was to test the hypothesis that perinatal CTM exposure (20 mg/kg/d) from postnatal day 1 (PN1) to PN10 leads to hyperexcited NE-LC
circuit function in adult rats (?PN90). Our single-neuron LC electrophysiological data demonstrated an increase in spontaneous and
The pathophysiology underlying major depressive disorder re-
mains poorly understood; however, selective serotonin reuptake
inhibitors (SSRIs), including citalopram (CTM), have been
widely prescribed and are preferred due to their low toxicity and
wide therapeutic index. So far, most of our knowledge regarding
the effects of antidepressant treatment has been obtained from
studies of adult human or rodent populations. A major conclu-
sion derived from these studies is that such exposure not only
upregulates the 5-HT-raphe system, but also downregulates the
noradrenergic locus ceruleus (NE-LC) system (Nestler et al.,
1990; Szabo et al., 1999; West et al., 2009). One of the potential
mechanisms behind such opposite effects on these two intercon-
nected modulatory systems (Cedarbaum and Aghajanian, 1978;
Luppi et al., 1995; Kim et al., 2004) is that 5-HT inhibits LC
function (McRae-Degueurce et al., 1982; Bobker and Williams,
1989; Haddjeri et al., 1997).
In addition to their traditional roles in adults, 5-HT and NE
are also known to play critical roles in neurodevelopment (Gas-
par et al., 2003; Sanders et al., 2005), and these roles may be sex
specific (Connell et al., 2004). Early antidepressant exposure in
male rats results in long-lasting behavioral effects, as well as a
reduction in expression of the 5-HT synthetic enzyme (trypto-
phan hydroxylase) within the raphe nuclear complex and of the
5-HT transporter (SERT) within their cortical efferent fibers
(Mirmiran et al., 1981; Maciag et al., 2006; Oberlander et al.,
al., 2011). In a recent study of adolescent [postnatal day 45
(PN45)] rats exposed to a SSRI, West et al. (2010) reported that
ing an opposite effect compared to adult treatment. It is still not
clear how SSRI exposure during early brain development affects
NE-LC function, but it appears that it is different from adult
exposure and that brief exposure can have dramatic effects that
are observed well into adulthood.
At present, prescription of SSRIs to children and pregnant
mothers is considered relatively safe (Cohen, 2007; Kendall-
Tackett and Hale, 2010), but adverse biological consequences of
such early exposure are suspected (Casper et al., 2003; Hendrick
et al., 2003; Moses-Kolko et al., 2005; Homberg et al., 2010),
including a suspected role in autism spectrum disorder (ASD)
(Chugani et al., 1999; Chandana et al., 2005; Whitaker-Azmitia,
2005; Croen et al., 2011). Interestingly, ASD is approximately
four times more prevalent in boys, suggesting a sexual dimor-
tigation was to explore the sex-specific electrophysiological and
and Psychiatry and Human Behavior, University of Mississippi Medical Center, Jackson, MS 39216. E-mail:
TheJournalofNeuroscience,November16,2011 • 31(46):16709–16715 • 16709
immunohistochemical consequences of perinatal SSRI exposure
on adult NE-LC circuit function.
Animals and drug application. Offspring from four timed-pregnant Long–
Evans rats purchased from Harlan Laboratories were cross-fostered on
from each treatment group, and no offspring were lost. A total of 21
Long–Evans rat pups (male ? 11, female ? 10) were used in this study
9) as described previously (Maciag et al., 2006). The dosing schedule (2
was selected to approximate the upper range of maternal and placental
serum reported in clinical reports of maternal antidepressant treatment
(Maciag et al., 2006). Maternal and offspring behavior were not overtly
affected by cross-fostering or injections of either saline or CTM to pups,
and we continue to examine behavioral effects associated with our drug
exposure (Maciag et al., 2006; Rodriguez-Porcel et al., 2011). At PN28,
pups were weaned and housed in groups of 2–3 per cage under standard
laboratory conditions with ad libitum access to food and water. When
iological and immunohistochemical studies. All experimental animal
ter Institutional Animal Care and Use Committee.
Electrophysiological recording. Animals were anesthetized with sodium
pentobarbital (50 mg/kg, i.p.) and positioned in a stereotaxic apparatus
with bregma 3.0 mm ventral to lambda. This position of the animal’s
head helps to increase successful location of the LC along its long axis as
well as to decrease the likelihood of penetrating the sinus rostral to the
cerebellum. The coordinates for LC (given this head position) were 3.5–
3.9 mm caudal to lambda and 1.0–1.2 mm lateral to midline (Paxinos
and Bloom, 1981; Berridge and Waterhouse, 2003). Either tungsten
(1.0–3.0 M?; FHC) or bundled (eight stainless steel) microwire arrays
(0.3–1.0 M?; Neurobiological Laboratory) were lowered into the LC
using a Kopf micropositioner. Neuronal signals were acquired and digi-
tized using Plexon hardware (MAP) and software (Sort Client 2.60).
?10 min followed by tail-pinch recordings for ?5 min, which included
?1 s tail pinch every 30 s throughout the recording session. Such com-
pression reliably elicits significant increases of LC firing and shows little
or no sensitization or habituation with repeated trials. Following exper-
iments, electrode locations were marked using a 15 s 75–150 ?A DC
current injection for histological verification.
Digitized unit recordings were further spike separated with Offline
Sorter version 2.88 (Plexon) using various waveform characteristics in-
cluding peak, valley, and principal components. Defined single units
were exported to NeuroExplorer version 3.266 (NEX Technologies) for
ings to calculate spontaneous activity of LC neurons and averaged to
determine the mean spike rate. Perievent interspike interval rasters with
histograms were created using the tail pinch recordings to visualize
pinch was used to quantify the stimulus-driven discharge rates across
groups. Stimulus-elicited bursting was quantified using the NeuroEx-
plorer burst analysis function with the following parameters: 0.1 s max-
imum interval to start burst, 0.1 s maximum interval to end burst,
0.2 s minimum interval between burst, 0.1 s minimum duration of
burst, and a minimum of two spikes in burst. These correspond to
typical criteria used to quantify bursts in the LC (Seager et al., 2004;
Devilbiss and Waterhouse, 2011). Baseline spike-rate averages, me-
dian interspike intervals, and burst analyses following tail pinch were
statistically analyzed using 2 ? 2 ANOVAs and post hoc simple main
effects tests (SPSS version 18).
Immunohistochemistry. After the electrophysiological recordings, ani-
mals were perfused through the ascending aorta with 0.9% saline fol-
lowed by 3.5% paraformaldehyde. Brains were sectioned with an AO
freezing microtome at 40 ?m. Tissues were processed in sets so that at
neocortex were collected and processed using an anti-rabbit tyrosine
hydroxylase (TH) antibody (1:1000 dilution; Millipore) for 48 h at 4°C.
The tissue was then incubated in a biotinylated anti-rabbit IgG
(BA9200, ABC Kit, Vector Laboratories) for 1 h, rinsed in PBS, and
then linked with Cy2-conjugated streptavidin (1:200 dilution, Jack-
son ImmunoResearch Laboratories) for 1 h at room temperature. To
determine whether TH-immunoreactive fibers in the neocortex are
noradrenergic in origin, double immunofluorescent methods were
conducted. For double immunostaining, brain tissues were processed
first for TH immunoreactivity as described, and then for either anti-
mouse dopamine-?-hydroxylase (DBH; 1:1000 dilution; Millipore) or
anti-mouse norepinephrine transporter (NET) antibody (1:1000 dilu-
tion; Mab Co) for 48 h at 4°C. The neural profiles were then visualized
using anti-mouse Cy3 (1:200 dilutions) for 1 h at room temperature.
To determine changes in the density and intensity of TH and DBH/
NET immunoreactivity attributable to SSRI exposure, standard analyses
were performed as described previously (Maciag et al., 2006; Weaver et
al., 2010; Zhang et al., 2011). Briefly, digital photomicrographs of sec-
tions containing LC neurons and neocortex (especially the somatosen-
sory cortex) were taken with consistent exposure times at 10 or 20
magnification, respectively, using a Nikon E800 epifluorescent micro-
analyzed with MetaMorph Imaging software (Molecular Devices; ver-
sion 6.2r6) using a threshold analysis to determine the average density
and intensity of immunostaining within (1) all usable LC soma regions
(typically only contralateral to recording sites) and (2) three cortical
columns of each hemisphere of neocortex. Values within animals were
age increase/decrease in staining by comparing experimental animals to
their corresponding saline controls. Statistical analysis was done using
one-sample t tests (SPSS version 18).
CTM-exposed male rats demonstrated approximately threefold
increase in spontaneous LC firing rate (Fig. 1A,B) with a signif-
icant sex by drug exposure interaction (F(1,83)? 4.279, p ?
0.042). Post hoc analysis revealed a simple main effect of drug
ing individual LC neurons, careful observation revealed a quali-
tative difference in the spike width of individual LC neurons
between male rats exposed to CTM and saline (Fig. 1C). There
was a significant sex by drug exposure interaction of spike width
main effect of drug exposure for males (F(1,120)? 38.181, p ?
0.001). This suggests a change in ion channel conductances dur-
with more precise (intracellular) recording techniques. Further-
exposed male rats compared to saline-exposed male rats (Fig.
2A,B), with a significant sex by drug exposure interaction
(F(1,108)? 8.831, p ? 0.004) and a simple main effect of drug
exposure for males (F(1,108)? 14.052, p ? 0.001). Individual LC
neurons are known to operate in distinct modes of tonic and
phasic firing (for review, see Berridge and Waterhouse, 2003;
Aston-Jones and Cohen, 2005). We found that the phasic burst-
ing response to tail pinch of CTM-exposed male rats also dem-
onstrated a statistically significant interaction between sex and
drug exposure in terms of bursts/min (F(1,108)? 6.821, p ?
mean burst duration (F(1,108)? 11.084, p ? 0.001), and mean
spikes in a burst (F(1,108)? 18.939, p ? 0.000) (Fig. 2C). Post hoc
16710 • J.Neurosci.,November16,2011 • 31(46):16709–16715 Darlingetal.•PerinatalCTMExposureEnhancesNE-LCFunction
analyses revealed simple main effects of drug exposure for males
in all of the above bursting characteristics (F(1,108)? 9.465, p ?
0.003; F(1,108)? 33.382, p ? 0.001; F(1,108)? 29.746, p ? 0.001;
and F(1,108)? 36.306, p ? 0.001, respectively).
ing intensity within the LC compared to saline exposure (t(6)?
3.026, p ? 0.023), but this difference was not observed in females
(t(4)? 0.422, p ? 0.694) (Fig. 3). Although a trend was noticed in
males, no significant change in TH immunostaining density within
1.573, p ? 0.167) or between female CTM and saline exposure
TH-positive fibers are typically found in the dopaminergic
prefrontal cortex, but expression is often
too low to be detected in the neocortex
expected, very few TH-positive fibers
were observed in saline-exposed animals.
In contrast, numerous TH-positive fibers
were found in the neocortex of CTM-
exposed male animals (Fig. 4). To deter-
fluorescent procedures were used, which
revealed that virtually every TH-positive
and/or NET (Fig. 4). Furthermore, an ap-
proximately fourfold increase in norad-
renergic TH-positive fiber density was
found in the neocortex of male CTM-
exposed animals compared to saline ex-
trend was not seen in females (t(4)?
0.200, p ? 0.851). Because the increased
density of noradrenergic TH-expressing
neocortical fibers could be explained by
an increased number of DBH-positive fi-
bers, we analyzed the percentage change
of DBH-positive fiber density and found
no significant change in DBH expression
in male CTM animals compared to saline
(t(5)? 0.008, p ? 0.994) or female CTM
animals compared to saline (t(4)? 0.344,
p ? 0.748).
We conducted both electrophysiological
and immunohistochemical techniques from
the same set of animals to elucidate the
neurodevelopmental effects of perinatal
We demonstrated that brief neonatal ex-
posure to one of the most selective SSRIs
(CTM) created long-lasting changes to
expression of TH in both the LC region
and their neocortical targets. Further-
more, male rats appeared to be more vul-
nerable to this early monoamine system
manipulation, suggesting a sex-specific response to early manip-
ulation of the monoamine system. Since our previous studies
have demonstrated that later exposure (PN8–PN21) to a smaller
quantity of CTM (10 mg/kg/d) downregulated 5-HT-raphe cir-
cuit function (Maciag et al., 2006; Weaver et al., 2010), and it is
well known that 5-HT and NE can exert inverse modulatory ac-
tions (for review, see Berridge and Waterhouse, 2003), it is not
surprising that the NE-LC system is upregulated. In brief, this
study provided the first demonstration of a sexually dimorphic
enhancement of the NE-LC system after perinatal exposure to
antidepressants such as CTM. Our novel findings could have
major implications for prescribing SSRIs to both children and
mental disorders such as ASD.
Darlingetal.•PerinatalCTMExposureEnhancesNE-LCFunction J.Neurosci.,November16,2011 • 31(46):16709–16715 • 16711
Manipulating the 5-HT system during early neurodevelop-
ment by exposure to SSRIs or through genetic means produces
al., 1999; Ansorge et al., 2004; Maciag et al., 2006; Oberlander et
2010; Rodriguez-Porcel et al., 2011), but little is known about
how this affects the adult NE-LC system [although blocking the
ilar alterations in behavior (Ansorge et al., 2008)]. As we stated
earlier, virtually all studies aimed at understanding the effects of
SSRIs on the NE-LC system have been reported using mature
The overall conclusion derived from those studies is that such
exposure in adults, especially chronic administration, leads to a
reduction in LC firing rates and a downregulation of LC neural
markers (e.g., TH). In contrast, West et al. (2010) reported that
paroxetine-induced increases of LC activity in adolescent rats
similar to adult exposure (decreased LC activity) after longer-
term treatment (8–14 d). This increased activity complements
our results, but with the apparent difference in chronic versus
acute exposure in the above-mentioned study, it is apparent that
less, it is clear that perinatal antidepressant exposure leads to
changes in LC activity and these effects persist into adulthood,
even after treatment has stopped.
tion that have begun to identify the specific contributions of the
tonic and phasic firing repertories of single LC neurons in various
contexts. These theories of NE-LC function include adaptive gain
modes that operate on cortical circuits to optimize behavioral re-
with a significant sex by drug exposure interaction (F(1,108)? 8.83, p ? 0.004) and a simple main effect of drug exposure for males (F(1,108)? 14.052, p ? 0.001). Perievent rasters and
(red)-labeled LC neurons (top) and in the corresponding intensity profiles (bottom). B, The data were analyzed using a threshold measurement in specified regions containing LC neurons to
determine the density (defined by percentage threshold area) and intensity (defined by average intensity of threshold area) of immunostaining. Histograms represent a percentage increase/
16712 • J.Neurosci.,November16,2011 • 31(46):16709–16715 Darlingetal.•PerinatalCTMExposureEnhancesNE-LCFunction
new behaviors to determine optimal responses (exploration,
tonic mode) (Aston-Jones and Cohen, 2005). Other theories fo-
cus more on arousal where the different modes serve to alter
responses of sensory systems for amplification of perithreshold
salient or novel subthreshold stimuli that would otherwise go
ilbiss and Waterhouse, 2011). Therefore, changes in LC physiol-
ogy could lead to altered behavioral flexibility and responding to
salient environmental cues.
Our present data with perinatally CTM-exposed male rats
revealed that the tonic firing rate of LC neurons was higher than
in controls, and exhibited more and longer bursts than in male
saline controls and females. Intriguingly, Usher et al. (1999) re-
tion task was correlated with faster tonic LC activity (?3 Hz),
whereas better performance was correlated with slower activity
(?2 Hz), suggesting that higher tonic LC activity may be detri-
mental to behavioral performance when behavioral flexibility
preferred over heightened selectivity (e.g., in stable and predict-
able environments). This is complementary to our recent behav-
rats exhibited neophobic-like behavior (Rodriguez-Porcel et al.,
circuit function in response to novelty.
The effects of antidepressant exposure on the adult NE-LC
circuit typically result in a reduction of LC firing rates with a
corresponding decrease in expression of the catalyzing catechol-
amine enzyme tyrosine hydroxylase (TH) within the LC (Nestler
LC was accompanied by a corresponding decrease in mRNA lev-
els, supporting the view that changes in LC neuronal firing cor-
responded to changes in protein and message expression within
the LC (Nestler et al., 1990). It has also been established that
with burst stimulation (Florin-Lechner et al., 1996). The data
presented here support this relationship between neuronal firing
and the expression of TH within the LC and their cortical targets
by demonstrating that perinatal exposure to SSRIs can increase
the expression of TH in the NE-LC circuitry with corresponding
increases in neuronal firing. The hyperexcitability of LC neurons
with an increase in phasic bursting could be the mechanism re-
sponsible for the observable levels of TH within NE neocortical
terminals, which typically are too weak to detect (Pickel et al.,
1975; Ho ¨kfelt et al., 1977).
ASD has a sexual bias, with males affected four times more
is unknown, but abnormally high levels of 5-HT during critical
tributing factor (Whitaker-Azmitia, 2005). For example, it has
been reported that human males have higher rates of 5-HT syn-
ing development is correlated with ASD (Chugani et al., 1999;
Chandana et al., 2005). Thus, it is of interest that studies have
demonstrated a sexual dimorphism in 5-HT receptor-mediated
behavior and 5-HT levels (Jones and Lucki, 2005), LC develop-
ment (Pinos et al., 2001), cortical monoamine levels (Connell et
tor (Curtis et al., 2006), and the sexual differentiation of mono-
aminergic neurons (Reisert and Pilgrim, 1991), all supporting a
possible mechanism for a sexually dimorphic response to altered
knowledge, the present findings provide the first indication that
perinatal exposure to CTM preferentially affects NE-LC circuit
Darlingetal.•PerinatalCTMExposureEnhancesNE-LCFunction J.Neurosci.,November16,2011 • 31(46):16709–16715 • 16713
function in males, but the precise biological function for such
sexually specific responses remains to be elucidated.
In animal models of depression, LC neural activity increases
(Simson and Weiss, 1988), and SSRI exposure reduces this effect
(West et al., 2009). Increased LC firing when SSRIs were admin-
istered during adolescence (PN45) was interpreted as a possible
explanation for the increased risk for suicide in younger popula-
tions taking SSRIs (Goodman et al., 2007; West et al., 2010). We
suggest that this earlier manipulation of the monoamine system
may result in long-lasting modifications of the NE-LC system in
males. Since perinatal antidepressant exposure alters both the
5-HT-raphe and NE-LC systems in adults, and both 5-HT and
NE have been implicated in depression, it may be premature to
both 5-HT and NE that contributes to the normal development
of the monoamine system, their neocortical sites, their firing
properties, and corresponding behaviors. Finally, our data are
done regarding the safety and/or side effects of antidepressant
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