adamec and Shallow [5] were the first to adapt the concept of risk
assessment to the EPM. This measure was scored when the rat poked its head and
forepaws into an open arm of the maze while keeping its hindquarters in a
closed arm. The frequency of risk assessment was measured and converted to
relative risk assessment by dividing these frequencies by the time spent in the
closed arms. Fecal boli deposited in the EPM were also counted.


#### 2.6.2. Startle Testing and Measures

Startle testing was conducted on the
same day as the hole board and EPM. The startle response was determined using a
standard startle chamber (San Diego Instruments). The apparatus was fitted with
a 20.32 cm Plexiglass cylinder used to hold the animal during the test, as well
as a speaker for producing the sound bursts. A piezoelectric transducer
positioned below the cylinder detected motion of the animal in the cylinder.
The output from this transducer was fed to a computer for sampling.Prior to startle testing, animals
were adapted to the apparatus for 10  minutes with a background white noise level of
60 dB. Then rats were subject to 40 trials (1/30 seconds) of 50 milliseconds bursts of 120 dB of white noise rising out of a background of 60 dB. Half the trials were
delivered while the chamber was dark while the other half were delivered with
an accompanying light (light intensity of 28 foot candles or 300 lux). The
light trials were randomly interspersed among the dark trials. During the light
trials, the lights would come on 2.95 seconds prior to the sound burst and remain
on for the duration of the sound burst, terminating at sound offset (lights on
for a total of 3 seconds). The chamber was in darkness between trials. A computer
attached to the transducer recorded 40 samples of output. Samples included a 20 milliseconds baseline and 250 milliseconds sample after onset of the noise burst. Average
transducer output just prior to noise burst was saved as a baseline (Vstart).
The computer then found the maximal startle amplitude within each of the
samples (Vmax). Both these measures were saved for later analysis. Peak startle
amplitude was expressed as
Vmax-Vstart
for analysis. At the end of the startle
session the rats were returned to their home cages. The apparatus was washed
between rats.


### 2.7. Electrophysiological Recording Procedure

Five days after HSV injection and
two days after treatment, all rats were anaesthetized with urethane (1.5 g/kg)
given in three divided doses separated by 10 minutes. Then the rats were placed in
a sterotaxic instrument and injected under the scalp with marcaine (2%
epinephrine) to locally anesthetize and reduce bleeding. The skull was exposed
and holes drilled to permit stereotaxically guided insertion of stimulating
electrodes into the central amygdala (CeA). Recording microelectrodes were
placed into the PAG. Stimulating
and recording electrode pairs were placed in both hemispheres. In addition,
skull screws were placed over the olfactory bulb to serve as a ground and
references. Stimulation electrodes were twisted bipolar stainless steel (0.125 mm in diameter, Plastics One) aimed at the CeA. Recording electrodes were
stainless steel microelectrodes (1μm tip diameter, 0.6–1 MΩ, Frederick Haer)
aimed at the PAG  (verified
coordinates appear in Table 1). Rats were placed in a shielded box for
stimulating and recording experiments. Temperature was maintained between 36-37°C by a rectal
thermistor connected to a digital thermometer and feedback control to a DC
heating pad (Frederick Haer) under the rat.
CeA was stimulated using a single biphasic constant current pulse (width
.2 milliseconds) at 1/5 seconds over a range of intensities (.025–2.5 mA), 10 stimulations per intensity. Evoked
potentials were sampled by computer and later analyzed from data stored on
computer using DataWave software (see  Adamec et al.  [27]
for further method details).Table 1
Mean
(and SEM) of electrode coordinates
averaged over group and hemisphere.
Brain areaMeanSEMCeA AP2.370.036CeA Lateral3.980.037CeA Vertical7.920.027PAG  AP6.10.061PAG  Lateral0.330.02PAG  Vertical5.620.034AP: Anterior-posterior plane (mm posterior to
Bregma);  Lateral: lateral plane (mm lateral to mid line);  Vertical: vertical plane (mm below Bregma) ; CeA: central amygdala;  PAG: periaqueductal grayAt the end of recording,  rats were overdosed with Chloral Hydrate
(1000 mg/mL, 1 mL,  IP) and perfused with cold phosphate buffered saline and 4%
Para-formaldehyde. Brains were extracted, sunk in 20% sucrose overnight at −4°C
and then stored at −70°C. Subsequently brains were examined  histologically for electrode locations, under
green fluorescence microscopy to visualize GFP production and
immunohistochemically to study pCREB  expression.


#### 2.7.1. Electrophysiology Analysis Methods

The
main measure of the size of the evoked potential was peak height (PH). The peak
height at each intensity was taken by computer from field potential averages as
illustrated in Figure4. The raw PH at each intensity was expressed as a ratio
of PH observed at threshold (see [23, 27]).


### 2.8. Immunocytochemistry

Thick
frozen coronal sections (40μm) were cut from 5.8 to 6.8 mm posterior to bregma
[44] to capture the same areas of the PAG studied in past predator stress experiments,
and to capture the targets of virus injection and electrophysiological
recording
. Anterior-posterior (AP) plane location was determined
by counting sections from the decussation of the anterior commissure (AP −0.26
from bregma, [44])  to the desired AP plane. This counting of
sections allowed for an estimation of the AP plane position to the nearest 40 μm during cutting. Every second section was saved, which provided 12 sections
from each brain for processing. To ensure even distribution a multiple of three
brains (one brain from each group) was cut and processed at the same time.After
sectioning, one section from each group was placed in a plastic tube with nylon
covering at one end and then immersed in a plastic well containing
phosphate-buffered saline (PBS). Each tube contained three sections, which were
processed at the same time. The tubes were removed, blotted, submerged in a
solution of normal goat serum and Triton X-100 and placed on a rocker for 1 hour.
The sections were washed with PBS, blotted and incubated at −4°C for either 24
or 48 hours (reused antibody) in the primary phospho CREB antibody (Upstate/Chemicon).  Consistent
with past work  [23, 32],
a dilution of 1/500 for the primary antibody was used. After incubation,
sections were washed again with PBS, blotted, and then immersed in the secondary
biotinylated antibody (goat antirabbit) for 1 h. Sections were washed, blotted,
and placed in the ABC (Vector Stain kit) solution for 1 h on a rocker. Finally,
sections were washed with PBS for a third time, blotted and submerged in
diaminobenzadine (DAB) solution
for 5–25 min,
monitoring for staining. Sections were then washed with PBS again, before
mounting onto slides, dehydrated and cover slipped.


#### 2.8.1. Image Analysis (Densitometry)

Stained sections were analyzed blind
to group assignment using image analysis software (MOKA software, Jandel).
Hemispheres were measured separately. The PAG  was divided into ventral, dorsal, and lateral areas to reflect the functional
columnar organization described by
Bandler and Depaulis [47].  This was done using the aqueduct of Sylvius
as a guide. Horizontal lines were drawn from the top of the aqueduct to the
outside edge of the PAG and from
the bottom of the aqueduct to the outside edge of the PAG for both left and right hemispheres (see also [23]).
The top sections were considered dorsal PAG,
the middle sections were lateral PAG and the bottom sections were ventral PAG.Raw pCREB  lir densitometry data of
each column in each hemisphere were converted to optical density (OD) units
relative to the whole section. This was done by converting the raw PAG and raw whole section densitometry data to OD
units via a calibrated step wedge. An image of the calibrated step wedge was
taken at the same time as section images for each rat. Exponential fits of raw
transmission values (x) to calibrated OD values were done by computer (Table
Curve program, Jandel). All fits were good (all df adjusted r2>.9, P < .01). The exponential was then used to interpolate and convert raw
transmission values to OD units. Analysis was performed on the ratio of average
OD values in particular PAG areas
to average OD values for the entire section.


## 2.1. Ethical Approval

The procedures involving animals
reported in this paper were reviewed  by
the Institutional Animal Care Committee of Memorial University and found to be
in compliance with the guidelines of the Canadian Council on Animal Care. Every
effort was made to minimize pain and stress to the test subjects while using as
few animals as possible.


## 2.2. Animals

Subjects were male hooded Long Evans
rats (Charles River Canada). Rats were housed singly in
clear polycarbonate cages measuring 46 cm× 24 cm × 20 cm for one week prior to
any testing. During this week, rats were acclimatized to their cage, and
handled. Handling involved picking up the rat and gently holding it on the
forearm. Minimal pressure was used if the rat attempted to escape, and grip was
released as soon as the rat became still. Rats were handled in the same room as
their home cage for one minute each day during the week long adaptation period.
Rats were given food and water ad lib and were exposed to a 12-hour light/dark cycle with lights on at seven a.m. The rats weighed
approximately 200 g on arrival and between 230 and 280 g on the day of testing.


## 2.3. Groups

After lab adaptation and handling,
the 12 subjects were randomly assigned to one of three groups of four. One
group served as a handled control (Handled GFP) while another was predator
stressed (Predator Stressed GFP).  Both
these groups were injected in the right lateral PAG (described in what follows) with the HSV-GFP vector before further treatment. This vector
consisted of an HSV virus carrying a green fluorescent protein gene (GFP), a
reporter used to visualize vector placement and virus induced gene expression.
This injection also served to control for any effects that GFP per se might
have. The third group was also handled (Handled CREB)
and before further treatment received an injection in the right lateral PAG  with an HSV-CREB  vector.  This vector included genes for
both CREB  and GFP.  The GFP served as a reporter of gene
expression, and the CREB  gene
elevated CREB  levels in the target
area.It is recognized that a group size
of four is small for behavioral studies of this nature. The small numbers were
necessitated by the availability of the virus. The implications of the small
group size are addressed further at the end of Section.


## 2.4. Surgical Microinfusion of Viral Vectors

Virus injections were done in the
lateral column of the right PAG,
where pCREB  increases in predator stressed rats have been observed [23, 32, 40]. The injections involved lowering a sterile
25 gauge needle attached to a microliter syringe into the brain using a
sterotaxically mounted microliter syringe holder. The coordinates for the
microinfusion according to the atlas of Paxinos and Watson [44] were, 6.3 mm posterior to bregma, 0.5 mm lateral
from the midline, and 5.5 mm below the skull. The injection of 0.5 μL
(in a concentration of 4.0 × 107 infectious units/mL supplied from
University of Texas South West Medical School) was given at a rate of 0.5 μL
per five minutes with the needle left in place for five minutes post injection.
This dose and rate were derived from the experience with the vector of one of
us (Berton). Moreover, in pilot studies with HSV-GFP, a 0.5 μl
injection at this rate produced GFP expression localized to the right lateral
column of the PAG  over an AP plane
range of.7 mm at three days post injection, the time of maximal protein
expression induced by this vector [43, 45].Injections  were performed under chloral hydrate
anesthesia (400 mg/kg, IP)  using
aseptic technique. Preanaesthetic doses of atropine were given (1.2 mg/kg).  Local anesthesia of wound edges was achieved
with marcaine and epinephrine (2%) infusion and supplemented as needed.  Holes in the skull were closed with sterile
gel foam and sealed with sterile bone wax and scalp wounds sutured. Rats were
kept warm under a lamp post surgery until they began to walk and groom, at
which time they were returned to their home cage. Surgery took approximately
one hour for each subject.


## 2.5. Cat Exposure and Handling Procedures

Three days after
virus (HSV) injection, when viral expression is peaking [43, 45],
rats were either handled or predator stressed. On the day of testing, predator
stressed rats were exposed to the same adult cat as described elsewhere [5].  The cat
exposure lasted 10  minutes  and was videotaped to capture the activities of both the
cat and the rat.  The cat generally
observed the rat at a distance with the intermittent approach and sniffing. On
occasion, the cat would mildly attack the rat but no injuries were ever
observed. At the end of the test, the rat was placed back into its home cage
and left undisturbed.  Rats in the other two groups whose treatment
included only handling did not come into contact with the cat, cat odors or
rats that had previously been exposed to cats. On the day of testing, rats in
these groups were weighed and handled as previously mentioned for 1 min. After
this handling period the rats were returned to their home cage and left
undisturbed. Handled and predator stressed rats home cages were kept in
separate rooms.


## 2.6. Behavioral Testing and Behavioral Measures

Four
days after HSV injection and one day after treatment, ALB was measured in the hole board, EPM and startle tests. The hole board test took
place just before the EPM as an independent test of activity and exploratory
tendency [46].


### 2.6.1. Hole Board and Elevated Plus Maze Testing and Measures

The hole board and EPM were constructed and
used as described elsewhere [5]. The behavior of
the rats in the hole board and EPM was videotaped remotely for later analysis.
Rats were first placed in the hold board for 5 minutes. At the end of this time
period they were transferred by gloved hand to the EPM for a further 5  minutes of
testing. At the end of this testing period the rats were returned to their home
cages.Several measures of activity and exploration
were taken while the rat was in the hole board. They included frequency of
rearing (activity), and head dips, a measure of exploratory tendency scored
when the rats placed its snout or head into a hole in the floor. Fecal boli
deposited were also counted.  A measure
of thigmotaxis was time spent near the wall of the hole board. This measure was
quantified as the rat having all four feet in the space between the holes for
head dipping and the wall. Time spent in the center of the hold board was also
recorded. A rat was considered to be in the middle when all four feet were in
the center space defined by a square drawn through the four holes in the floor
of the box.In the EPM, exploration and activity
were scored as the number of entries into the closed arms of the maze (closed
arm entries). An entry was only recorded when the rat had all four feet inside
one arm of the maze. Other measures of exploration included head dips, scored
when a rat  placed its snout or head over
the side of an open arm, and rearing as a measure of activity. These behaviors
were divided into three types: protected (rat had all four feet in closed arm
for rearing or hindquarters in the closed arm for head dips), center (rat has
all four feet in center of maze), and unprotected (rat has all four feet in an
open arm). Time spent grooming was also recorded using the same three
subdivisions.Measures of anxiety-like behavior
were also taken. Two measures assessed open arm exploration: ratio time and
ratio entry. Ratio time was the time spent in the open arms of the maze divided
by the total time spent in any arm of the maze. The smaller the ratio the less
open arm exploration indicating a more “anxious” rat. Ratio entry was the
number of entries into the open arms of the maze divided by the total entries
into any arm of the maze. Again, the smaller the ratio, the less the open arm
exploration experienced, the more “anxious” the rat.Adamec and Shallow [5] were the first to adapt the concept of risk
assessment to the EPM. This measure was scored when the rat poked its head and
forepaws into an open arm of the maze while keeping its hindquarters in a
closed arm. The frequency of risk assessment was measured and converted to
relative risk assessment by dividing these frequencies by the time spent in the
closed arms. Fecal boli deposited in the EPM were also counted.


### 2.6.2. Startle Testing and Measures

Startle testing was conducted on the
same day as the hole board and EPM. The startle response was determined using a
standard startle chamber (San Diego Instruments). The apparatus was fitted with
a 20.32 cm Plexiglass cylinder used to hold the animal during the test, as well
as a speaker for producing the sound bursts. A piezoelectric transducer
positioned below the cylinder detected motion of the animal in the cylinder.
The output from this transducer was fed to a computer for sampling.Prior to startle testing, animals
were adapted to the apparatus for 10  minutes with a background white noise level of
60 dB. Then rats were subject to 40 trials (1/30 seconds) of 50 milliseconds bursts of 120 dB of white noise rising out of a background of 60 dB. Half the trials were
delivered while the chamber was dark while the other half were delivered with
an accompanying light (light intensity of 28 foot candles or 300 lux). The
light trials were randomly interspersed among the dark trials. During the light
trials, the lights would come on 2.95 seconds prior to the sound burst and remain
on for the duration of the sound burst, terminating at sound offset (lights on
for a total of 3 seconds). The chamber was in darkness between trials. A computer
attached to the transducer recorded 40 samples of output. Samples included a 20 milliseconds baseline and 250 milliseconds sample after onset of the noise burst. Average
transducer output just prior to noise burst was saved as a baseline (Vstart).
The computer then found the maximal startle amplitude within each of the
samples (Vmax). Both these measures were saved for later analysis. Peak startle
amplitude was expressed as
Vmax-Vstart
for analysis. At the end of the startle
session the rats were returned to their home cages. The apparatus was washed
between rats.


## 2.6.1. Hole Board and Elevated Plus Maze Testing and Measures

The hole board and EPM were constructed and
used as described elsewhere [5]. The behavior of
the rats in the hole board and EPM was videotaped remotely for later analysis.
Rats were first placed in the hold board for 5 minutes. At the end of this time
period they were transferred by gloved hand to the EPM for a further 5  minutes of
testing. At the end of this testing period the rats were returned to their home
cages.Several measures of activity and exploration
were taken while the rat was in the hole board. They included frequency of
rearing (activity), and head dips, a measure of exploratory tendency scored
when the rats placed its snout or head into a hole in the floor. Fecal boli
deposited were also counted.  A measure
of thigmotaxis was time spent near the wall of the hole board. This measure was
quantified as the rat having all four feet in the space between the holes for
head dipping and the wall. Time spent in the center of the hold board was also
recorded. A rat was considered to be in the middle when all four feet were in
the center space defined by a square drawn through the four holes in the floor
of the box.In the EPM, exploration and activity
were scored as the number of entries into the closed arms of the maze (closed
arm entries). An entry was only recorded when the rat had all four feet inside
one arm of the maze. Other measures of exploration included head dips, scored
when a rat  placed its snout or head over
the side of an open arm, and rearing as a measure of activity. These behaviors
were divided into three types: protected (rat had all four feet in closed arm
for rearing or hindquarters in the closed arm for head dips), center (rat has
all four feet in center of maze), and unprotected (rat has all four feet in an
open arm). Time spent grooming was also recorded using the same three
subdivisions.Measures of anxiety-like behavior
were also taken. Two measures assessed open arm exploration: ratio time and
ratio entry. Ratio time was the time spent in the open arms of the maze divided
by the total time spent in any arm of the maze. The smaller the ratio the less
open arm exploration indicating a more “anxious” rat. Ratio entry was the
number of entries into the open arms of the maze divided by the total entries
into any arm of the maze. Again, the smaller the ratio, the less the open arm
exploration experienced, the more “anxious” the rat.Adamec and Shallow [5] were the first to adapt the concept of risk
assessment to the EPM. This measure was scored when the rat poked its head and
forepaws into an open arm of the maze while keeping its hindquarters in a
closed arm. The frequency of risk assessment was measured and converted to
relative risk assessment by dividing these frequencies by the time spent in the
closed arms. Fecal boli deposited in the EPM were also counted.


## 2.6.2. Startle Testing and Measures

Startle testing was conducted on the
same day as the hole board and EPM. The startle response was determined using a
standard startle chamber (San Diego Instruments). The apparatus was fitted with
a 20.32 cm Plexiglass cylinder used to hold the animal during the test, as well
as a speaker for producing the sound bursts. A piezoelectric transducer
positioned below the cylinder detected motion of the animal in the cylinder.
The output from this transducer was fed to a computer for sampling.Prior to startle testing, animals
were adapted to the apparatus for 10  minutes with a background white noise level of
60 dB. Then rats were subject to 40 trials (1/30 seconds) of 50 milliseconds bursts of 120 dB of white noise rising out of a background of 60 dB. Half the trials were
delivered while the chamber was dark while the other half were delivered with
an accompanying light (light intensity of 28 foot candles or 300 lux). The
light trials were randomly interspersed among the dark trials. During the light
trials, the lights would come on 2.95 seconds prior to the sound burst and remain
on for the duration of the sound burst, terminating at sound offset (lights on
for a total of 3 seconds). The chamber was in darkness between trials. A computer
attached to the transducer recorded 40 samples of output. Samples included a 20 milliseconds baseline and 250 milliseconds sample after onset of the noise burst. Average
transducer output just prior to noise burst was saved as a baseline (Vstart).
The computer then found the maximal startle amplitude within each of the
samples (Vmax). Both these measures were saved for later analysis. Peak startle
amplitude was expressed as
Vmax-Vstart
for analysis. At the end of the startle
session the rats were returned to their home cages. The apparatus was washed
between rats.


## 2.7. Electrophysiological Recording Procedure

Five days after HSV injection and
two days after treatment, all rats were anaesthetized with urethane (1.5 g/kg)
given in three divided doses separated by 10 minutes. Then the rats were placed in
a sterotaxic instrument and injected under the scalp with marcaine (2%
epinephrine) to locally anesthetize and reduce bleeding. The skull was exposed
and holes drilled to permit stereotaxically guided insertion of stimulating
electrodes into the central amygdala (CeA). Recording microelectrodes were
placed into the PAG. Stimulating
and recording electrode pairs were placed in both hemispheres. In addition,
skull screws were placed over the olfactory bulb to serve as a ground and
references. Stimulation electrodes were twisted bipolar stainless steel (0.125 mm in diameter, Plastics One) aimed at the CeA. Recording electrodes were
stainless steel microelectrodes (1μm tip diameter, 0.6–1 MΩ, Frederick Haer)
aimed at the PAG  (verified
coordinates appear in Table 1). Rats were placed in a shielded box for
stimulating and recording experiments. Temperature was maintained between 36-37°C by a rectal
thermistor connected to a digital thermometer and feedback control to a DC
heating pad (Frederick Haer) under the rat.
CeA was stimulated using a single biphasic constant current pulse (width
.2 milliseconds) at 1/5 seconds over a range of intensities (.025–2.5 mA), 10 stimulations per intensity. Evoked
potentials were sampled by computer and later analyzed from data stored on
computer using DataWave software (see  Adamec et al.  [27]
for further method details).Table 1
Mean
(and SEM) of electrode coordinates
averaged over group and hemisphere.
Brain areaMeanSEMCeA AP2.370.036CeA Lateral3.980.037CeA Vertical7.920.027PAG  AP6.10.061PAG  Lateral0.330.02PAG  Vertical5.620.034AP: Anterior-posterior plane (mm posterior to
Bregma);  Lateral: lateral plane (mm lateral to mid line);  Vertical: vertical plane (mm below Bregma) ; CeA: central amygdala;  PAG: periaqueductal grayAt the end of recording,  rats were overdosed with Chloral Hydrate
(1000 mg/mL, 1 mL,  IP) and perfused with cold phosphate buffered saline and 4%
Para-formaldehyde. Brains were extracted, sunk in 20% sucrose overnight at −4°C
and then stored at −70°C. Subsequently brains were examined  histologically for electrode locations, under
green fluorescence microscopy to visualize GFP production and
immunohistochemically to study pCREB  expression.


### 2.7.1. Electrophysiology Analysis Methods

The
main measure of the size of the evoked potential was peak height (PH). The peak
height at each intensity was taken by computer from field potential averages as
illustrated in Figure4. The raw PH at each intensity was expressed as a ratio
of PH observed at threshold (see [23, 27]).


## 2.7.1. Electrophysiology Analysis Methods

The
main measure of the size of the evoked potential was peak height (PH). The peak
height at each intensity was taken by computer from field potential averages as
illustrated in Figure4. The raw PH at each intensity was expressed as a ratio
of PH observed at threshold (see [23, 27]).


## 2.8. Immunocytochemistry

Thick
frozen coronal sections (40μm) were cut from 5.8 to 6.8 mm posterior to bregma
[44] to capture the same areas of the PAG studied in past predator stress experiments,
and to capture the targets of virus injection and electrophysiological
recording
. Anterior-posterior (AP) plane location was determined
by counting sections from the decussation of the anterior commissure (AP −0.26
from bregma, [44])  to the desired AP plane. This counting of
sections allowed for an estimation of the AP plane position to the nearest 40 μm during cutting. Every second section was saved, which provided 12 sections
from each brain for processing. To ensure even distribution a multiple of three
brains (one brain from each group) was cut and processed at the same time.After
sectioning, one section from each group was placed in a plastic tube with nylon
covering at one end and then immersed in a plastic well containing
phosphate-buffered saline (PBS). Each tube contained three sections, which were
processed at the same time. The tubes were removed, blotted, submerged in a
solution of normal goat serum and Triton X-100 and placed on a rocker for 1 hour.
The sections were washed with PBS, blotted and incubated at −4°C for either 24
or 48 hours (reused antibody) in the primary phospho CREB antibody (Upstate/Chemicon).  Consistent
with past work  [23, 32],
a dilution of 1/500 for the primary antibody was used. After incubation,
sections were washed again with PBS, blotted, and then immersed in the secondary
biotinylated antibody (goat antirabbit) for 1 h. Sections were washed, blotted,
and placed in the ABC (Vector Stain kit) solution for 1 h on a rocker. Finally,
sections were washed with PBS for a third time, blotted and submerged in
diaminobenzadine (DAB) solution
for 5–25 min,
monitoring for staining. Sections were then washed with PBS again, before
mounting onto slides, dehydrated and cover slipped.


### 2.8.1. Image Analysis (Densitometry)

Stained sections were analyzed blind
to group assignment using image analysis software (MOKA software, Jandel).
Hemispheres were measured separately. The PAG  was divided into ventral, dorsal, and lateral areas to reflect the functional
columnar organization described by
Bandler and Depaulis [47].  This was done using the aqueduct of Sylvius
as a guide. Horizontal lines were drawn from the top of the aqueduct to the
outside edge of the PAG and from
the bottom of the aqueduct to the outside edge of the PAG for both left and right hemispheres (see also [23]).
The top sections were considered dorsal PAG,
the middle sections were lateral PAG and the bottom sections were ventral PAG.Raw pCREB  lir densitometry data of
each column in each hemisphere were converted to optical density (OD) units
relative to the whole section. This was done by converting the raw PAG and raw whole section densitometry data to OD
units via a calibrated step wedge. An image of the calibrated step wedge was
taken at the same time as section images for each rat. Exponential fits of raw
transmission values (x) to calibrated OD values were done by computer (Table
Curve program, Jandel). All fits were good (all df adjusted r2>.9, P < .01). The exponential was then used to interpolate and convert raw
transmission values to OD units. Analysis was performed on the ratio of average
OD values in particular PAG areas
to average OD values for the entire section.


## 2.8.1. Image Analysis (Densitometry)

Stained sections were analyzed blind
to group assignment using image analysis software (MOKA software, Jandel).
Hemispheres were measured separately. The PAG  was divided into ventral, dorsal, and lateral areas to reflect the functional
columnar organization described by
Bandler and Depaulis [47].  This was done using the aqueduct of Sylvius
as a guide. Horizontal lines were drawn from the top of the aqueduct to the
outside edge of the PAG and from
the bottom of the aqueduct to the outside edge of the PAG for both left and right hemispheres (see also [23]).
The top sections were considered dorsal PAG,
the middle sections were lateral PAG and the bottom sections were ventral PAG.Raw pCREB  lir densitometry data of
each column in each hemisphere were converted to optical density (OD) units
relative to the whole section. This was done by converting the raw PAG and raw whole section densitometry data to OD
units via a calibrated step wedge. An image of the calibrated step wedge was
taken at the same time as section images for each rat. Exponential fits of raw
transmission values (x) to calibrated OD values were done by computer (Table
Curve program, Jandel). All fits were good (all df adjusted r2>.9, P < .01). The exponential was then used to interpolate and convert raw
transmission values to OD units. Analysis was performed on the ratio of average
OD values in particular PAG areas
to average OD values for the entire section.


## 3. Results

### 3.1. Anxiety-Like Behavior in the EPM

Groups
differed in the measures of open arm exploration-ratio time and ratio entry
(allP (2,9)  ≥  14.78, P <.002). Predator stress reduced
ratio time and ratio entry in the EPM, consistent with many past studies
(Figure 1, upper right panel, ratio time only is shown, ratio entry findings
were very similar). Predator stress reduced open arm exploration (increased
anxiety) the most relative to controls (Handled GFP).  Injection of HSV-CREB  in the right PAG alone was also
anxiogenic in the EPM, reducing ratio time and entries in Handled-CREB rat to a level between Handled GFP and
Predator Stressed GFP rats (Figure 1, Tukey Kramer test, P < .05).Plotted over groups are mean±  SEM of  EPM and hole board behaviors. Means marked
with a different letter differ from each other (P < .05). (a), shows frequency of closed arm entries (left)
and ratio time (right) in the EPM. (b),  shows the frequency of rears
(left) and head dips (right) in the hole board test.
(a)(b)With
regard to ratio frequency of risk assessment, though there was no group effect
(F (2,9)  =  2.58, P <.13), a planned t-test
contrasting the predator stressed group with the two handled groups combined
(which did not differ) revealed that predator stress reduced risk assessment relative
to both Handled groups (Figure 2; t (9) = 2.19, P < .029, 1 tailed).
This finding of reduced risk assessment following predator stress is consistent
with many previous studies.Figure 2
Plotted over groups are mean±  SEM of risk assessment behavior in the EPM. Means marked with a different letter
differ from each other (P < .05).


### 3.2. Exploration and Activity in EPM and Hole Board

There
were no differences between groups in closed arm entries (activity) in the EPM
(Figure1, (a) left panel).  Similarly,
groups did not differ in rears (activity) and head dips (exploration) in the
hole board (Figure 1, (b) two panels). These data indicate that group
differences in open arm exploration seen in the EPM are not the result of
changes in activity or exploration.


### 3.3. Acoustic Startle Response

Startle in the light and dark trials
did not differ so analyses across light and dark trials were combined.


#### 3.3.1. Startle Amplitude

Between groups startle data were not
normally distributed (Omnibus Normality Test = 148.07,P < .0001). Therefore, Kruskal-Wallis one way non-parametric
ANOVA on medians of peak startle amplitude over trials was used. Groups
differed (χ2 (2) = 119.90, P  < .001). Planned comparisons
(Kruskal-Wallis multiple comparison
z-test z > 3.98,
P < .01)
revealed that predator stress increased startle over both handled groups
(Figure 3, bottom left panel).
Nevertheless startle amplitude of
Handled CREB  rats was also
higher than Handled GFP, but lower than predator stressed animals (Figure 3,
(b) left panel).(a) shows example fits (solid line) to an FFT smoothed (20%) function (dashed
line) of the means of peak startle amplitude (dotted line) over 20 trials for
Handled GFP control (left) and predator stressed (right) rats. Plotted in (b) are median peak startle amplitudes (left) andτ±SE
(right),
estimated from declining exponential functions, for rats in each experimental
group. Medians and Tau values marked with a different letter differ from each
other (P < .05).
(a)(b)In the lower right is a computer average of a CeA-PAG  evoked potential illustrating how peak height (PH) was measured by computer.
Plotted in the graphs are means± SEM of PH of CeA-PAG  evoked potentials
expressed as a ratio of threshold PH versus intensity of stimulation in
μC/pulse (calculated as intensity in μA times pulse width in microsecond to take pulse
width into the intensity measure). Means are plotted separately by group and
within a group separately by hemisphere.
(a)(b)(c)


#### 3.3.2. Habituation of Acoustic Startle Response

Predator stress prolongs habituation
to startle [6, 15, 16, 48].  Therefore,
habituation to startle in the three groups was determined and
compared.  Exponential decline functions
of the form(1)y=y0+ae−t/τ were fit to the peak startle amplitude mean
data from each group across 20 trials (combined light and dark startle trials)
using Jandel table curve V 4.0. In (1),
y and y0 are peak
startle amplitude, a is a constant, e is the base of the natural logarithm, t is the trial number and τ is the trial constant, or the number
of trials to decline to 37% of the maximal peak startle amplitude. To improve
the fit, an FFT smoothing function provided by the program (20% FFT smooth) was
applied. Care was taken to ensure the smoothing did not distort the data
(Figure 3(a)). All fits were good (degrees of freedom adjusted   r2>.84; all fits F (2,17) >  58.3, P < .001; t (38)  ≥  6.18, P < .01 for all t-tests
of differences from zero of τ). The
estimate of τ included a standard
error of estimate. These standard errors were used to perform planned two
tailed t-tests between groups using
the different τ  values (Figure 3(b),  right panel).  The pattern of the
findings from this analysis was surprising. Both the Handled CREB  and predator stressed group took significantly
longer to habituate than Handled GFP controls. While this result was expected
for the predator stressed group given previous work, the fact that the Handled CREB  group took longer to habituate than the
predator stressed group was uncharacteristic of the amplitude findings.


### 3.4. Electrophysiology

A
three way ANOVA was done on ratio PH of the CeA-PAG  evoked potential data. The factors examined were Group (Handled GFP control,
predator stressed, and Handled CREB),
Hemisphere (right and left) and Intensity of stimulation. There was a
significant Groupx Hemisphere x
Intensity interaction (F (12,54) = 2.24, P <.04).
The interaction is displayed in Figure 4. Intensity of stimulation was expressed
in μC (micro-coulombs) per pulse. All groups were stimulated using the same
intensity series, so group differences cannot be attributed to difference in
the intensity of stimulation.Planned
comparisonst-test mean contrasts
were used to examine the interaction by comparing the three groups at each
intensity in each hemisphere. All groups showed the same ratio PH values at
intensity 1 in both hemispheres. Moreover, ratio PH in Handled GFP controls
were equal in both hemispheres and unchanged over intensity of CeA stimulation
(Figure 4(c)). Similarly, left hemisphere ratio PH of Handled CREB  rats did not change over intensity and did not
differ from ratio PH in right or left hemisphere of Handled GFP controls. In
contrast right hemisphere ratio PH of Handled CREB  rats rose over intensity (t(54) =
5.61, P < .01, comparing intensity 1
and 10) and differed from their own left hemisphere ratio PH over intensities
2–10 (all t(54) >  2.80, P < .05; Figure 4(b), top right panel).
Therefore, CREB  injection per se
selectively potentiated right hemisphere CeA-PAG  evoked potentials relative to the left hemisphere and relative to Handled GFP
controls, which did not differ from Handled CREB  rats in the left hemisphere.As
might be expected from previous work, predator stress potentiated right and
left hemisphere CeA-PAG  evoked
potentials (Figure4(a), upper left panel). Ratio PH in left and right hemispheres
rose over intensity (all t(54) >
4.68, P < .01, comparing intensity 1
and 10). However, right hemisphere response exceeded the left at intensities
4–9 (all t(54) > 2.09, P < .05). This suggests that left
hemisphere potentiation in predator stressed rats was fading relative to right
hemisphere potentiation two days after treatment.  Nevertheless, predator stress potentiated
left CeA-PAG ratio PH over that
seen in the left hemisphere of Handled CREB  rats or in the left or right hemispheres of Handled GFP control rats, in that
left ratio PH of predator stressed rats exceeded left ratio PH of Handled CREB  rats (and left and right ratio PH of Handled
GFP control) rats at intensities 3, 5,  9-10 (all t(54) > 2.04, P < .05).Comparing
right hemisphere ratio PH of Handled CREB  and predator stress rats suggests nearly equal potentiation.  Groups did not differ at intensities 1-2 and
4–8, but Handled CREB ratio PH did
exceed that of predator stressed at intensities 3, 9 and 10 (allt(54) > 2.15, P < .05).  Therefore right PAG CREB  injection per se is as effective, or even more effective, than predator stress
in potentiating right CeA-PAG  evoked potentials.


### 3.5. Histological Verification of Electrode and Cannula Placements

Tips
of stimulating and recording electrodes were visualized microscopically from
tissue sections and plotted onto rat atlas sections [44].
Rats from all three groups had correctly placed electrodes, allowing the use of
each subject for data analysis. Two way ANOVAs were done examining group and
hemisphere factors with separate analyses for the coordinates of each plane
(AP, lateral and vertical) for each electrode target. Lateral and vertical
coordinates were taken from the atlas sections while AP plane was calculated
from section number. No group, hemisphere, or groups x hemisphere interactions
were observed. The CeA stimulating electrodes were correctly placed in the
medial central nucleus while the recording electrodes were in the lateral
columns of the right and left PAG.
Average location of tips for both the stimulating and recording electrodes
appear in Table 1. Verification of cannula placement was completed in much the
same way, average coordinates appear in Table 2. Furthermore, the absolute
distance from the recording electrode was very small (Table 2) indicating that
electrophysiological recordings were taken from a position close to viral injection.Table 2
Mean
(and SEM) of PAG  cannula coordinates and in relation to PAG  electrodes.
Right cannulaMeanSEMMean absolute distance (mm)SEMfrom the right
PAG  electrodePAG  AP6.130.0680.280.034PAG  Lateral0.360.0320.090.026PAG  Vertical5.620.040.110.028AP: Anterior-Posterior Plane (mm posterior to
Bregma)Lateral: Lateral Plane (mm lateral to mid line)Vertical: Vertical Plane (mm below Bregma)PAG: Periaqueductal Gray


### 3.6. pCREB lir Immunohistochemistry Densitometry Analysis

Relative
OD data were analyzed separately for each of the three columns in the PAG. The lateral column was of primary interest
since this was the area where  CREB protein expression was enhanced (Figure5(a),  top left panel). A one way ANOVA of right hemisphere data revealed a significant
difference between the groups (F (2,41)
= 3.30, P < .05). In contrast,
groups did not differ in the left hemisphere (F (2,41) = 1.88, P <.17).
Predator stressed rats had significantly more pCREB  lir than Handled GFP
controls with the Handled CREB  rats falling in between these two groups, differing from neither (Tukey-Kramer
Test, P < .05).  The mean of pCREB  lir in Handled CREB  rats measured here at 5 days post HSV
injection is likely an underestimate of its value at peak expression of CREB, which occurs at three days after HSV
injection, when treatments occurred (stress or handling), and which fades
thereafter [43].Mean± SEM relative optical density units (PAG optical density units divided by total PAG section optical density units) in all three columns for all experimental groups
are plotted. The left side of each panel displays data from the right
hemisphere while the right side of the panel illustrates left hemisphere data.
For a given column,  means marked with
the same letter do not differ, but differ from those with different letters,
while means marked with two letters do not differ from means marked with either
of the letters (Tukey-Kramer tests, P < .05).  Means marked with “@” show a
within group difference between hemispheres (P < .05 1 tailed test).
(a)(b)(c)One tailedt-tests were used to compare within groups across hemispheres based on the
prediction that right column pCREB-lir would be increased in predator stressed
rats based on previous findings, and on the prediction that increased CREB  expression in Handled CREB  rats would increase pCREB-lir.  Both the
predator stressed and Handled CREB  rats exhibited more pCREB  lir in the right over the left (all t, P <.04,
1 tailed), whereas there were no hemisphere differences in the Handled GFP
control group.Data from the dorsal column of the PAG were analyzed in the same way with somewhat
differing results (Figure5(b), top right panel). A one way ANOVA of right hemisphere
data revealed a significant difference between groups (F (2,41) = 3.66, P <.04)
while the left hemisphere again showed no group difference (F (2,41) = 0.74, P <.49). Comparison of the groups in the right hemisphere
revealed that the predator stressed rats showed elevated pCREB  lir which was
greater than the Handled groups which did not differ (Tukey-Kramer tests, P < .05). Furthermore, comparison of
groups across the two hemispheres revealed that, like the lateral column, both
the stressed and Handled CREB  group had elevated pCREB  lir in the right hemisphere as compared to the left
(all t,
P <.04 t tailed) with the Handled GFP control group again showing
no difference between hemispheres.Expression of pCREB  in the ventral
column of the PAG presented
another pattern of results (Figure5(c), bottom panel). A one way ANOVA in the
right hemisphere revealed a significant difference between groups (F (2,41) = 6.93, P <.003). In this case however, the stressed rats had
significantly lower pCREB  expression than Handled CREB  rats with the Handled GFP control group falling in between, differing from
neither (Tukey-Kramer tests, P < .05). Much like the other two columns, no difference was seen between
groups in the left hemisphere (F (2,41)
= 1.36, P <.28). Comparisons within
groups across hemispheres showed that both the Handled GFP control and Handled CREB  rats had increased pCREB  in the right over the
left hemisphere (all t, P < .01), while there was no hemisphere
difference in predator stressed rats. These ventral column results mirror
previous findings with the exception of the hemisphere differences [40]. The
fact that the Handled CREB  group
did not differ from the Handled GFP controls indicates that CREB may not be having an effect in this column.
This also suggests that regional differences in the pathways controlling
phosphorylation of CREB may be
dependent on predator stress.


### 3.7. Visualization of GFP

Verification of gene expression was
achieved by examining all PAG sections taken for green flourescence as evidence of expression of the reporter
GFP. Green flourescence in the right PAG verified gene expression of GFP occurred after HSV injection in the vicinity of
the injection cannulas and PAG recording electrodes (Figure6 , e.g., five days after HSV injection).
Since  flourescence ranges from cannula
to PAG  electrodes, one can derive
a sense of the AP plane range of gene expression from PAG electrode position relative to cannulas. Referring to  Table 2, evidence of gene expression five
days post HSV injection appears over a range of ±.28  ±.034 mm (mean ± SEM) from the cannula in the AP plane.  This represents a range nearly as extensive
in previous pilot work which found that at
the time of peak gene expression (three days post HSV injection),  GFP expression was localized to the right lateral
column of the PAG  over an AP plane
range of  ±.35 mm from the cannula.Figure 6
Depicted in the figure are fluorescence
photomicrographs illustrating green florescent protein (GFP) localized in the
right lateral column of the periaqueductal gray (PAG)
5 days after HSV injection in the right lateral PAG.
Magnifications are 6.5 and 25 times (×).


### 3.8. Power Associated with Significant Results

Given the small n of groups, power (α =.05) of all significant findings was calculated. Significant behavioral and
electrophysiological findings all had power values in excess of.90.  Power associated with pCREB  expression
analyses varied with column of the PAG,
ranging from .82 to .91 in the dorsal and ventral columns to a reduced power for
the lateral column results of .60.The power of a
test depends on the value of the type I error (hereα=.05), the sample size, the standard
deviation, and the magnitude of the effect being tested reflected here in
magnitude of mean differences.  Most
findings appear quite robust with power values in excess of .80, suggesting
robust effects of predator stress and virally induced CREB  expression on brain and behavior.  The
reduced power for the lateral column pCREB  findings suggests a fading effect in
this column.


## 3.1. Anxiety-Like Behavior in the EPM

Groups
differed in the measures of open arm exploration-ratio time and ratio entry
(allP (2,9)  ≥  14.78, P <.002). Predator stress reduced
ratio time and ratio entry in the EPM, consistent with many past studies
(Figure 1, upper right panel, ratio time only is shown, ratio entry findings
were very similar). Predator stress reduced open arm exploration (increased
anxiety) the most relative to controls (Handled GFP).  Injection of HSV-CREB  in the right PAG alone was also
anxiogenic in the EPM, reducing ratio time and entries in Handled-CREB rat to a level between Handled GFP and
Predator Stressed GFP rats (Figure 1, Tukey Kramer test, P < .05).Plotted over groups are mean±  SEM of  EPM and hole board behaviors. Means marked
with a different letter differ from each other (P < .05). (a), shows frequency of closed arm entries (left)
and ratio time (right) in the EPM. (b),  shows the frequency of rears
(left) and head dips (right) in the hole board test.
(a)(b)With
regard to ratio frequency of risk assessment, though there was no group effect
(F (2,9)  =  2.58, P <.13), a planned t-test
contrasting the predator stressed group with the two handled groups combined
(which did not differ) revealed that predator stress reduced risk assessment relative
to both Handled groups (Figure 2; t (9) = 2.19, P < .029, 1 tailed).
This finding of reduced risk assessment following predator stress is consistent
with many previous studies.Figure 2
Plotted over groups are mean±  SEM of risk assessment behavior in the EPM. Means marked with a different letter
differ from each other (P < .05).


## 3.2. Exploration and Activity in EPM and Hole Board

There
were no differences between groups in closed arm entries (activity) in the EPM
(Figure1, (a) left panel).  Similarly,
groups did not differ in rears (activity) and head dips (exploration) in the
hole board (Figure 1, (b) two panels). These data indicate that group
differences in open arm exploration seen in the EPM are not the result of
changes in activity or exploration.


## 3.3. Acoustic Startle Response

Startle in the light and dark trials
did not differ so analyses across light and dark trials were combined.


### 3.3.1. Startle Amplitude

Between groups startle data were not
normally distributed (Omnibus Normality Test = 148.07,P < .0001). Therefore, Kruskal-Wallis one way non-parametric
ANOVA on medians of peak startle amplitude over trials was used. Groups
differed (χ2 (2) = 119.90, P  < .001). Planned comparisons
(Kruskal-Wallis multiple comparison
z-test z > 3.98,
P < .01)
revealed that predator stress increased startle over both handled groups
(Figure 3, bottom left panel).
Nevertheless startle amplitude of
Handled CREB  rats was also
higher than Handled GFP, but lower than predator stressed animals (Figure 3,
(b) left panel).(a) shows example fits (solid line) to an FFT smoothed (20%) function (dashed
line) of the means of peak startle amplitude (dotted line) over 20 trials for
Handled GFP control (left) and predator stressed (right) rats. Plotted in (b) are median peak startle amplitudes (left) andτ±SE
(right),
estimated from declining exponential functions, for rats in each experimental
group. Medians and Tau values marked with a different letter differ from each
other (P < .05).
(a)(b)In the lower right is a computer average of a CeA-PAG  evoked potential illustrating how peak height (PH) was measured by computer.
Plotted in the graphs are means± SEM of PH of CeA-PAG  evoked potentials
expressed as a ratio of threshold PH versus intensity of stimulation in
μC/pulse (calculated as intensity in μA times pulse width in microsecond to take pulse
width into the intensity measure). Means are plotted separately by group and
within a group separately by hemisphere.
(a)(b)(c)


### 3.3.2. Habituation of Acoustic Startle Response

Predator stress prolongs habituation
to startle [6, 15, 16, 48].  Therefore,
habituation to startle in the three groups was determined and
compared.  Exponential decline functions
of the form(1)y=y0+ae−t/τ were fit to the peak startle amplitude mean
data from each group across 20 trials (combined light and dark startle trials)
using Jandel table curve V 4.0. In (1),
y and y0 are peak
startle amplitude, a is a constant, e is the base of the natural logarithm, t is the trial number and τ is the trial constant, or the number
of trials to decline to 37% of the maximal peak startle amplitude. To improve
the fit, an FFT smoothing function provided by the program (20% FFT smooth) was
applied. Care was taken to ensure the smoothing did not distort the data
(Figure 3(a)). All fits were good (degrees of freedom adjusted   r2>.84; all fits F (2,17) >  58.3, P < .001; t (38)  ≥  6.18, P < .01 for all t-tests
of differences from zero of τ). The
estimate of τ included a standard
error of estimate. These standard errors were used to perform planned two
tailed t-tests between groups using
the different τ  values (Figure 3(b),  right panel).  The pattern of the
findings from this analysis was surprising. Both the Handled CREB  and predator stressed group took significantly
longer to habituate than Handled GFP controls. While this result was expected
for the predator stressed group given previous work, the fact that the Handled CREB  group took longer to habituate than the
predator stressed group was uncharacteristic of the amplitude findings.


## 3.3.1. Startle Amplitude

Between groups startle data were not
normally distributed (Omnibus Normality Test = 148.07,P < .0001). Therefore, Kruskal-Wallis one way non-parametric
ANOVA on medians of peak startle amplitude over trials was used. Groups
differed (χ2 (2) = 119.90, P  < .001). Planned comparisons
(Kruskal-Wallis multiple comparison
z-test z > 3.98,
P < .01)
revealed that predator stress increased startle over both handled groups
(Figure 3, bottom left panel).
Nevertheless startle amplitude of
Handled CREB  rats was also
higher than Handled GFP, but lower than predator stressed animals (Figure 3,
(b) left panel).(a) shows example fits (solid line) to an FFT smoothed (20%) function (dashed
line) of the means of peak startle amplitude (dotted line) over 20 trials for
Handled GFP control (left) and predator stressed (right) rats. Plotted in (b) are median peak startle amplitudes (left) andτ±SE
(right),
estimated from declining exponential functions, for rats in each experimental
group. Medians and Tau values marked with a different letter differ from each
other (P < .05).
(a)(b)In the lower right is a computer average of a CeA-PAG  evoked potential illustrating how peak height (PH) was measured by computer.
Plotted in the graphs are means± SEM of PH of CeA-PAG  evoked potentials
expressed as a ratio of threshold PH versus intensity of stimulation in
μC/pulse (calculated as intensity in μA times pulse width in microsecond to take pulse
width into the intensity measure). Means are plotted separately by group and
within a group separately by hemisphere.
(a)(b)(c)


## 3.3.2. Habituation of Acoustic Startle Response

Predator stress prolongs habituation
to startle [6, 15, 16, 48].  Therefore,
habituation to startle in the three groups was determined and
compared.  Exponential decline functions
of the form(1)y=y0+ae−t/τ were fit to the peak startle amplitude mean
data from each group across 20 trials (combined light and dark startle trials)
using Jandel table curve V 4.0. In (1),
y and y0 are peak
startle amplitude, a is a constant, e is the base of the natural logarithm, t is the trial number and τ is the trial constant, or the number
of trials to decline to 37% of the maximal peak startle amplitude. To improve
the fit, an FFT smoothing function provided by the program (20% FFT smooth) was
applied. Care was taken to ensure the smoothing did not distort the data
(Figure 3(a)). All fits were good (degrees of freedom adjusted   r2>.84; all fits F (2,17) >  58.3, P < .001; t (38)  ≥  6.18, P < .01 for all t-tests
of differences from zero of τ). The
estimate of τ included a standard
error of estimate. These standard errors were used to perform planned two
tailed t-tests between groups using
the different τ  values (Figure 3(b),  right panel).  The pattern of the
findings from this analysis was surprising. Both the Handled CREB  and predator stressed group took significantly
longer to habituate than Handled GFP controls. While this result was expected
for the predator stressed group given previous work, the fact that the Handled CREB  group took longer to habituate than the
predator stressed group was uncharacteristic of the amplitude findings.


## 3.4. Electrophysiology

A
three way ANOVA was done on ratio PH of the CeA-PAG  evoked potential data. The factors examined were Group (Handled GFP control,
predator stressed, and Handled CREB),
Hemisphere (right and left) and Intensity of stimulation. There was a
significant Groupx Hemisphere x
Intensity interaction (F (12,54) = 2.24, P <.04).
The interaction is displayed in Figure 4. Intensity of stimulation was expressed
in μC (micro-coulombs) per pulse. All groups were stimulated using the same
intensity series, so group differences cannot be attributed to difference in
the intensity of stimulation.Planned
comparisonst-test mean contrasts
were used to examine the interaction by comparing the three groups at each
intensity in each hemisphere. All groups showed the same ratio PH values at
intensity 1 in both hemispheres. Moreover, ratio PH in Handled GFP controls
were equal in both hemispheres and unchanged over intensity of CeA stimulation
(Figure 4(c)). Similarly, left hemisphere ratio PH of Handled CREB  rats did not change over intensity and did not
differ from ratio PH in right or left hemisphere of Handled GFP controls. In
contrast right hemisphere ratio PH of Handled CREB  rats rose over intensity (t(54) =
5.61, P < .01, comparing intensity 1
and 10) and differed from their own left hemisphere ratio PH over intensities
2–10 (all t(54) >  2.80, P < .05; Figure 4(b), top right panel).
Therefore, CREB  injection per se
selectively potentiated right hemisphere CeA-PAG  evoked potentials relative to the left hemisphere and relative to Handled GFP
controls, which did not differ from Handled CREB  rats in the left hemisphere.As
might be expected from previous work, predator stress potentiated right and
left hemisphere CeA-PAG  evoked
potentials (Figure4(a), upper left panel). Ratio PH in left and right hemispheres
rose over intensity (all t(54) >
4.68, P < .01, comparing intensity 1
and 10). However, right hemisphere response exceeded the left at intensities
4–9 (all t(54) > 2.09, P < .05). This suggests that left
hemisphere potentiation in predator stressed rats was fading relative to right
hemisphere potentiation two days after treatment.  Nevertheless, predator stress potentiated
left CeA-PAG ratio PH over that
seen in the left hemisphere of Handled CREB  rats or in the left or right hemispheres of Handled GFP control rats, in that
left ratio PH of predator stressed rats exceeded left ratio PH of Handled CREB  rats (and left and right ratio PH of Handled
GFP control) rats at intensities 3, 5,  9-10 (all t(54) > 2.04, P < .05).Comparing
right hemisphere ratio PH of Handled CREB  and predator stress rats suggests nearly equal potentiation.  Groups did not differ at intensities 1-2 and
4–8, but Handled CREB ratio PH did
exceed that of predator stressed at intensities 3, 9 and 10 (allt(54) > 2.15, P < .05).  Therefore right PAG CREB  injection per se is as effective, or even more effective, than predator stress
in potentiating right CeA-PAG  evoked potentials.


## 3.5. Histological Verification of Electrode and Cannula Placements

Tips
of stimulating and recording electrodes were visualized microscopically from
tissue sections and plotted onto rat atlas sections [44].
Rats from all three groups had correctly placed electrodes, allowing the use of
each subject for data analysis. Two way ANOVAs were done examining group and
hemisphere factors with separate analyses for the coordinates of each plane
(AP, lateral and vertical) for each electrode target. Lateral and vertical
coordinates were taken from the atlas sections while AP plane was calculated
from section number. No group, hemisphere, or groups x hemisphere interactions
were observed. The CeA stimulating electrodes were correctly placed in the
medial central nucleus while the recording electrodes were in the lateral
columns of the right and left PAG.
Average location of tips for both the stimulating and recording electrodes
appear in Table 1. Verification of cannula placement was completed in much the
same way, average coordinates appear in Table 2. Furthermore, the absolute
distance from the recording electrode was very small (Table 2) indicating that
electrophysiological recordings were taken from a position close to viral injection.Table 2
Mean
(and SEM) of PAG  cannula coordinates and in relation to PAG  electrodes.
Right cannulaMeanSEMMean absolute distance (mm)SEMfrom the right
PAG  electrodePAG  AP6.130.0680.280.034PAG  Lateral0.360.0320.090.026PAG  Vertical5.620.040.110.028AP: Anterior-Posterior Plane (mm posterior to
Bregma)Lateral: Lateral Plane (mm lateral to mid line)Vertical: Vertical Plane (mm below Bregma)PAG: Periaqueductal Gray


## 3.6. pCREB lir Immunohistochemistry Densitometry Analysis

Relative
OD data were analyzed separately for each of the three columns in the PAG. The lateral column was of primary interest
since this was the area where  CREB protein expression was enhanced (Figure5(a),  top left panel). A one way ANOVA of right hemisphere data revealed a significant
difference between the groups (F (2,41)
= 3.30, P < .05). In contrast,
groups did not differ in the left hemisphere (F (2,41) = 1.88, P <.17).
Predator stressed rats had significantly more pCREB  lir than Handled GFP
controls with the Handled CREB  rats falling in between these two groups, differing from neither (Tukey-Kramer
Test, P < .05).  The mean of pCREB  lir in Handled CREB  rats measured here at 5 days post HSV
injection is likely an underestimate of its value at peak expression of CREB, which occurs at three days after HSV
injection, when treatments occurred (stress or handling), and which fades
thereafter [43].Mean± SEM relative optical density units (PAG optical density units divided by total PAG section optical density units) in all three columns for all experimental groups
are plotted. The left side of each panel displays data from the right
hemisphere while the right side of the panel illustrates left hemisphere data.
For a given column,  means marked with
the same letter do not differ, but differ from those with different letters,
while means marked with two letters do not differ from means marked with either
of the letters (Tukey-Kramer tests, P < .05).  Means marked with “@” show a
within group difference between hemispheres (P < .05 1 tailed test).
(a)(b)(c)One tailedt-tests were used to compare within groups across hemispheres based on the
prediction that right column pCREB-lir would be increased in predator stressed
rats based on previous findings, and on the prediction that increased CREB  expression in Handled CREB  rats would increase pCREB-lir.  Both the
predator stressed and Handled CREB  rats exhibited more pCREB  lir in the right over the left (all t, P <.04,
1 tailed), whereas there were no hemisphere differences in the Handled GFP
control group.Data from the dorsal column of the PAG were analyzed in the same way with somewhat
differing results (Figure5(b), top right panel). A one way ANOVA of right hemisphere
data revealed a significant difference between groups (F (2,41) = 3.66, P <.04)
while the left hemisphere again showed no group difference (F (2,41) = 0.74, P <.49). Comparison of the groups in the right hemisphere
revealed that the predator stressed rats showed elevated pCREB  lir which was
greater than the Handled groups which did not differ (Tukey-Kramer tests, P < .05). Furthermore, comparison of
groups across the two hemispheres revealed that, like the lateral column, both
the stressed and Handled CREB  group had elevated pCREB  lir in the right hemisphere as compared to the left
(all t,
P <.04 t tailed) with the Handled GFP control group again showing
no difference between hemispheres.Expression of pCREB  in the ventral
column of the PAG presented
another pattern of results (Figure5(c), bottom panel). A one way ANOVA in the
right hemisphere revealed a significant difference between groups (F (2,41) = 6.93, P <.003). In this case however, the stressed rats had
significantly lower pCREB  expression than Handled CREB  rats with the Handled GFP control group falling in between, differing from
neither (Tukey-Kramer tests, P < .05). Much like the other two columns, no difference was seen between
groups in the left hemisphere (F (2,41)
= 1.36, P <.28). Comparisons within
groups across hemispheres showed that both the Handled GFP control and Handled CREB  rats had increased pCREB  in the right over the
left hemisphere (all t, P < .01), while there was no hemisphere
difference in predator stressed rats. These ventral column results mirror
previous findings with the exception of the hemisphere differences [40]. The
fact that the Handled CREB  group
did not differ from the Handled GFP controls indicates that CREB may not be having an effect in this column.
This also suggests that regional differences in the pathways controlling
phosphorylation of CREB may be
dependent on predator stress.


## 3.7. Visualization of GFP

Verification of gene expression was
achieved by examining all PAG sections taken for green flourescence as evidence of expression of the reporter
GFP. Green flourescence in the right PAG verified gene expression of GFP occurred after HSV injection in the vicinity of
the injection cannulas and PAG recording electrodes (Figure6 , e.g., five days after HSV injection).
Since  flourescence ranges from cannula
to PAG  electrodes, one can derive
a sense of the AP plane range of gene expression from PAG electrode position relative to cannulas. Referring to  Table 2, evidence of gene expression five
days post HSV injection appears over a range of ±.28  ±.034 mm (mean ± SEM) from the cannula in the AP plane.  This represents a range nearly as extensive
in previous pilot work which found that at
the time of peak gene expression (three days post HSV injection),  GFP expression was localized to the right lateral
column of the PAG  over an AP plane
range of  ±.35 mm from the cannula.Figure 6
Depicted in the figure are fluorescence
photomicrographs illustrating green florescent protein (GFP) localized in the
right lateral column of the periaqueductal gray (PAG)
5 days after HSV injection in the right lateral PAG.
Magnifications are 6.5 and 25 times (×).


## 3.8. Power Associated with Significant Results

Given the small n of groups, power (α =.05) of all significant findings was calculated. Significant behavioral and
electrophysiological findings all had power values in excess of.90.  Power associated with pCREB  expression
analyses varied with column of the PAG,
ranging from .82 to .91 in the dorsal and ventral columns to a reduced power for
the lateral column results of .60.The power of a
test depends on the value of the type I error (hereα=.05), the sample size, the standard
deviation, and the magnitude of the effect being tested reflected here in
magnitude of mean differences.  Most
findings appear quite robust with power values in excess of .80, suggesting
robust effects of predator stress and virally induced CREB  expression on brain and behavior.  The
reduced power for the lateral column pCREB  findings suggests a fading effect in
this column.


## 4. Discussion

The primary purpose of this study
was to examine the functional significance of pCREB  changes within the right
lateral column of PAG.  This was accomplished by genetically inducing
an increased expression of CREB,
through viral vectoring, and determining the behavioral, electrophysiological
and pCREB  expression changes in comparison to predator stressed and Handled GFP
control rats.


### 4.1. Behavioral Effects of Viral Vectoring CREB

Viral vectoring to induce CREB  expression in the right lateral column of the PAG produced behavioral effects resembling those
seen in predator stressed rats. Handled CREB  rats showed increased open arm avoidance in the EPM (decreased ratio time and
entry) as compared to Handled GFP controls. However, predator stress was even
more effective, increasing open arm avoidance over that seen in Handled CREB  rats. Despite, this graded change in anxiety
between groups, measures of activity and exploration in the plus maze or hole
board did not differ (Figure1). This pattern of results suggests changes in
open arm exploration (anxiety) in EPM are not due to changes in activity or
exploratory tendencies,  consistent with
previous findings using predator stressed rats in similar testing situations [5, 9, 15, 23, 25, 32].The ability of CREB per se to increase open arm avoidance in the
absence of any predator stress is a remarkable finding. It suggests a direct
role for CREB  and possibly pCREB
expression [32] in behavioral changes produced by
stress. Predator stress likely induces CREB signaling change, and then behavioral changes via NMDA receptor activation in
the PAG [7, 23, 25, 40].
In the present study, stress effects were mimicked by bypassing the NMDA
receptor activation and directly activating CREB mediated processes.Not all effects of predator stress
were mimicked by PAG CREB  induction, however. Normally predator stress
reduces ratio frequency risk assessment in an NMDA receptor-dependent manner [5, 7, 16, 25, 26]. While predator stress in the present study
also reduced risk assessment, the risk assessment of Handled CREB  rats was unaffected and did not differ from
Handled GFP controls (Figure 2). The lack of a predator stress type response in
the Handled CREB  rats suggests
that increasing CREB  expression in
PAG may not be the only factor
that mediates suppression of risk assessment, or alternatively may only affect
some EPM behaviors. Other necessary factors at play could include changes in
amygdala pCREB  expression and potentiation of ventral hippocampal to BLA
transmission, both of which follow predator stress [32].  In addition, risk assessment changes produced
by predator stress are highly predicted by right hemisphere changes in
transmission in both CeA-PAG and
hippocampal to BLA pathways [9].  Since only PAG  was manipulated in Handled CREB  rats, it is likely these other factors were not engaged, but were engaged in
predator stressed rats. Perhaps changes in risk assessment require all changes
to occur.  A change in hippocampal
spatial information transfer to BLA might make sense, since risk assessment is
described as a form of sampling the immediate environment for potential threats
[49]. Other possible reasons include the
following. Handled CREB  rats were
more anxious than Handled GFP controls in EPM, but their level of anxiety was
not as great as predator stressed rats. Greater levels of anxiety may be
associated with less risk assessment [49], and so
the more anxious predator stressed rats displayed reduced risk assessment.
Further testing will be required to decide between these possibilities.Handled CREB  rats also had elevated median peak startle amplitude in comparison to the
Handled GFP control group. Moreover, the predator stressed group showed startle
amplitudes that surpassed those of the Handled CREB  rats. This graded response of enhanced startle over groups is reminiscent of
open arm avoidance in the EPM, and supports the notion that inducing CREB  expression per se induces an anxious state
which is milder than that produced by predator stress. Reasons for the milder
effects of direct PAG manipulation
in comparison to predator stress may parallel those raised above to explain
risk assessment discrepancies. Finally, the enhancement of startle amplitude in
predator stressed rats is consistent with past studies [6, 23, 26, 27].Predator stress also reliably
decreases rate of habituation of the acoustic startle response [15, 16, 48]. Present data are consistent with these
findings in that predator stressed rats took significantly longer to habituate
than Handled GFP controls (Figure 3). This replication furthers the validity of
predator stress as a model of hyperarousal aspects of PTSD, since delayed
habituation to startle is also observed in PTSD patients [50–53].Surprisingly, the Handled CREB  group took even longer than the predator
stressed rats to habituate to startle.
This finding implicates CREB dependent mechanisms in delay of startle habituation, which are likely NMDA
receptor-dependent, given that CPP
administered 30 minutes prior to predator stress blocks delay of startle
habituation as well as increased right lateral PAG pCREB  expression [16, 40]. However, this finding
also suggests some difference in mechanisms of induction of neural changes by CREB  in PAG underlying enhanced startle amplitude and delay of habituation. Delay in
startle habituation has been observed in the absence of increased startle
amplitude making it likely that different neural circuits/mechanisms mediate
changes in these two responses to acoustic startle [7, 16].  Additionally, recent studies suggest that
separate portions of the CeA-PAG  pathway mediate the stress induced changes in startle amplitude and startle
habituation [7]. Another possible explanation
could be the following.  Though NMDA
receptor-dependent potentiation of efferent transmission from amygdala to PAG mediates increases in startle amplitude [9, 23, 26],  it is
homosynaptic depression in brain stem startle pathways that underlies
habituation [54], and  direct CREB  expression in PAG more powerfully
engaged such depression than predator stress per second.


### 4.2. Effects of Viral Vectoring CREB on CeA-PAG Transmission

A fascinating finding was that viral
vectoring of CREB  induced a
potentiation of the CeA-PAG  pathway in the right hemisphere (Figure4) analogous to that seen after
predator stress. Moreover, potentiation in this group was restricted to the
same hemisphere as injection. In fact the evoked potentials in the left
hemisphere of the Handled CREB  rats did not differ from those observed in Handled GFP controls. This implies
that any behavioral changes observed in this group can be attributed to the
change in transmission due to CREB  induction in the right hemisphere.In past studies,
CeA-PAG  potentiation by predator
stress has been shown to be NMDA receptor-dependent.  CPP administration prior to predator stress
blocks both anxiogenic effects and and CeA-PAG  potentiation  [7, 16].
Moreover, given that predator stress induces NMDA receptor-dependent right PAG pCREB  expression, it has also been suggested
that long lasting right CeA-PAG  pathway potentiation is dependent on pCREB  expression [7, 40].  Present findings in Handled CREB  rats
support this hypothesis.The present study also adds new data
on the time course of CeA-PAG  pathway potentiation in predator stressed rats.
Current results show that, as expected, predator stressed rats exhibited
potentiation in the right CeA-PAG  pathway two days after predator stress (Figure4), complementing those studies
that have replicated this finding at 1, 9 and up to 12 days post predator
stress [9, 23, 32].
A novel finding was the fading, but still present, potentiation in the
left CeA-PAG  of predator stressed
rats. The presence of potentiation in the left hemisphere adds to previous
studies showing left CeA-PAG  one day after predator stress [27], but fading completely by 9 days [7]. Present findings suggest a left hemisphere
potentiation lasting at least two days.The presence of bilateral CeA-PAG  pathway potentiation in predator stressed rats
and the unilateral induced right CeA-PAG  pathway potentiation in Handled CREB  rats at the time of anxiety testing may account for some of the differences in
open arm avoidance, risk assessment, and startle response between groups. This
especially concerns the absence of reduced risk assessment in the Handled CREB  group, since NMDA block in the left
dorsolateral amygdala 30  minutes  prior to predator stress prevents stress effects
on risk assessment [26]. Moreover path analysis
suggest that changes in open arm exploration and risk assessment may depend on
bihemispheric changes in limbic transmission in the early stages after predator
stress [27].Long lasting potentiation in the
right CeA-PAG  pathway by predator
stress has been suggested to reflect some, but not all, of the anxiogenic
neuroplastic changes after predator stress [9, 23].  Taken together present findings lend strong
support to this view.


### 4.3. Effects of Viral Vectoring CREB on pCREB lir

Given that predator stress increases
pCREB  lir selectively in the right lateral column of the PAG, and that CeA-PAG  potentiation persists longer in the right hemisphere, it has been suggested
that increased production of pCREB  underlies right CeA-PAG  potentiation. Furthermore, degree of
pCREB  expression and right CeA-PAG  potentiation correlate highly with the same measures of the predator stress
experience suggesting a strong relationship between these two phenomena [23].In
the present study, densitometry analysis revealed a right over left lateral PAG  increase in pCREB  lir in both Handled CREB  and predator stressed groups. Thus increasing CREB  expression directly and genetically in the
right lateral PAG also increased
pCREB  in a pattern similar to predator stress in a group which had not been
predator stressed. Moreover, in Handled CREB  rats, the increase of pCREB  in the right but not left lateral column of the PAG  is consistent
with potentiation in the right but not left CeA-PAG  pathway in this group. The fact that pCREB  expression in the right lateral
column in the Handled CREB  group
was intermediate, neither differing from the predator stressed group nor the
Handled GFP controls, is consistent with their milder than predator stressed
rats increase in anxiety in the EPM and acoustic startle tests. Taken together,
these results support the suggestion that elevated pCREB  leads to neuroplastic
changes that induce right CeA-PAG  potentiation and increased anxiety [23, 32].This
conclusion must be tempered by the reduced power associated with lateral column
significant findings.  The reduced power
here likely reflects a reduced effect evidenced in the small mean differences
encountered in the analyses.  As pointed
out above (Section3.6) the mean of pCREB  lir in Handled CREB  rats measured at 5 days post HSV injection is
likely an underestimate of its value at peak expression of CREB, which occurs at three days after HSV
injection, when treatments occurred (stress or handling), and which fades
thereafter [43].  Moreover, effects of
predator stress on pCREB  expression are evident at 20  minutes  post stress and fade
thereafter (20 and unpublished observations). Since transient NMDA receptor
block prevents predator stress effects on brain and behavior and suppresses
pCREB  expression [7, 25, 26, 40],  it is
likely that changes in brain and behavior depend on immediate effects of
increased pCREB  expression, which in this study would have likely begun before
the time of pCREB  measurement. Further studies examining CREB  and pCREB  expression in lateral PAG  at 1–3 days post HSV injection are required to
clarify present findings.Present findings mirror those seen
in previous work with respect to the lateral column of the PAG. However, the dorsal column results in
comparison require greater interpretation. The pattern of dorsal column pCREB
changes stand in contrast to findings that predator stress alone does not alter
pCREB  lir in this column when measured 20 minutes  after predator stress [23, 40]. In the current study predator stressed rats
had elevated pCREB  expression in the right dorsal PAG,
while the two Handled groups had lower and similar levels of expression two
days after treatment (Figure 5). A right over left hemisphere expression effect
was observed in both the predator stressed and Handled CREB  groups, similar to the lateral column. The fact that the right exceeds the left
in the Handled CREB  rats suggests
that right lateral column pCREB  enhancement may have spread to the dorsal
column, but not enough to differ from the Handled GFP control. Other
explanations include a potential leak up the cannula tract or the possibility
that this is a function of CREB  induction, since the predator stressed group demonstrated similar effects
though more pronounced. The increase of right over left pCREB  expression in
predator stressed rats suggests that the EPM is having an effect on the dorsal
column up to 24 hours later. This extends previous findings which showed that
dorsal column pCREB  was elevated bilaterally in predator stressed rats 20–25  minutes
after exposure to the EPM which took place 7 days after predator stress [55]. Previous and present findings differ, however, in
that in the present study, there was no pCREB  increase over control in the left
hemisphere in predator stressed rats. This suggests that an increased time
interval between the predator stress experience and EPM testing may allow for
left hemisphere pCREB  levels to increase. Conversely, in the present study 24
hours elapsed between EPM testing and pCREB  testing.  Perhaps left dorsal column pCREB  expression
faded over this time interval. Further research into time course of pCREB
changes following predator stress and EPM exposure seems warranted.Though lateral and dorsal column
findings are somewhat in line with previous wok, the results of the ventral
column are not. In the present study pCREB  expression in predator stressed rats
was decreased in comparison to both Handled groups in the right hemisphere, and
right and left hemisphere expression did not differ in predator stressed
rats.  Moreover, Handled groups displayed
increased pCREB  expression in right over left hemispheres (Figure5). There are
discrepancies and similarities with previous work examining pCREB  expression 20 minutes after handling or predator stress. Previous work showed no differences in
pCREB  expression between predator stressed and handled controls in ventral PAG of both hemispheres, with right hemisphere
expression elevated over the left  [23]. Perhaps differences in time of sampling pCREB
expression accounts for the discrepancies between past and present findings,
since pCREB  in the present study was measured two days after treatment.If decreases of pCREB  expression in
ventral PAG are normally delayed
after predator stress (for which we have preliminary evidence, unpublished
data), then present findings suggest such decreases are independent of enhanced
pCREB  expression in lateral PAG  induced  by direct genetic induction at
least. If increase in lateral column and decrease in ventral column pCREB
expression parallel enhancement and suppression of normal functioning, then one
might suspect a shifting of defensive response bias toward avoidance of
threatening stimuli and away from a relaxed immobility, along the lines of
functional columnar differences in the dorsolateral and ventral PAG  described by Depaulis and Bandler [47].  Further
time course studies of shifting defensive response bias following predator
stress seem warranted.


### 4.4. Summary and Conclusions

In summary, the present study
demonstrated that directly inducing CREB (and pCREB) expression  in the right
lateral PAG reproduced behavioral,
brain, and molecular changes that closely resemble those seen in predator
stressed rats. These findings suggest increased CREB  (and perhaps
pCREB) expression in the lateral PAG  is at least sufficient to produce brain and behavioral changes normally induced
by a brief predator stress.  Moreover,
similar effects of inducing CREB  expression in basolateral amygdala on EPM anxiety at least, have been reported
by Wallace et al. [43].  Together these data support the idea that the
CREB-pCREB  pathways in the right
lateral PAG, and perhaps amygdala,
are important entry level molecular paths to lasting anxiogenic effects of
predator stress.  To the extent that
predator stress models some aspects of PTSD, present finding point to CREB  and pCREB  pathways as possible new therapeutic
targets.


## 4.1. Behavioral Effects of Viral Vectoring CREB

Viral vectoring to induce CREB  expression in the right lateral column of the PAG produced behavioral effects resembling those
seen in predator stressed rats. Handled CREB  rats showed increased open arm avoidance in the EPM (decreased ratio time and
entry) as compared to Handled GFP controls. However, predator stress was even
more effective, increasing open arm avoidance over that seen in Handled CREB  rats. Despite, this graded change in anxiety
between groups, measures of activity and exploration in the plus maze or hole
board did not differ (Figure1). This pattern of results suggests changes in
open arm exploration (anxiety) in EPM are not due to changes in activity or
exploratory tendencies,  consistent with
previous findings using predator stressed rats in similar testing situations [5, 9, 15, 23, 25, 32].The ability of CREB per se to increase open arm avoidance in the
absence of any predator stress is a remarkable finding. It suggests a direct
role for CREB  and possibly pCREB
expression [32] in behavioral changes produced by
stress. Predator stress likely induces CREB signaling change, and then behavioral changes via NMDA receptor activation in
the PAG [7, 23, 25, 40].
In the present study, stress effects were mimicked by bypassing the NMDA
receptor activation and directly activating CREB mediated processes.Not all effects of predator stress
were mimicked by PAG CREB  induction, however. Normally predator stress
reduces ratio frequency risk assessment in an NMDA receptor-dependent manner [5, 7, 16, 25, 26]. While predator stress in the present study
also reduced risk assessment, the risk assessment of Handled CREB  rats was unaffected and did not differ from
Handled GFP controls (Figure 2). The lack of a predator stress type response in
the Handled CREB  rats suggests
that increasing CREB  expression in
PAG may not be the only factor
that mediates suppression of risk assessment, or alternatively may only affect
some EPM behaviors. Other necessary factors at play could include changes in
amygdala pCREB  expression and potentiation of ventral hippocampal to BLA
transmission, both of which follow predator stress [32].  In addition, risk assessment changes produced
by predator stress are highly predicted by right hemisphere changes in
transmission in both CeA-PAG and
hippocampal to BLA pathways [9].  Since only PAG  was manipulated in Handled CREB  rats, it is likely these other factors were not engaged, but were engaged in
predator stressed rats. Perhaps changes in risk assessment require all changes
to occur.  A change in hippocampal
spatial information transfer to BLA might make sense, since risk assessment is
described as a form of sampling the immediate environment for potential threats
[49]. Other possible reasons include the
following. Handled CREB  rats were
more anxious than Handled GFP controls in EPM, but their level of anxiety was
not as great as predator stressed rats. Greater levels of anxiety may be
associated with less risk assessment [49], and so
the more anxious predator stressed rats displayed reduced risk assessment.
Further testing will be required to decide between these possibilities.Handled CREB  rats also had elevated median peak startle amplitude in comparison to the
Handled GFP control group. Moreover, the predator stressed group showed startle
amplitudes that surpassed those of the Handled CREB  rats. This graded response of enhanced startle over groups is reminiscent of
open arm avoidance in the EPM, and supports the notion that inducing CREB  expression per se induces an anxious state
which is milder than that produced by predator stress. Reasons for the milder
effects of direct PAG manipulation
in comparison to predator stress may parallel those raised above to explain
risk assessment discrepancies. Finally, the enhancement of startle amplitude in
predator stressed rats is consistent with past studies [6, 23, 26, 27].Predator stress also reliably
decreases rate of habituation of the acoustic startle response [15, 16, 48]. Present data are consistent with these
findings in that predator stressed rats took significantly longer to habituate
than Handled GFP controls (Figure 3). This replication furthers the validity of
predator stress as a model of hyperarousal aspects of PTSD, since delayed
habituation to startle is also observed in PTSD patients [50–53].Surprisingly, the Handled CREB  group took even longer than the predator
stressed rats to habituate to startle.
This finding implicates CREB dependent mechanisms in delay of startle habituation, which are likely NMDA
receptor-dependent, given that CPP
administered 30 minutes prior to predator stress blocks delay of startle
habituation as well as increased right lateral PAG pCREB  expression [16, 40]. However, this finding
also suggests some difference in mechanisms of induction of neural changes by CREB  in PAG underlying enhanced startle amplitude and delay of habituation. Delay in
startle habituation has been observed in the absence of increased startle
amplitude making it likely that different neural circuits/mechanisms mediate
changes in these two responses to acoustic startle [7, 16].  Additionally, recent studies suggest that
separate portions of the CeA-PAG  pathway mediate the stress induced changes in startle amplitude and startle
habituation [7]. Another possible explanation
could be the following.  Though NMDA
receptor-dependent potentiation of efferent transmission from amygdala to PAG mediates increases in startle amplitude [9, 23, 26],  it is
homosynaptic depression in brain stem startle pathways that underlies
habituation [54], and  direct CREB  expression in PAG more powerfully
engaged such depression than predator stress per second.


## 4.2. Effects of Viral Vectoring CREB on CeA-PAG Transmission

A fascinating finding was that viral
vectoring of CREB  induced a
potentiation of the CeA-PAG  pathway in the right hemisphere (Figure4) analogous to that seen after
predator stress. Moreover, potentiation in this group was restricted to the
same hemisphere as injection. In fact the evoked potentials in the left
hemisphere of the Handled CREB  rats did not differ from those observed in Handled GFP controls. This implies
that any behavioral changes observed in this group can be attributed to the
change in transmission due to CREB  induction in the right hemisphere.In past studies,
CeA-PAG  potentiation by predator
stress has been shown to be NMDA receptor-dependent.  CPP administration prior to predator stress
blocks both anxiogenic effects and and CeA-PAG  potentiation  [7, 16].
Moreover, given that predator stress induces NMDA receptor-dependent right PAG pCREB  expression, it has also been suggested
that long lasting right CeA-PAG  pathway potentiation is dependent on pCREB  expression [7, 40].  Present findings in Handled CREB  rats
support this hypothesis.The present study also adds new data
on the time course of CeA-PAG  pathway potentiation in predator stressed rats.
Current results show that, as expected, predator stressed rats exhibited
potentiation in the right CeA-PAG  pathway two days after predator stress (Figure4), complementing those studies
that have replicated this finding at 1, 9 and up to 12 days post predator
stress [9, 23, 32].
A novel finding was the fading, but still present, potentiation in the
left CeA-PAG  of predator stressed
rats. The presence of potentiation in the left hemisphere adds to previous
studies showing left CeA-PAG  one day after predator stress [27], but fading completely by 9 days [7]. Present findings suggest a left hemisphere
potentiation lasting at least two days.The presence of bilateral CeA-PAG  pathway potentiation in predator stressed rats
and the unilateral induced right CeA-PAG  pathway potentiation in Handled CREB  rats at the time of anxiety testing may account for some of the differences in
open arm avoidance, risk assessment, and startle response between groups. This
especially concerns the absence of reduced risk assessment in the Handled CREB  group, since NMDA block in the left
dorsolateral amygdala 30  minutes  prior to predator stress prevents stress effects
on risk assessment [26]. Moreover path analysis
suggest that changes in open arm exploration and risk assessment may depend on
bihemispheric changes in limbic transmission in the early stages after predator
stress [27].Long lasting potentiation in the
right CeA-PAG  pathway by predator
stress has been suggested to reflect some, but not all, of the anxiogenic
neuroplastic changes after predator stress [9, 23].  Taken together present findings lend strong
support to this view.


## 4.3. Effects of Viral Vectoring CREB on pCREB lir

Given that predator stress increases
pCREB  lir selectively in the right lateral column of the PAG, and that CeA-PAG  potentiation persists longer in the right hemisphere, it has been suggested
that increased production of pCREB  underlies right CeA-PAG  potentiation. Furthermore, degree of
pCREB  expression and right CeA-PAG  potentiation correlate highly with the same measures of the predator stress
experience suggesting a strong relationship between these two phenomena [23].In
the present study, densitometry analysis revealed a right over left lateral PAG  increase in pCREB  lir in both Handled CREB  and predator stressed groups. Thus increasing CREB  expression directly and genetically in the
right lateral PAG also increased
pCREB  in a pattern similar to predator stress in a group which had not been
predator stressed. Moreover, in Handled CREB  rats, the increase of pCREB  in the right but not left lateral column of the PAG  is consistent
with potentiation in the right but not left CeA-PAG  pathway in this group. The fact that pCREB  expression in the right lateral
column in the Handled CREB  group
was intermediate, neither differing from the predator stressed group nor the
Handled GFP controls, is consistent with their milder than predator stressed
rats increase in anxiety in the EPM and acoustic startle tests. Taken together,
these results support the suggestion that elevated pCREB  leads to neuroplastic
changes that induce right CeA-PAG  potentiation and increased anxiety [23, 32].This
conclusion must be tempered by the reduced power associated with lateral column
significant findings.  The reduced power
here likely reflects a reduced effect evidenced in the small mean differences
encountered in the analyses.  As pointed
out above (Section3.6) the mean of pCREB  lir in Handled CREB  rats measured at 5 days post HSV injection is
likely an underestimate of its value at peak expression of CREB, which occurs at three days after HSV
injection, when treatments occurred (stress or handling), and which fades
thereafter [43].  Moreover, effects of
predator stress on pCREB  expression are evident at 20  minutes  post stress and fade
thereafter (20 and unpublished observations). Since transient NMDA receptor
block prevents predator stress effects on brain and behavior and suppresses
pCREB  expression [7, 25, 26, 40],  it is
likely that changes in brain and behavior depend on immediate effects of
increased pCREB  expression, which in this study would have likely begun before
the time of pCREB  measurement. Further studies examining CREB  and pCREB  expression in lateral PAG  at 1–3 days post HSV injection are required to
clarify present findings.Present findings mirror those seen
in previous work with respect to the lateral column of the PAG. However, the dorsal column results in
comparison require greater interpretation. The pattern of dorsal column pCREB
changes stand in contrast to findings that predator stress alone does not alter
pCREB  lir in this column when measured 20 minutes  after predator stress [23, 40]. In the current study predator stressed rats
had elevated pCREB  expression in the right dorsal PAG,
while the two Handled groups had lower and similar levels of expression two
days after treatment (Figure 5). A right over left hemisphere expression effect
was observed in both the predator stressed and Handled CREB  groups, similar to the lateral column. The fact that the right exceeds the left
in the Handled CREB  rats suggests
that right lateral column pCREB  enhancement may have spread to the dorsal
column, but not enough to differ from the Handled GFP control. Other
explanations include a potential leak up the cannula tract or the possibility
that this is a function of CREB  induction, since the predator stressed group demonstrated similar effects
though more pronounced. The increase of right over left pCREB  expression in
predator stressed rats suggests that the EPM is having an effect on the dorsal
column up to 24 hours later. This extends previous findings which showed that
dorsal column pCREB  was elevated bilaterally in predator stressed rats 20–25  minutes
after exposure to the EPM which took place 7 days after predator stress [55]. Previous and present findings differ, however, in
that in the present study, there was no pCREB  increase over control in the left
hemisphere in predator stressed rats. This suggests that an increased time
interval between the predator stress experience and EPM testing may allow for
left hemisphere pCREB  levels to increase. Conversely, in the present study 24
hours elapsed between EPM testing and pCREB  testing.  Perhaps left dorsal column pCREB  expression
faded over this time interval. Further research into time course of pCREB
changes following predator stress and EPM exposure seems warranted.Though lateral and dorsal column
findings are somewhat in line with previous wok, the results of the ventral
column are not. In the present study pCREB  expression in predator stressed rats
was decreased in comparison to both Handled groups in the right hemisphere, and
right and left hemisphere expression did not differ in predator stressed
rats.  Moreover, Handled groups displayed
increased pCREB  expression in right over left hemispheres (Figure5). There are
discrepancies and similarities with previous work examining pCREB  expression 20 minutes after handling or predator stress. Previous work showed no differences in
pCREB  expression between predator stressed and handled controls in ventral PAG of both hemispheres, with right hemisphere
expression elevated over the left  [23]. Perhaps differences in time of sampling pCREB
expression accounts for the discrepancies between past and present findings,
since pCREB  in the present study was measured two days after treatment.If decreases of pCREB  expression in
ventral PAG are normally delayed
after predator stress (for which we have preliminary evidence, unpublished
data), then present findings suggest such decreases are independent of enhanced
pCREB  expression in lateral PAG  induced  by direct genetic induction at
least. If increase in lateral column and decrease in ventral column pCREB
expression parallel enhancement and suppression of normal functioning, then one
might suspect a shifting of defensive response bias toward avoidance of
threatening stimuli and away from a relaxed immobility, along the lines of
functional columnar differences in the dorsolateral and ventral PAG  described by Depaulis and Bandler [47].  Further
time course studies of shifting defensive response bias following predator
stress seem warranted.


## 4.4. Summary and Conclusions

In summary, the present study
demonstrated that directly inducing CREB (and pCREB) expression  in the right
lateral PAG reproduced behavioral,
brain, and molecular changes that closely resemble those seen in predator
stressed rats. These findings suggest increased CREB  (and perhaps
pCREB) expression in the lateral PAG  is at least sufficient to produce brain and behavioral changes normally induced
by a brief predator stress.  Moreover,
similar effects of inducing CREB  expression in basolateral amygdala on EPM anxiety at least, have been reported
by Wallace et al. [43].  Together these data support the idea that the
CREB-pCREB  pathways in the right
lateral PAG, and perhaps amygdala,
are important entry level molecular paths to lasting anxiogenic effects of
predator stress.  To the extent that
predator stress models some aspects of PTSD, present finding point to CREB  and pCREB  pathways as possible new therapeutic
targets.



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*Source: 904568-2009-04-01.xml*