activity and opiate withdrawal behaviors by selectively manipulating CREB activity in the LC using viral vectors encoding genes for
CREBGFP (wild-type CREB tagged with green fluorescent protein), caCREBGFP (a constitutively active CREB mutant), dnCREBGFP (a
dominant-negative CREB mutant), or GFP alone as a control. Our results show that in vivo overexpression of CREBGFP in the LC
cantly faster and had a more depolarized resting membrane potential compared with GFP-expressing control cells. Conversely, LC
neuronal activity was decreased by dnCREBGFP, and the neurons were hyperpolarized by this treatment. Together, these data provide
direct evidence that CREB plays an important role in controlling the electrical excitability of LC neurons and that morphine-induced
rons in the brain, has served as a useful model of opiate action
(Nestler and Aghajanian, 1997). Acutely, opiates decrease LC
opiates, as demonstrated by the return of their firing rates and
cAMP signaling activity toward pretreatment levels during con-
tinuous opiate exposure, as well as dependence on opiates, as
levels after opiate withdrawal (Rasmussen et al., 1990; Kogan et
al., 1992; Nestler, 1992; Ivanov and Aston-Jones, 2001). Several
lines of evidence indicate that these opiate-induced adaptations
in the LC contribute to opiate physical dependence and with-
drawal. Local infusion of an opioid receptor antagonist into the
LC induces opiate withdrawal (Koob et al., 1992). Moreover,
ically within the LC attenuates opiate physical dependence and
withdrawal (Punch et al., 1997; Taylor et al., 1998). The LC is
thought to exert these effects via its widespread projections to
numerous CNS areas (Aston-Jones and Harris, 2004). Although
the precise role of the LC in opiate dependence and withdrawal
the molecular basis of opiate action in the nervous system, with
documented in numerous brain regions (Nestler, 2004).
regulated transcription factor cAMP response element-binding
protein (CREB) in opiate-induced neural plasticity. Opiates
acutely decrease CREB phosphorylation (required for CREB ac-
continuous opiate exposure, and CREB phosphorylation and
itation of opiate withdrawal (Guitart et al., 1992; Shaw-
Lutchman et al., 2002). This phenomenon is partly mediated by
Correspondence should be addressed to Dr. Eric J. Nestler, Department of Psychiatry, The University of Texas
Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9070. E-mail:
4624 • TheJournalofNeuroscience,April26,2006 • 26(17):4624–4629
et al. (1996) showed that mice deficient in CREB exhibit mark-
edly attenuated signs of physical opiate withdrawal, and we
showed that local knockdown of CREB activity in the LC specif-
ically, using CREB antisense oligonucleotides, caused a similar
attenuation of certain withdrawal behaviors (Lane-Ladd et al.,
1997). This antisense oligonucleotide treatment also prevented
the opiate withdrawal-induced increase in LC neuronal activity
(Lane-Ladd et al., 1997). However, these findings are limited by
legitimate concerns about the potential toxicity and lack of spec-
ificity of antisense oligonucleotide treatments in vivo and by the
lack of knowledge of what effect induction of CREB, as seen in
In the present study, we used viral-mediated gene transfer to
more definitively investigate the effect of CREB in the LC on
opiate physical dependence and withdrawal and on LC neuronal
activity by selectively increasing or decreasing CREB function in
Animals. Adult male Sprague Dawley rats (Charles River, Kingston, NY)
were used for in vivo viral injections and drug treatments (initial weight,
200–250 g), and younger rats were used for slice cultures (25–30 d old).
Surgical procedures. Stereotaxic surgery and intra-LC injections were
performed as described previously (Lane-Ladd et al., 1997). Rats were
herpes simplex virus (HSV)–green fluorescent protein (GFP) or HSV-
LacZGFP as controls; and HSV-CREBGFP (CREB tagged with GFP),
133; also known as mCREB), and HSV-caCREBGFP (a constitutively
vectors have been used successfully in previous studies to produce the
expected changes in CREB-mediated transcription (Barrot et al., 2002;
Chao et al., 2002; Olson et al., 2005).
Drug treatments. After bilateral injection of HSV vectors, morphine-
dependent rats were produced as described by Rasmussen et al. (1990).
Briefly, rats were given daily subcutaneous implantations of sham or
withdrawal behaviors exactly as described previously (Rasmussen et al.,
1990; Lane-Ladd et al., 1997).
Slice cultures. Slice cultures were prepared according to Stoppini et al.
(1991). Acute brain slices (250 ?m) containing the LC were obtained in
sucrose–artificial CSF (ACSF) (see below, Electrophysiological record-
medium used in this study was MEM (Invitrogen, Gaithersburg, MD)
containing 30 mM HEPES, 20 mM
ture media used previously by other investigators (Stoppini et al., 1991).
However, the tonic firing of LC neurons was lost in those media. Our
culture medium successfully maintained stable firing of these neurons.
Electrophysiological recordings. Recordings were obtained from LC
neurons in slice cultures and in acute brain slices from HSV vector-
injected rats. Acute brain slices at the level of the LC were prepared as
described previously (Lane-Ladd et al., 1997). The ACSF contained the
following (in mM): 128 NaCl, 3 KCl, 1.25 NaH2PO4, 10 D-glucose, 24
NaHCO3, 2 CaCl2, and 2 MgSO4, oxygenated with 95% O2and 5% CO2
cell and partial access recordings (see below, Validation of partial access
recording) were filled with an internal solution containing the following
(in mM): 115 potassium gluconate, 20 KCl, 10 HEPES, 1.0 EGTA, 4
ATP-Mg, and 0.3 GTP (pH 7.2, 280–290 mOsm). The firing rate and
resting membrane potential (RMP) of LC neurons were recorded in
CA), and data acquisition was realized with pClamp 8.2 (Molecular De-
vices). All data in this study are presented as means ? SEM. Except for
D-glucose, 5% B27, 5.0 mM
forskolin (Calbiochem, La Jolla, CA), all reagents were purchased from
Sigma (St. Louis, MO).
Validation of partial access recording. In this study, the partial access
recording mode was used to measure the firing rate and membrane po-
tential (MP) of LC neurons, because it was associated with less distur-
bance of the neuronal milieu. This recording mode was obtained as de-
scribed by Alreja and Aghajanian (1995). After making a giga seal, the
partial access recording mode was established by maintaining the giga
seal for a short period (3–5 min). Regular spontaneous spikes of LC
neurons could be seen clearly after the mode establishment. The partial
access mode was characterized with a high access resistance (?40 M?)
recordings in LC neurons (Alreja and Aghajanian, 1991). The internal
solution in the recording pipette had full access to cytoplasm in whole-
cell recordings (access resistance, 4–6 M?; spike amplitude, 80–100
recordings, because of a time-dependent dialysis of cytoplasm by the
pipette solution, as observed in previous studies (Alreja and Aghajanian,
1991, 1995). Therefore, the whole-cell recording mode was not suitable
for measuring the effect of CREB on the frequency of pacemaker activity
solution, and the highly stable basal firing of these neurons could be
neurons could be reliably measured after the partial access was stable
(after 10–15 min) and remained constant throughout the remainder of
the recording period (Fig. 1B,C). The MP measured with the partial
access mode was not significantly different from the RMP measured by
whole-cell recordings (55.9 ? 0.9 mV, n ? 16 vs 55.5 ? 1.5 mV, n ? 11;
p ? 0.5) (Fig. 1D). These results suggest that the MP obtained with
partial access was a close measurement of true whole-cell RMP in LC
neurons. It should be pointed out that the MP recorded in partial access
tom, Sample trace of on-line firing rate analysis with pClamp (analysis on at ?4 min after
making giga-seal). The firing rate remained stable during the measurement (?60 min), al-
Hanetal.•CREBintheLCandOpiateWithdrawalJ.Neurosci.,April26,2006 • 26(17):4624–4629 • 4625
was not derived during the resting state of the
neurons. This is because tetrodotoxin (TTX)
induced obvious depolarization of LC neurons
as seen by other investigators (Ishimatsu et al.,
2002; Jedema and Grace, 2004). Moreover,
there were calcium spikes in LC neurons in the
presence of TTX (Ivanov and Aston-Jones,
2001; Han and Nestler, 2005). The MP and fir-
ing rates measured here were in the normal
range for LC neurons (Alreja and Aghajanian,
LC neurons varied as a function of recording
time during partial access, with spike ampli-
tudes increasing over time presumably as a
consequence of gradually decreasing access re-
sistance. However, Figure 1C also clearly dem-
neurons remained extremely stable despite the
changing spike amplitude during the course of
the experiment. Indeed, the opposite effect of
ing rate and MP were apparent throughout the
entire recording period (data not shown).
In vivo manipulations of CREB activity
In a previous study, intra-LC infusion of
antisense oligonucleotides to CREB was shown to attenuate cer-
tain behaviors of opiate withdrawal (Lane-Ladd et al., 1997).
However, it has remained unknown whether morphine with-
LC. This is an important question, because chronic opiate expo-
sure and induction of withdrawal robustly increases CREB func-
tion in the LC (see Introduction). To systematically investigate
the role of CREB in the regulation of opiate withdrawal, we used
nipulate CREB activity in the LC. The activity of these vectors on
CREB-mediated transcription has been verified both in vivo and
in vitro (see Materials and Methods). Here, we further verified
these vectors by showing that HSV-CREBGFP increased (134 ?
19% of HSV-GFP) whereas HSV-dnCREBGFP decreased (60 ?
25% of HSV-GFP) expression of tyrosine hydroxylase, a well
established CREB target, in the LC (n ? 4; p ? 0.05).
jection of vehicle, HSV-GFP, HSV-CREBGFP, or HSV-dnCREB
into the LC. This method allows the highly selective targeting of
HSV vectors to the LC (Fig. 2A–D). Opiate withdrawal was then
induced by administration of naltrexone, an opioid receptor an-
shakes and ptosis) were aggravated by CREBGFP expression
compared with vehicle and GFP control, whereas these with-
drawal behaviors showed significant attenuation after dnCREB-
GFP expression (Fig. 2E). Teeth chatter, another sign of with-
drawal, showed similar regulation: worsening by CREBGFP
controls. Other signs, including lacrimation (Fig. 2E) and irrita-
bility, piloerection, and diarrhea (data not shown), were not af-
fected by these treatments. The attenuated opiate withdrawal
caused by dnCREBGFP is consistent with the previous findings
by CREBGFP demonstrated that upregulation of CREB in the
LC is capable of worsening opiate physical dependence and
In vivo dnCREBGFP expression decreases the firing rate of
opiate withdrawal behaviors by CREB, electrophysiological re-
cordings were performed using brain slices obtained from drug-
naive rats 24 h after bilateral viral injection into the LC. Here, we
focused on LC neuronal firing rates, which have been shown to
reflect aspects of opiate tolerance, dependence, and withdrawal
(see Introduction). Our results showed that HSV-mediated ex-
ing neurons in the same slices, or to neurons in slices from unin-
neurons from the same brain slices or the other controls men-
tioned above (Fig. 3). This finding is consistent with previous
observations using CREB antisense oligonucleotides (Lane-Ladd
no effect on their firing rate compared with controls (Fig. 3).
by in vivo expression of CREBGFP
Several groups have shown that activation of the cAMP signaling
pathway in LC neurons increases neuronal firing (North et al.,
1987; Alreja and Aghajanian, 1995; Ivanov and Aston-Jones,
2001). Accordingly, evidence suggests that upregulation of the
tributes to the increased firing of the neurons seen under these
conditions (Kogan et al., 1992; Ivanov and Aston-Jones, 2001).
ity of the cAMP pathway in the LC (Lane-Ladd et al., 1997), we
examined the sensitivity of the cAMP pathway in LC neurons
4626 • J.Neurosci.,April26,2006 • 26(17):4624–4629Hanetal.•CREBintheLCandOpiateWithdrawal
of adenylyl cyclase) increased the basal firing rate of LC neurons
significantly more in CREBGFP?cells than in CREBGFP?neu-
rons (Fig. 4A,B). In contrast, forskolin increased basal LC firing
to an identical extent in LacZGFP-expressing or nonexpressing
described below): responses of LC neu-
rons to forskolin were sensitized by CRE-
BGFP expression over a dose range of the
drug (Fig. 4D). These results demonstrate
that, although CREB overexpression does
not alter basal activity of LC neurons, it is
sufficient for enhancing the excitatory ef-
fect of forskolin on these cells.
In vitro expression of caCREBGFP and
To further investigate the relationship be-
tween CREB and LC neuronal activity, we
developed a slice culture system in which
infected with HSV vectors. Acute brain slices could not be used
for this purpose, because HSV vectors require 12–24 h to express
their encoded transgenes and LC neurons do not remain healthy
brain regions were not suitable for the LC, because LC neurons
completely lost their pacemaker activity after a few hours in cul-
and Methods), we succeeded in preparing slice cultures of LC
neurons in which the cells showed a stable level of regular tonic
firing for up to 48 h (Fig. 5A–D). As a control experiment, the
firing rate of LC neurons in untreated slice cultures was com-
pared with that in LC neurons expressing GFP. There was no
difference between these two groups of neurons (Fig. 5D,E). Ex-
pression of dnCREBGFP in LC neurons dramatically decreased
the effect seen with dnCREBGFP in vivo (Fig. 5D,E,G). Con-
versely, LC neuronal firing rates were greatly increased by over-
expression of caCREBGFP (Fig. 5D,E,G). The RMP of the neu-
rons was also measured in each group. Expression of caCREB
depolarized the cells compared with controls, whereas dnCREB
hyperpolarized the LC neurons (Fig. 5D,F,H).
The involvement of CREB in the chronic actions of opiates was
hypothesized originally based on the observation that prolonged
exposure to opiates upregulates the cAMP signaling pathway in
the LC (Guitart et al., 1992; Nestler, 1992). Because CREB is
known to mediate many of the effects of cAMP on gene expres-
sion, it was hypothesized that CREB may be an important medi-
ator of opiate-induced changes in gene expression in the LC that
contribute to a state of opiate dependence (Nestler and Aghaja-
nian, 1997; Nestler, 2004). In the present study, using viral-
evidence to support this scheme. We demonstrate that upregu-
lating CREB activity in the LC aggravates opiate dependence and
withdrawal, enhances the excitatory effect of forskolin on LC
neurons, increases the LC neuronal firing rate, and depolarizes
these neurons. In contrast, downregulating CREB activity in the
LC opposes opiate dependence and withdrawal, decreases LC
firing rate, and hyperpolarizes the neurons. These observations
support a scheme whereby the activity of CREB is a key control
point for the excitability of LC neurons and raise the possibility
that chronic opiates, by altering CREB function, induce changes
dependence and withdrawal.
*p ? 0.05. C, In vivo expression of LacZGFP has no effect, with similar forskolin-induced in-
neurons from untreated slices (n ? 42 for untreated control; n ? 7–12 in each other group). ***p ? 0.001. C, Cumulative
Hanetal.•CREBintheLCandOpiateWithdrawal J.Neurosci.,April26,2006 • 26(17):4624–4629 • 4627
The observation that CREB overexpression increases
forskolin-induced activation of LC neurons suggests that among
the many target genes for CREB in these cells are components of
that chronic opiate administration upregulates the cAMP path-
way in the LC, including increased expression of two isoforms of
adenylyl cyclase, ACI and ACVIII, and of the catalytic and regu-
latory subunits of protein kinase A (Lane-Ladd et al., 1997; Chao
et al., 2002). Of these various cAMP signaling proteins, only AC-
VIII appears to be a direct target for CREB (Lane-Ladd et al.,
1997). Consistent with these observations, CREB, or caCREB to
an even greater extent, induces ACVIII promoter activity,
whereas dnCREB inhibits promoter activity, both in vitro and
within the brain in vivo (Chao et al., 2002). Because activation of
the cAMP pathway excites LC neurons (Wang and Aghajanian,
1987; Alreja and Aghajanian, 1995; Ivanov and Aston-Jones,
2001), these observations support the hypothesis that chronic
opiate exposure, through the induction of CREB and the conse-
quent induction of ACVIII, increases LC neuronal excitability.
Additional work is needed to directly investigate this possibility.
It is likely that many additional genes are involved in the ef-
fects of CREB on LC neuronal excitability. A recent microarray
study identified numerous genes that are upregulated or down-
regulated in the rat and mouse LC in response to chronic opiate
administration (McClung et al., 2005). Interestingly, ?20% of
these opiate-regulated genes are known to be regulated by cAMP
or CREB. Several of these genes (e.g., the ?1subunit of Na?/K?
ATPase and the GluR1 AMPA glutamate receptor subunit)
would be expected, like ACVIII, to alter LC excitability. Addi-
CREB on the LC. The ionic mechanisms underlying the tonic
firing activity of these neurons, and their activation after stimu-
lation of the cAMP pathway, remain unclear (North et al., 1987;
Wang and Aghajanian, 1990; Ivanov and Aston-Jones, 2001; Al-
reja and Aghajanian, 1993). We were unable to identify the ionic
mechanisms by which CREB regulates LC neuronal activity, be-
down of the cAMP pathway occurs under these conditions (Al-
reja and Aghajanian, 1995). This important question, therefore,
must await future investigations.
Despite the large number of studies that have implicated di-
verse roles for CREB in the regulation of neural and behavioral
studies that have directly examined the influence of CREB on
neuronal activity per se. A recent study by Marie et al. (2005),
using viral vector techniques similar to those used here, demon-
strated that CREB promotes glutamatergic transmission medi-
ated via NMDA glutamate receptors at excitatory synapses in the
hippocampus. A preliminary study of nucleus accumbens neu-
rons indicates that CREB also promotes the excitability of these
cells, although via distinct mechanisms possibly involving regu-
2006). Our findings show that CREB exerts a similar, excitatory
anisms. These studies are important, because they show that
CREB tends to exert the same net functional effect (enhancing
excitability) in numerous neuronal cell types, although perhaps
via distinct mechanisms in each cell type. As we identify these
ionic mechanisms, we will gain an increasingly complete under-
standing, at the detailed molecular level, by which chronic per-
These studies are a critical step in our efforts to identify the mo-
lecular and cellular basis of neural and behavioral plasticity.
nal excitability. A, Photograph under dissection microscope showing an overnight-cultured
0.1). The firing rate of caCREBGFP-expressing neurons was dramatically increased compared
creased the LC firing rate (n ? 18 in dnCREBGFP group). F, The RMP was also reciprocally
regulated by caCREBGFP and dnCREBGFP (n ? 16–32 neurons per group). G, Cumulative
18–43). H, Cumulative probability plot of the RMP from dnCREBGFP-, GFP-, and caCREBGFP-
4628 • J.Neurosci.,April26,2006 • 26(17):4624–4629Hanetal.•CREBintheLCandOpiateWithdrawal
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