Telemetry: C-Fos and Dfosb Immunoreactivity in Rat Brain JT Cunningham Et Al

c-Fos and DFosB immunoreactivity in rat brain
JT Cunningham et al
1885
and Blessing, 1994; Naritoku et al, 1995; Osharina et al,

2006; Rutherfurd et al, 1992; Yousfi-Malki and Puizillout,
1994). All these studies measured the ability of acute VNS,
for 30180 min, to increase Fos protein in brain of either
rats or rabbits. In all but one study (Gieroba and Blessing,
1994), stimulation was carried out in anesthetized animals.
Anesthesia can increase Fos protein throughout brain
(Takayama et al, 1994), which could mask effects of vagal
stimulation. Anesthesia can also change the threshold for
activation of different types of fibers in the vagal bundle
(Woodbury and Woodbury, 1990). Further, in all but one
study (Naritoku et al, 1995), stimulation of the vagus was
carried out using either electrodes or stimulation parameters that resulted in alterations of peripheral autonomic
function, (eg mean arterial pressure, MAP; heart rate, HR;
and/or respiratory frequency, RF). Such changes would be
expected to produce reflexes that could activate brain
regions thereby complicating the interpretation of the
results.
In view of these considerations, it seemed worthwhile
to reexamine this issue using clinically relevant stimulation parameters in conscious rats. Also, given the
long-term nature of VNS, it also appeared useful to examine
effects of VNS after more chronic stimulation. Consequently, in nonanesthetized rats we studied the effects of
both acute (2 h) or more chronic (3 weeks) VNS using c-Fos
or DFosB as markers of activation. Fos is an immediate
early gene product and has been used to indicate acute
activation of cells, usually peaking within 13 h of stimulus
exposure (Kovacs, 1998). By contrast, FosB and its splice
variant DFosB show a more delayed activation but persist
longer than c-Fos; consequently, they have been suggested
to be markers of chronic neuronal activation (Nestler,
2004).
Controls received a dummy simulator pack (n 14) that

was the same size and weight (48 mm " 33 mm " 7.1 mm;
16 g).
Telemetry
Some rats were also instrumented with a radio telemetry
transmitter (Data Sciences Instr.) so as to monitor systolic
and diastolic blood pressure, HR, RF, and activity. Systolic
and diastolic blood pressures were used to calculate an MAP
that was used for statistical analysis. Physical activity was
assessed by measuring changes in the animals transmitter
signal strength. When the animal changed position, the
change in transmitter signal strength relative to the
reference point was measured as an increase in counts/
min. Low counts indicated reduced physical activity in the
animal. The activity data provided by this system is strictly
a measure of locomotor activity and has been used widely
for this purpose (Ansah et al, 1996; Howarth et al, 2005;
Kawashima et al, 1996; Meerlo et al, 1999; Zhang et al,
2004). For the acute study, radio telemetry signals were
recorded continuously from each individual at a rate of
64 Hz. Signals were averaged during a 1 h baseline period
before stimulation, during the first hour of VNS or sham
stimulation, the second hour of VNS or sham stimulation,
and for a 30 min poststimulation or sham recovery period.
In the chronic study, radio telemetry signals were sampled
continuously for 10 s every 10 min, 24 h a day for the
duration of the experiment. Signals were recorded for 5 days
prior to stimulation and throughout the 3-week stimulation
period. The 10 s samples were used to create hourly
averages that were further averaged over the 24 h period
to generate daily means.
Acute VNS
MATERIALS AND METHODS
Animals
Experiments were carried out using adult male Sprague
Dawley rats, 250350 g (Charles River). Rats were individually housed and maintained in a temperaturecontrolled environment on a 14 : 10 h lightdark cycle.
Rats had ad libitum access to food and water. Experimental protocols were approved by the IACUC in accordance with the guidelines of the Public Health Service,
American Physiological Society, and Society for Neuroscience.
Vagus nerve electrodes were implanted on the left vagus
nerve under aseptic conditions. The surgical procedure was
similar to that described by Dorr and Debonnel (2006)
except that the anesthetic was 2% isofluorane. Electrodes
were connected to a stimulator pack that was sutured in and
placed in a subcutaneous pouch created on the back of the
rat. Both the electrode and stimulator were supplied by
Cyberonics Inc. (Houston, TX). The bipolar stimulating
electrode was configured with the cathode as the proximal
lead and the anode at the distal lead to preferentially direct
action potential propagation toward the CNS by creating
anodal block at the distal lead. Rats that received VNS
(n 15) were instrumented with an operational stimulator
pack that was programmed by a handheld computer.
Seven days after implantation, the vagus nerve was

stimulated for 2 h (one burst of 20 Hz, 250 ms pulse width,
0.25 mA output current for 30 s every 5 min) in six rats,
whereas five rats served as sham controls. These stimulation
parameters are very similar to those used clinically (Rush
et al, 2005a, b). Thirty minutes after the 2 h vagus nerve or
sham stimulation period, rats were anesthetized and
perfused intracardially. All rats in this study were instrumented with radio telemetry transmitters.
Chronic VNS Stimulation

Rats (n 9) received continuous VNS for 3 weeks with the
same duty cycle and stimulus parameters used in the acute
study. Nine rats served as sham controls. Six rats from the
VNS group and six rats from the sham group received radio
telemetry transmitters. Rats were perfused 30 min after the
end of the experiment.
Fos and DFosB Immunohistochemistry

All rats were anesthetized with pentobarbital (50 mg/kg i.p.)
and perfused with 0.1 M phosphate-buffered saline (PBS)
followed by 300500 ml of 4% paraformadlehyde in PBS.
Brains were removed and placed in PBS with 30% sucrose
for 34 days. Each brain was marked on the left side
Neuropsychopharmacology
c-Fos and DFosB immunoreactivity in rat brain

JT Cunningham et al
1886
(ipsilateral to the VNS implant) and sectioned in a cryostat.

Three serial sets of 40 mm coronal sections from each brain
were placed in cryoprotectant and stored at #201C until
processed for immunohistochemistry (Cunningham et al,
2002).
Separate sets of serial sections were stained for either
c-Fos (Rabbit anti-c-Fos Ab5, Calbiochem, San Diego, CA)
or FosB (Goat anti-FosB (102; sc-46 g), Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (Howe et al,
2004; Ji et al, 2005). The primary antibody used in this study
does not discriminate between FosB and its splice variant
DFosB. However, DFosB accumulates with chronic stimuli
as a result of its long half-life, particularly the 37 kDa
isoform (Nestler, 2004), which is the isoform detected by the
antibody used. For this reason, we refer to chronic
stimulation increasing DFosB in this study although a
contribution from FosB cannot be excluded. For c-Fos
immunohistochemistry, sections were incubated with the
primary antibody (1 : 30 000) for 72 h at 41C. Sections
processed for FosB staining were incubated with the
primary antibody (1 : 5000) for 72 h at 41C. The sections
were incubated in biotyinlated horse anti-rabbit IgG or
horse anti-goat IgG (Vector Laboratories, Berlingame, CA)
diluted 1 : 200 in PBS for 2 h at room temperature. Sections
were reacted with an avidinperoxidase conjugate (Vectastain ABC Kit; Vector Laboratories) and PBS containing
0.04% 3,30 -diaminobenzidine hydrochloride and 0.04%
nickel ammonium sulfate for 10 to 11 min. Brain stem
sections were then processed for dopamine-b-hydroxylase
(DBH) immunofluorescence (Curtis et al, 1999; Grindstaff
et al, 2000; Ji et al, 2005). Sections were mounted on gelatincoated slides, which were air-dried for 12 days, and
coverslipped with Permount.
Forebrain regions included in the analysis were the
paraventricular nucleus of the hypothalamus, supraoptic
nucleus, amygdala, bed nucleus of the stria terminalis
(BNST), paraventricular nucleus of the thalamus, vertical
limb of the diagonal band of Broca, cingulate cortex, and
insular cortex. The following brain stem regions were also
analyzed: the dorsal raphe, LC, NTS, area postrema, caudal
ventrolateral medulla, rostral ventrolateral medulla, and
parabrachial nucleus. DBH immunofluorescence was used
anatomically to define catecholamine-containing regions
and ensure that sections were obtained from the same
rostral-caudal plane for each set of sections from each rat.
Tissue sections containing regions of interest were inspected using an Olympus microscope (IX 50) equipped for
epifluorescence. Digital images were acquired using a Spot
camera (SPOT RT Slider, Diagnostic Instruments, Sterling
Heights, MI) connected to a Pentium computer running
Spot Imaging software (v. 3.24). Some images were adjusted
for brightness and contrast in order to standardize them for
analysis. Regions of interest were identified using the rat
brain stereotaxic atlas of Paxinos and Watson (1986). Three
to six images were taken from each region for each animal
bilaterally as previously described (Cunningham et al, 2002,
2007; Howe et al, 2004; Ji et al, 2005). The number of c-Fos
or DFosB positive cells were recorded for each image and
averaged for each animal. Counts were generated by
observers who were blind to the treatment condition
associated with the images (Cunningham et al, 2002, 2007;
Howe et al, 2004; Ji et al, 2005).
Neuropsychopharmacology
Forced Swim Test

After 1 week of recovery, rats were trained and tested in the
forced swim test (FST) using the method of Lucki (1997),
with minor modifications. Training and test sessions were
recorded by a video camera positioned above the swimming
tank. On the training day, the rats were placed into a
cylindrical tank (60 " 30 cm) containing 251C water at a
depth of 35 cm for 15 min. The first 5 min of the 15 min
session were recorded on videotape. Water was changed
between subjects. After 24 h, the test session was carried out
in the same manner for 5 min, and recorded in its entirety.
One-half hour after the rats were removed from the water
on the training day, they were injected either with saline or
desipramine (DMI), or the vagal nerve stimulators were
turned on (using the handheld computer). Rats received
three sessions of VNS, using the stimulation parameters
described above, for 2 h each, starting 30 min after the
training session and then at 6.5 and 2.5 h before the test
session. This sequence mimics the way drugs are usually
given in the FST (Cryan et al, 2002a). Sham VNS controls
were implanted with a dummy simulator and electrodes
placed around the left vagus nerve as described above. DMI,
used at a dose of 15 mg/kg, s.c., was injected 23.5, 2.5, and
0.5 h before the test session. A control group of rats received
injections of 0.9% NaCl at the same intervals. The
videotaped behavior of the rats was scored using a timesampling technique to rate the predominant behavior over a
5 s interval as described by Lucki (1997). Immobility was
defined as floating or no active movements made other than
that necessary to keep the nose above the water. Swimming
consisted of active motions throughout the swim tank and
crossing into another quadrant, but not having the forepaws
break the surface of the water. Climbing was defined as
upward-directed movements of the forepaws against the
wall and/or having the forepaws break or churn the surface
of the water in vigorous swimming.
Statistical Analysis
Telemetry data from the acute and chronic studies were
analyzed using separate two-way mixed effect analyses of
variance (ANOVAs) and NewmanKeuls t-tests were used
for post hoc analysis (SigmaStat, v. 2.03, Systat Software Inc.,
Point Richmond, CA). Data from the cell counts were
analyzed by one-way ANOVA with NewmanKeuls t-test for
post hoc analysis. Statistical analyses of the FST results were
performed by Students t-tests. All values are presented as
meanSEM. Po0.05 was considered statistically significant.
RESULTS
Telemetry
In the acute study, baseline values for rats receiving VNS
were slightly higher for average MAP and somewhat lower
for HR and RF compared with values for the sham group,
but these differences were not statistically significant. Acute
VNS did not significantly affect MAP, HR, RF, or activity as
compared with the prestimulation baseline values or values
for the sham group (Figure 1). Activity, as measured by the

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c-Fos and DFosB immunoreactivity in rat brain

and Blessing, 1994; Naritoku et al, 1995; Osharina et al,

Controls received a dummy simulator pack (n 14) that

Seven days after implantation, the vagus nerve was

Chronic VNS Stimulation

Fos and DFosB Immunohistochemistry

c-Fos and DFosB immunoreactivity in rat brain

(ipsilateral to the VNS implant) and sectioned in a cryostat.

Forced Swim Test

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