Pain-Induced Negative Affect Is

Mediated via Recruitment of The

Nucleus Accumbens Kappa Opioid
System
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 Report

Pain-Induced Negative Affect Is Mediated via

Recruitment of The Nucleus Accumbens Kappa
Opioid System
Graphical Abstract Authors
Nicolas Massaly, Bryan A. Copits,
Adrianne R. Wilson-Poe, ...,
Ream Al-Hasani, Michael R. Bruchas,
Jose A. Morón

Correspondence
al-hasanir@wustl.edu (R.A.-H.),
mbruchas@uw.edu (M.R.B.),
jmoron-concepcion@wustl.edu (J.A.M.)

In Brief
Massaly et al. identify a pain-induced
enhancement in the kappa opioid system
within nucleus accumbens, which drives
pain-associated negative emotional
states. These results provide a functional
substrate for therapies that would
circumvent pain-induced affective
disorders.

Highlights
d Pain recruits the dynorphin-kappa opioid receptor system in
the nucleus accumbens

d Inhibitory inputs onto dynorphin cells are reduced during

inflammatory pain

d Increase in dynorphin tone mediates inflammatory pain-

induced negative affect

Massaly et al., 2019, Neuron 102, 1–10

May 8, 2019 ª 2019 Elsevier Inc.
https://doi.org/10.1016/j.neuron.2019.02.029
 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Neuron

Report

Pain-Induced Negative Affect Is Mediated

via Recruitment of The Nucleus Accumbens
Kappa Opioid System
Nicolas Massaly,1 Bryan A. Copits,1 Adrianne R. Wilson-Poe,1 Lucia Hipólito,2 Tamara Markovic,1 Hye Jean Yoon,1
Shiwei Liu,3 Marie C. Walicki,1,4,5 Dionnet L. Bhatti,1 Sunil Sirohi,6 Amanda Klaas,7 Brendan M. Walker,6 Rachael Neve,8
Catherine M. Cahill,3 Kooresh I. Shoghi,7,9 Robert W. Gereau IV,1,9 Jordan G. McCall,1,4,5 Ream Al-Hasani,1,4,5,*
Michael R. Bruchas,1,9,* and Jose A. Morón1,9,10,*
1Department of Anesthesiology, Washington University Pain Center, Washington University in St. Louis, School of Medicine, St. Louis, MO

63110, USA
2Department of Pharmacy and Pharmaceutical Technology and Parasitology, University of Valencia, Valencia 46100, Spain
3Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, Los Angeles, CA 90095, USA
4Department of Pharmaceutical and Administrative Sciences, St. Louis College of Pharmacy, St. Louis, MO 63110, USA
5Center for Clinical Pharmacology, St. Louis College of Pharmacy and Washington University in St. Louis School of Medicine, St. Louis, MO

63110, USA
6Department of Psychology, Washington State University, Pullman, WA 99164-4820, USA
7Department of Radiology, Washington University in St. Louis, School of Medicine, St. Louis, MO 63110, USA
8Department of Brain and Cognitive Science, Viral Gene Transfer Core, MIT, Cambridge, MA 02139-4307, USA
9Department of Neuroscience, Washington University in St. Louis, St. Louis, MO 63110, USA
10Lead Contact

*Correspondence: al-hasanir@wustl.edu (R.A.-H.), mbruchas@uw.edu (M.R.B.), jmoron-concepcion@wustl.edu (J.A.M.)

https://doi.org/10.1016/j.neuron.2019.02.029

SUMMARY states largely drives anxiety- and stress-induced disorders and

Negative affective states affect quality of life for
patients suffering from pain. These maladaptive
emotional states can lead to involuntary opioid over-
dose and many neuropsychiatric comorbidities.
Uncovering the mechanisms responsible for pain-
induced negative affect is critical in addressing these
comorbid outcomes. The nucleus accumbens (NAc)
shell, which integrates the aversive and rewarding
valence of stimuli, exhibits plastic adaptations in
the presence of pain. In discrete regions of the
NAc, activation of the kappa opioid receptor (KOR)
decreases the reinforcing properties of rewards and
induces aversive behaviors. Using complementary
techniques, we report that in vivo recruitment of
NAc shell dynorphin neurons, acting through KOR,
is necessary and sufficient to drive pain-induced
negative affect. Taken together, our results provide
evidence that pain-induced adaptations in the kappa
opioid system within the NAc shell represent a func-
tional target for therapeutic intervention that could
circumvent pain-induced affective disorders.
INTRODUCTION
Pain is a major, growing epidemic in the U.S., afflicting more than
30% of the population (Elman et al., 2013). In this maladaptive
condition, the presence of under- or untreated negative affective
involuntary opioid overdoses (Elman and Borsook, 2016; Volkow
and McLellan, 2016). Despite the drive to improve pain treatment
and prevent tragic outcomes, the mechanisms underlying these
negative affective states are not yet clear. Recent human studies
show reduced nucleus accumbens (NAc) activity, alterations
in reward evaluation, decision making, and motivation in pain
patients (Apkarian et al., 2004; Verdejo-Garcı́a et al., 2009). In
rodent models, transient activation of kappa opioid receptors
(KORs) in a discrete subregion of the NAc abates the reinforcing
and motivational values of rewards through presynaptic inhibi-
tion of dopamine, glutamate, and serotonin release (Crowley
and Kash, 2015; Narita et al., 2005; Tejeda et al., 2017). This
behavioral adaptation is also observed during inflammatory
pain, which represents a characteristic feature of negative affec-
tive states (Al-Hasani et al., 2015; Castro and Berridge, 2014;
Narita et al., 2005; Shippenberg et al., 1988; Verdejo-Garcı́a
et al., 2009). We hypothesized that KORs in a subregion of the
NAc (NAc shell cold spot [NAcShCS] for rats and ventromedial
NAc shell [vNAcSh] for mice) represent a key mechanism for
generating pain-induced negative affective states.
RESULTS
Kappa Opioid Receptors Are Both Necessary and
Sufficient to Drive Pain-Induced Negative Affect
Recent evidence from pain and depression studies report that
decreased motivation in goal-directed behavior represents a
characteristic feature of pain-induced negative affect (Hipólito
et al., 2015; Schwartz et al., 2014). Therefore, to determine the
role of the KORs in these maladaptive states, we combined local
microinjection of the long-acting selective KOR antagonist

Neuron 102, 1–10, May 8, 2019 ª 2019 Elsevier Inc. 1

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

(legend on next page)

2 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

norbinaltorphimine (norBNI) with a progressive ratio (PR)
schedule of reinforcement for sucrose rewards (Figure 1A). In
agreement with previous literature (Leitl et al., 2014; Narita
et al., 2005; Schwartz et al., 2014), inflammatory pain decreased
the motivational state of rats as measured by a lower number of
rewards earned (Figures 1B and 1C). Local pharmacological
blockade of KORs, using bilateral micro-injections of norBNI
(2 mg/side) into the NAcShCS, was sufficient to prevent this
pain-induced decrease in motivation (Figures 1B and 1C). This
result cannot be attributed to an intrinsic effect of norBNI as
blockade of KORs alone, in sham pain conditions, did not alter
the rat’s motivational state (norBNI-Saline, Figures 1B and 1C).
Furthermore, inflamed rats did not exhibit any difference in
reward acquisition when lower efforts were required (i.e., the
initial slope of reward earning during PR test [Figure 1C] or in a
fixed ratio schedule of reinforcement [Hipólito et al., 2015]).
This demonstrates that inflammatory pain did not affect the abil-
ity for rats to interact with the reward-paired lever but rather
impaired their motivation when high efforts were required to
obtain the reward. Additionally, while injection of norBNI in the
NAcShCS reversed pain-induced negative affective state, com-
plete Freund’s adjuvant (CFA)-induced hyperalgesia, measured
using Hargreaves thermal test, was not affected (Figure 1D).
These results demonstrate that KOR blockade in the NAcShCS
reverses pain-induced negative affective states without affecting
thermal hyperalgesia. It has been widely shown that pain-
induced decrease in motivated behavior occurs across species
(Okun et al., 2016; Schwartz et al., 2014). Therefore, to determine
the extent to which these findings apply to other species, we
conducted additional studies to further evaluate a potential
role for KOR in pain-induced negative affect in both male and
female mice (Figures 1E–1H). Local micro-injection of norBNI
(2 mg/side) in the vNAcSh reversed the pain-induced decrease
in motivation observed 2 days after CFA treatment (Figures
1E–1H) in both sexes. Importantly, this result emphasizes a
KOR-dependent mechanism in the vNAcSh to drive pain-
induced negative affect across species and sexes. Overall, our
data suggest that KORs are necessary within the NAcShCS/
vNAcSh for reducing motivated behavior induced by inflamma-
tory pain without directly impacting the nociceptive component
of pain (Figures 1B–1H). In order to examine whether activation
of KOR within the NAc would be sufficient to drive a decrease
in motivation, rats received a local bilateral micro-injection of
the short-acting KOR agonist U50,488 (1 mg/side) (Figure 1I).
Selective engagement of KOR signaling within the NAcShCS
induced a significant decrease in motivation for sucrose self-
administration in naive rats (Figures 1J and 1K). Moreover, resto-
ration in sucrose seeking was observed during a third PR test run
24 h after pharmacological treatment, a time point at which
U50,488 is washed off (Figures 1J and 1K). Interestingly, local
KOR stimulation did not impact sucrose self-administration on
a fixed ratio schedule of reinforcement or impact the initial shape
of the PR test (Figures S1A–S1C and Figure 1K, respectively).
These data further support the specific involvement of KORs in
the NAcShCS in decreasing motivated behavior rather than
causing a generalizable behavioral impairment.
Activation of Dynorphin-Containing Neurons in the
vNAcSh Is Sufficient to Drive Negative Affective States
and Aversive Behavior
In the NAcSh, a large population of MSNs contain dynorphin and
locally control presynaptic neurotransmitter release (Al-Hasani
et al., 2015; Nestler and Carlezon, 2006). To investigate
the role of these dynorphin-containing neurons on negative
affective states, we selectively expressed channelrhodopsin-2
(ChR2) in the vNAcSh of dynorphin-cre+ mice (Figure 2A). Here
we observe that stimulation of dynorphin-containing neurons in
the vNAcSh is sufficient to decrease motivation to self-admin-
ister sucrose (Figures 2B–2D). In addition, we previously re-
ported that stimulation of dynorphin-containing neurons in the
vNAcSh was sufficient to drive a KOR-dependent real-time place
aversion (RTPA) (Al-Hasani et al., 2015). We combined RTPA-
induced photo-stimulation of dynorphin-containing neurons in
the vNacSh with local KOR blockade using norBNI (2 mg and
4 mg in 0.5 mL) to test their involvement in mediating negative
affective states and aversive behavior during inflammatory
pain. We found that local NAc norBNI pretreatment (2 mg
and 4 mg per side) blocks dynorphin-neuron-stimulated RTPA

Figure 1. Kappa Opioid Receptors Are Both Necessary and Sufficient to Drive Pain-Induced Negative Affect
(A) Schematic representation of the behavioral methodology.
(B) Blockade of KORs in the NAcShCS prevented pain-induced decrease in motivation (two-way ANOVA for repeated measures: time: F2,116 = 69.51, p < 0.0001;
interaction: F6,116 = 7.349, p < 0.0001. Post hoc during PR3: aCSF-Saline [n = 16] versus aCSF-CFA [n = 19], p < 0.0001, norBNI-Saline [n = 9] versus aCSF-CFA
[n = 19], p < 0.0001 and aCSF-CFA [n = 19] versus norBNI-CFA [n = 18], p = 0.0225).
(C) Representation of the average number of rewards obtained every minute across the 2-h PR3 test session.
(D) Paw withdrawal latency was unchanged in inflamed animals treated with norBNI (two-way ANOVA for repeated measures: time: F2,116 = 35.95, p < 0.0001;
treatment: F3,58 = 25.08, p < 0.0001; interaction: F6,116 = 74.93, p < 0.0001. Post hoc during PR3: aCSF-CFA [n = 19] versus norBNI-CFA [n = 18], p = 0.9998).
(E) Schematic representation of the behavioral methodology.
(F) Representation of training process across experimental days.
(G) Blockade of KORs in the vNAcSh prevented the pain-induced decrease in motivation (two-way ANOVA for repeated measures: time: F2,76 = 0.5201,
p < 0.0001; treatment: F2,38 = 3.432, p = 0.0426. Post hoc: norBNI-Saline [n = 14] versus aCSF-CFA [n = 13], p = 0.0023, and aCSF-CFA [n = 13] versus
norBNI-CFA [n = 14], p = 0.0408).
(H) Representation of the average number of rewards obtained every minute across the PR3 session.
(I) Schematic representation of the behavioral methodology.
(J) Stimulation of KORs in the NAcShCS by U50,488 injection is sufficient to decrease motivation (two-way ANOVA for repeated measures: time: F2,24 = 4.767, p =
0.0181; treatment: F1,12 = 6.005, p = 0.0306. Post hoc after treatment [PR2]: aCSF [n = 7] versus U50,488 [n = 7], p = 0.0064, and 24 h after injection: p > 0.9999).
(K) Representation of the average number of rewards obtained every minute across the 2-h test PR2 session.
(L) During Hargreaves test, the paw withdrawal latency is slightly increased after U50,488 injection in the NAcShCS (two-way ANOVA for repeated measures: time
F2,28 = 9,246, p = 0.0008; interaction F2,28 = 4.060, p = 0.0283; Post hoc test: aCSF [n = 7] versus U50,488 [n = 7] on PR2: p = 0.0326).

Neuron 102, 1–10, May 8, 2019 3

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Figure 2. Activation of Dynorphin-Containing Neurons in the vNACSh Is Sufficient to Drive Negative Affective States and Aversive Behavior
(A) Schematic representation of the behavioral paradigm.
(B) Representation of the training process across experimental days for mice.
(C) Photo-stimulation of dynorphin-containing neurons in the vNAcSh is sufficient to decrease motivation for sucrose self-administration. Mann-Whitney: Ctrl
(n = 7) versus Chr2 (n = 6), p = 0.0385.
(D) Representation of the average number of rewards obtained every minute across the 1-h test session (Kolmogorov-Smirnov: p < 0.0001).
(E) Schematic representation of the behavioral paradigm for real-time place preference testing (RTPT).
(F) Representative heatmaps of the animal activity during the 20 min RTPT.
(G) During baseline test (no pain), aCSF-dyn-cre+ mice demonstrated an aversion for the photo-stimulated compartment (one-sample t test compared to 50%:
n = 13, p = 0.0352). This effect was fully prevented by norBNI pretreatment (one-sample t test compared to 50%: norBNI-dyn-cre+ 2 mg: n = 9, p = 0.6833, norBNI-
dyn-cre+ 4 mg: n = 7, p = 0.3014). Two days after pain induction, 2 mg of norBNI did not reverse the aversive properties of dynorphin-containing neurons
stimulation (one-sample t test compared to 50%: n = 9, p = 0.0122). In contrast, 4 mg of norBNI successfully prevented photo-stimulation-induced aversion
(one-sample t test compared to 50%: n = 7, p = 0.0807).
(H) Locomotor activity was similar throughout all groups of mice during RTPT (two-way ANOVA for repeated measures: time: F1,33 = 30.16, p < 0.0001; treatment:
F3,33 = 0.7857, p = 0.5105; interaction: F3,33 = 1.342, p = 0.5722).

(Figures 2E–2G). Interestingly, 48 h after CFA injection, pretreat- ditions. Importantly, KOR blockade did not impact locomotor
ment with 2 mg of norBNI no longer prevented KOR-mediated activity (Figure 2H) or have an effect on RTPT behavior in
aversion (Figures 2F and 2G). However, local vNAcSh infusion eYFP-expressing controls (Figures S1D–S1F). Furthermore, we
of 4 mg of norBNI blocked the aversion in inflammatory pain con- demonstrated that norBNI micro-injection in the vNAcSh did

4 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Figure 3. Inflammatory Pain Increases KOR Functional Activity and Recruits Dynorphin-Containing Neurons in the NAcSh through a
Disinhibition Mechanism
(A) Dynorphin A stimulation dose-dependently increases GTPgS incorporation in NAc tissue from CFA-injected (n = 6) and saline-injected (n = 6) rats (two-way
mixed-model ANOVA: dose: F1,30 = 8.717, p = 0.0066). GTPgS incorporation is significantly higher in CFA-treated animals, suggesting an increase in KOR
functional activity (two-way mixed-model ANOVA: pain effect: F2,30 = 29.98, p < 0.0001).
(B) Representative GTPgS autoradiography of slices incubated with U69,593 in saline (top) and CFA ± JdTic (bottom) injected animals.
(C and D) In conditions of pain, KOR functional activity was increased in the NAc shell (C; unpaired two-way t test: p = 0.0027, n = 8) but not in the NAc core
(D; unpaired two-way t test: p = 0.1450, n = 8).
(E) Representative pictures of dynorphin A expression in the NAcShCS in either saline-injected (top) and CFA-injected (bottom) animals. ACA, anterior
commissure; NAcSh, nucleus accumbens shell; NAcCr, nucleus accumbens core.
(F) Dynorphin A content in the NAcShCS is increased after CFA-induced inflammation (Mann-Whitney test for unpaired values; p = 0.0022, n = 6).
(G) Schematic representation of electrophysiology methodology. Lower panel: representative picture of a patch pipette onto the somatic area of an Ai14+
dynorphin neuron in the vNACsh.
(H) Representative traces of current response from vNacSh dynorphin neurons obtained from either saline or CFA mice.
(legend continued on next page)

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 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

not impact the inflammatory pain-induced anxiety as measured
in an open field chamber (Figures S1G–S1I), consistent with a
role of KORs in anxiety in the amygdala (Al-Hasani et al., 2015;
Bruchas et al., 2009; Crowley et al., 2016; Knoll and Carlezon,
2010). Our results indicate that pain does not potentiate the aver-
sive behavior induced by dynorphin-containing neuron photo-
stimulation but that KORs are required for the modulation of
pain-induced negative affect. These findings identify a key role
for the KOR-dynorphin system in pain-induced negative affect.
Inflammatory Pain Increases KOR Functional Activity
and Recruits Dynorphin-Containing Neurons in the
NAcSh through a Disinhibition Mechanism
In order to determine how inflammatory pain engages the KOR
system in the NAcShCS, we used in situ hybridization in rat tissue
to measure the expression of KOR mRNA (Oprk1) (Tejeda et al.,
2017). No significant differences in KOR mRNA expression were
observed between saline- and CFA-injected animals 48 h after
pain induction (Figures S2A and S2B). In addition, we assessed
KOR functional activity using a radiolabeled non-hydrolysable
[35S] GTPgS to measure KOR-dependent G-protein coupling.
Dynorphin-A-stimulated [35S] GTPgS binding was significantly
elevated in the NAc of CFA-injected animals compared to con-
trols (Figure 3A), indicating enhanced KOR G-protein coupling
in inflammatory pain. For additional anatomical resolution, we
utilized [35S] GTPgS autoradiography in brain slices (Liu et al.,
2016). In CFA-injected rats, KOR-induced G-protein activation
was enhanced in the NAc shell (Figures 3B–3D). This increase
was dependent on KOR activation, as no radioactive incorpora-
tion was visualized when slices were with both KOR agonist
and a selective KOR antagonist. Interestingly, only a slight
non-significant increase in [35S] GTPgS radiolabeling was
observed in the NAc core and in the band of Broca, suggesting
that enhanced KOR-mediated activation of Ga signaling in pain
conditions is tightly localized to the NAc shell (Figure 3D).
Compensatory changes in the KOR system may also be medi-
ated via recruitment of dynorphin-containing medium spiny neu-
rons (MSNs). We hypothesized that changes in NAc dynorphin
tone may underlie the KOR-dependent decrease in motivation
and adaptive changes in KOR activity observed during pain
(Liu et al., 2016; Muschamp and Carlezon, 2013; Narita et al.,
2005). To test this, we examined the expression of the dynorphin
A peptide in the NAc shell using immunohistochemistry. Rats
experiencing inflammatory pain exhibited a robust increase in
dynorphin A expression within the NAcShCS (Figures 3E and
3F) compared to sham pain controls. To investigate whether
this increase in dynorphin A expression was correlated with an
increase in dynorphin neuron excitability, we conducted whole-
cell patch-clamp electrophysiological recordings on dynorphin-
containing neurons in the vNAcSh in dynorphin-cre+ reporter
mice (Figure 3G) (Al-Hasani et al., 2015; Krashes et al., 2014).
Dynorphin-containing neurons from CFA-treated reporter mice
exhibited enhanced excitability, as measured by a depolarized
resting membrane potential and a lower rheobase (Figures 3H–
3J). These results demonstrate that inflammatory pain signifi-
cantly increases the excitability of dynorphin-containing vNAcSh
neurons (Figures 3H–3J). To determine the potential cellular
mechanisms underlying this increase in dynorphinergic tone
within the NAcSh, we examined spontaneous synaptic input to
the vNAcSh using dynorphin-cre reporter mice. In inflammatory
pain, as compared to sham pain controls, a decrease in the fre-
quency and amplitude of spontaneous inhibitory postsynaptic
currents was specifically observed in dynorphin-containing
neurons (Figures 3K–3P). Interestingly, a shift in the distribution
toward increased inhibitory tone onto non-dynorphin neurons
in vNAcSh was also observed (Figures 3N–3P). Together, these
results support the conclusion that a pain-induced selective
disinhibition of dynorphin-containing neurons leads to an
enhancement of their excitability in the vNAcSh.
Inflammatory Pain Mediates Negative Affective States
through Recruitment of Dynorphin-Containing Neurons
in the NAcShCS
To further determine whether pain-induced increase in dynor-
phin cell excitability results in an enhancement in dynorphin
tone, we used in vivo positron emission tomography (PET) imag-
ing (Figure 4A). In anesthetized rats, the binding efficacy of a
radioactive competitive antagonist for KORs, 11C-LY2795050
(Zheng et al., 2013), was measured before (baseline) or 48 h
after a saline or a CFA injection as an indirect measure of recep-
tor occupancy. Compared to baseline, the distribution volume of
11
C-LY2795050 was significantly decreased when rats were
in pain while it remained unchanged in sham pain control animals
(Figures 4B and 4C). This selective reduction in 11C-LY2795050
binding suggests an elevation in endogenous dynorphin onto
KORs during inflammatory pain.
We then examined whether dynorphin-containing neuronal
activation is necessary for the observed effects of inflammatory
pain on motivated behavior, using a chemogenetic approach
(Roth, 2016) to selectively silence the activity of NAc dynor-
phin-containing neurons. Following a baseline PR test, wild-
type rats were bilaterally micro-injected in the NAcShCS with a

(I and J) CFA-injected animals display a higher resting membrane potential (I; unpaired two-way t test: p = 0.032, ncells/animals = 7–9/4) and (J) a lower rheobase
(J; unpaired two-way t test: p = 0.019, ncells/animals = 7–9/4).
(K) Representative traces of sIPSCs from dyn+ neurons.
(L and M) Amplitude (L) and frequency (M) of sIPSC onto dyn+ neurons are decreased in conditions of inflammatory pain as compared to saline control (frequency:
two-tailed t test for unpaired values p = 0.0406; amplitude: two-tailed Mann-Whitney for unpaired values, p = 0.0140, ncells/animals = 7–8/4). The cumulative
probability plots demonstrate a significant shift toward smaller and less frequent events in dynorphin+ neurons after CFA (Kolmogorov-Smirnov test, p < 0.0001,
ncells/animals = 7–8/4).
(N) Representative traces of sIPSCs from Dyn– neurons.
(O and P) Neither the mean amplitude (O) nor frequency (P) of sIPSCs onto dyn– neurons are affected by inflammatory pain (frequency: two-tailed Mann-
Whitney for unpaired values, p = 0.6277; amplitude: two-tailed Mann-Whitney for unpaired values, p = 0.4242, ncells/animals = 5–6/4). A plot of the cumulative
probability revealed a significant shift toward larger and more frequent events in dynorphin-negative neurons after CFA (Kolmogorov-Smirnov test,
p < 0.0001, ncells/animals = 5–6/4).

6 Neuron 102, 1–10, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Figure 4. Inflammatory Pain Mediates Negative Affective States through Recruitment of Dynorphin-Containing Neurons in the NAcShCS
(A) Schematic representation of the PET imaging methodology in rats.
(B) Inflammatory pain, but not saline control, decreases the distribution volume of 11C-LY2795050 observed in rat’s brain 2 days after CFA injection (two-way
ANOVA for repeated measures: time: F1,12 = 26.88, p = 0.0002; interaction: F1,12 = 10.02, p = 0.0081; post hoc tests: baseline CFA versus test CFA: p < 0.0001;
baseline saline versus test saline: p = 0.3260; test CFA versus test saline: p = 0.0282, n = 7).
(C) Representative images of PET imaging before (left) and after (right) inflammation.
(D) Schematic representation of the behavioral methodology.
(E) Silencing dynorphin neurons in the NAcShCS prevented the inflammatory pain-induced decrease in motivation for sucrose self-administration (two-way
ANOVA for repeated measures: time: F1, 38 = 75.81, p < 0.0001; interaction: F4,38 = 15.84, p < 0.0001. Post hoc test: aCSF-Veh.-CNO versus hM4Di-CFA-Sal.:
p = 0.0006; aCSF-Veh.-CNO versus Ctrl-CFA-CNO: p = 0.0003; Ctrl-CFA-CNO versus hM4Di-CFA-CNO: p = 0.0255; hM4Di-CFA-Sal. versus hM4Di-CFA-CNO:
p = 0.0417; hM4Di-CFA-Sal. versus hM4Di-Veh.-CNO: p = 0.0046; Ctrl-CFA-CNO versus hM4Di-Veh.-CNO: p = 0.0027; hM4Di-CFA-CNO versus
hM4Di-Veh.-CNO: p = 0.9658; hM4Di-CFA-Sal. versus Ctrl-CFA-CNO: p = 0.9988; aCSF-Veh.-CNO versus hM4Di-Veh.-CNO: p = 0.9706; and aCSF-Veh.-CNO
versus hM4Di-CFA-CNO: p = 0.7173, n = 8–9).

herpes simplex virus (HSV) containing inhibitory designer DISCUSSION

receptors exclusively activated by designer drugs (DREADDs)
(Dyn2.0-hM4Di-IRES-mCherry) or its control virus (Dyn2.0-
IRES-mCherry) (Figure 4D). This approach allowed us to specif-
ically target NAcShCS dynorphin-containing neurons (Figures
S3A and S3B) and decrease their excitability upon CNO admin-
istration (Figures S3C–S3F). Importantly, CNO alone did not
impact neuronal excitability (Figures S3G–S3J). As previously
described, rats experiencing inflammatory pain display a signif-
icant decrease in motivation compared to sham pain animals.
Chemogenetic silencing of NAcShCS dynorphin-containing
neurons blocked the decrease in motivation observed in
CFA-treated animals (Figure 4E). Importantly, the sole expres-
sion of Gai-coupled DREADDs, not acute CNO administration
(1mg.kg 1), impacted pain-induced decrease in motivated
behavior (Figure 4E). Lastly, silencing dynorphin-containing neu-
rons did not increase motivated behavior in non-pain conditions
(Figure 4E). These results establish that neither DREADD
expression nor CNO administration have non-specific actions
on these behaviors (Figure 4E). Altogether, these results demon-
strate that dynorphin-containing neuronal activity is necessary
for generating inflammatory pain-induced negative affect.
In the present study, we identified a critical role for the kappa
opioid system in the modulation of negative affect associated
with inflammatory pain. We report that pain enhances dynorphin
expression and recruits dynorphin-containing neurons in a
discrete subregion of the NAc shell through a disinhibition mech-
anism. In addition, we further report that pain increases KOR
function and occupancy using both GTPgS binding and in vivo
PET imaging. Finally, using a complementary series of pharma-
cological, optogenetic, and chemogenetic approaches, we
report that both dynorphin-containing neurons and KOR activity
in the NAc shell are necessary and sufficient to drive pain-
induced negative affective states.
Previous evidence has uncovered a role for the dynorphin
peptide in decreasing opioid-mediated goal-directed behaviors
and opioid-induced dopamine release in the NAc during pain
states (Narita et al., 2005; Verdejo-Garcı́a et al., 2009). However,
the source of endogenous peptide and its consequent mecha-
nism of action have remained elusive. In the current work,
we uncover that pain is sufficient to increase dynorphin expres-
sion in the NAcShCS (Figures 3E and 3F) and recruit local

Neuron 102, 1–10, May 8, 2019 7

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029
dynorphin-containing neuron activity (Figures 3G–3I). We deter-
mine that this recruitment is due to a substantial decrease in
IPSCs onto these NAc dynorphin-containing neurons, leading
to a hyperexcitable state for those neurons (Figures 3G–3M).
We demonstrate for the first time using PET scan imaging in ro-
dents, that pain induces increases in dynorphin tone in vivo (Fig-
ures 4A–4C). In light of the overall increase in KOR occupancy
observed in our study, it is important to consider the possibility
that several hubs of dynorphin-containing neurons, such as
ventral pallidum or lateral hypothalamus (Baldo et al., 2003;
Peyron et al., 1998), are also recruited during pain conditions.
Through the development of a gen 2.0-specific and efficient
inhibitory DREADDs (Figure S3) in rats, we uncover the role
of the NAcShCS dynorphin-containing neuron recruitment in
pain-induced decreases in motivational states (Figures 4D and
4E). Together, these results reveal a source of endogenous
dynorphin in the vNAcSh/NAcShCS that drives pain-induced
negative affect, resolving an important gap in our understanding
of the impact of pain on the dynorphin-KOR system.
Recent studies have identified alterations in excitatory trans-
mission onto D2-expressing MSNs in the NAc during inflamma-
tory and neuropathic pain (Ren et al., 2016; Schwartz et al.,
2014). While these studies describe increased excitatory inputs
(Ren et al., 2016) that can trigger long-term plasticity at synapses
onto D2R-expressing neurons (Schwartz et al., 2014), we report
here that dynorphin-containing neurons are specifically disinhi-
bited in inflammatory pain (Figures 3K–3M). Furthermore, while
NAc receives KOR-enriched synaptic terminals from brain struc-
tures involved in affect and motivation (Bruchas et al., 2009;
Land et al., 2009; Margolis et al., 2003; Muschamp and Carlezon,
2013), we demonstrate here that local KOR blockade in the NAc
reverses pain-induced negative affect in both mice and rats
(Figure 1). Local release of dynorphin, activating KORs in the
NAc, may thus negatively regulate the release of serotonin,
dopamine, glutamate and/or GABA, leading to altered NAc func-
tion in pain-induced behaviors, including reward seeking.
Despite this evidence, the role of other peptides and neurotrans-
mitters in pain-induced negative affect cannot be ignored.
Similarly, activation of the KOR system located outside of the
NAc may be necessary to drive pain-induced adaptations in
negative affect. Nonetheless, in addition to previous reports
(Ren et al., 2016; Schwartz et al., 2014), our work provides a
novel allostatic mechanism through which pain impacts the
nucleus accumbens microcircuitry to induce negative affective
states.
Using a combination of pharmacological studies in both rats
and mice, we show that adaptations in the dynorphin-KOR sys-
tem occur when aversive behaviors are measured in conditions
of pain (Figure 2). Lastly, a recent study from Yang and collabo-
rators has uncovered two distinct accumbens shell subregions
projecting to the ventral tegmental area (VTA) to regulate motiva-
tional states (Yang et al., 2018). Future studies will further
examine how the dynorphin-containing neuronal population
described in our findings integrates with those specific circuits
to drive motivational state adaptations in pain conditions.
While other studies describe a potential therapeutic role for
KOR antagonists (Al-Hasani et al., 2015; Castro and Berridge,
2014; Chavkin, 2011), our current findings provide further in-
8 Neuron 102, 1–10, May 8, 2019
sights into neurobiological targets for future pharmacother-
apies that attenuate unwanted negative outcomes during
pain. Emerging therapies such as focused ultrasound technol-
ogy (Elias et al., 2016), the development of photoactivable
compounds (Banghart and Sabatini, 2012), and drugs specif-
ically targeting the activated-KOR structure (Che et al., 2018)
could allow for selective and localized treatments. Together
with a better understanding of dynorphin-KOR system in
pain-induced negative affect, these studies open new avenues
for targeting KOR as a site for treating pain-induced emotional
states.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENT AND RESOURCE SHARING
d EXPERIMENTAL MODEL AND SUBJECT DETAILS
d METHOD DETAILS
B Surgeries
B Chemogenetics, Optogenetics and Behavioral Assays
B Receptor Function Assessment
B Electrophysiology
B Immunohistochemistry
B Positron Emission Tomography (Pet) Imaging
B In Situ Hibridization
B Histology
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
SUPPLEMENTAL INFORMATION
Supplemental Information can be found with this article online at https://doi.
org/10.1016/j.neuron.2019.02.029.
ACKNOWLEDGMENTS
We would like to thank all members from the Moron-Concepcion, Bruchas and
Al-Hasani laboratories for their help throughout the completion of the current
study and Lindsay Lueptow for the complementary analysis of GTPgammaS
slices. This work was supported by US National Institutes of Health (NIH) grant
DA041781 (J.A.M.), DA042581 (J.A.M.), DA042499 (J.A.M.), DA041883
(J.A.M.), DA045463 (J.A.M.), NARSAD Independent Investigator Award from
the Brain and Behavior Research Foundation (J.A.M.), DA033396 (M.R.B.),
DA037152 (M.R.B.), R01-NS106953 (R.W.G.), AA020394 (B.M.W.), K99/R00-
DA038725 (R.A.), K99-DA041467 (A.R.W.-P.), Philippe Foundation (N.M.),
the ‘‘Spanish Ministerio de Economia y Competitividad’’ MINECO PSI2016-
77895-R (L.H.), the Mallinckrodt Institute of Radiology at Washington Univer-
sity from pilot grant 16-014 to support the positron emission tomography
studies, the Hope Center Viral Vectors Core at Washington University School
of Medicine.
AUTHOR CONTRIBUTIONS
Conceptualization, N.M., L.H., R.W.G., K.I.S., R.A., M.R.B., and J.A.M.; Meth-
odology, N.M., R.A., M.R.B., and J.A.M.; Formal Analysis, N.M., and J.A.M.;
Investigation, N.M., B.A.C., A.R.W.-P., M.C.W., J.G.M., T.M., L.H., H.J.Y.,
B.M.W., S.S., S.L., C.M.C., D.L.B., R.A., and A.K.; Writing – Original Draft,
N.M., R.A., M.R.B., and J.A.M.; Writing – Review & Editing, N.M., R.A.,
 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029
M.R.B., and J.A.M.; Funding Acquisition, R.A., M.R.B., and J.A.M.; Resources,
R.N., R.A., M.R.B., and J.A.M.; Supervision, N.M., R.A., M.R.B., and J.A.M.
DECLARATION OF INTERESTS
The authors declare no competing financial interests.
Received: May 22, 2018
Revised: September 20, 2018
Accepted: February 14, 2019
Published: March 13, 2019
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 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit Anti-dynorphin A Peninsula Laboratories International Inc. Cat# T-4266.0500; RRID: AB_518292
Mouse Anti-mCherry DHBS Cat# DSHB-mCherry-3A11; RRID:
AB_2617430
Biotin conjugated anti-rabbit Bio-Rad Cat# 170-6515
Alexa 488 conjugated streptavidin Thermo Fisher Scientific Cat# S32354; RRID: AB_2336881
Goat anti-Mouse - Secondary Antibody, Thermo Fisher Scientific Cat# A-11019; RRID: AB_143162
Alexa Fluor 568
Endogenous Biotin Blocking Kit Thermo Fisher Scientific Cat# E21390
Goat anti-Rabbit, Biotin-XX Thermo Fisher Scientific Cat# B-2770; RRID: AB_2536431
Bacterial and Virus Strains
HSV-Dyn2.0-hM4Di-IRES-mCherry Obtained from Rachael Neve, M.I.T. N/A
HSV-Dyn2.0-IRES-mCherry Obtained from Rachael Neve, M.I.T. N/A
AAV-EF1a-DIO-hChR2(H134R)-EYFP UNC Vector Core N/A
AAV-EF1a-DIO-EYFP UNC Vector Core N/A
Chemicals, Peptides, and Recombinant Proteins
11
C-LY2795050 Washington University Cyclotron Facility N/A
Complete Freund’s adjuvant Thermo Fisher Scientific Cat# 77140
JDTic Obtained from Ivy Carroll, Reserach N/A
Triangle Institute
Nor-Binaltorphimine dihydrochloride Sigma-Aldrich CAS Number 105618-26-6; Cat# N1771
U-50488 Sigma-Aldrich Cat# D8040
U-69593 Sigma-Aldrich CAS Number 96744-75-1; Cat#U103
Clozapine-N-oxide Sigma-Aldrich CAS Number 34233-69-7; Cat# C0882
Experimental Models: Organisms/Strains
Rat: Sprague Dawley Envigo N/A
Mouse: C57BL/6J The Jackson Laboratory IMSR Cat# JAX:000664; RRID:
IMSR_JAX:000664
Mouse: C57BL/6J - preprodynorphin-IRES-cre Obtained from Bradford Lowell Laboratory N/A
(dyn-Cre)
Mouse: B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J - The Jackson Laboratory IMSR Cat# JAX:007909; RRID:
Ai9-reporter IMSR_JAX:007909
Mouse: B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J - The Jackson Laboratory IMSR Cat# JAX:007914; RRID:
Ai14-reporter IMSR_JAX:007914
Software and Algorithms
GraphPad Prism 8 GraphPad RRID: SCR_002798
Illustrator Adobe RRID: SCR_010279
Ethovision XT 10.0 Noldus RRID: SCR_000441

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jose A.
Morón (jmoron-concepcion@wustl.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

All procedures were approved by the Washington University Institutional Animal Care and Use Committee (IACUC) in accordance
with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Adult male Sprague Dawley rats

Neuron 102, 1–10.e1–e6, May 8, 2019 e1

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

(250-300 g), adult male and females dynorphin-cre (dyn-Cre) mice (25-30 g) (Al-Hasani et al., 2015), adult male and females Ai14 or
Ai9 preprodynorphin reporter mice (Al-Hasani et al., 2015; Krashes et al., 2014) (25-30 g) and adult C57BL/6J males and females mice
(25-30 g) were used for this study. All animals were 10 to 12 weeks at the beginning of the experiments. Two to three rats were housed
together with a 12/12 h dark/light cycle (lights on at 7:00 AM) and acclimated to the animal facility holdings for at least 7 days before
any manipulation. Rats received food ad libitum until 2 days before starting the behavioral studies, when food restriction (17 g of rat
chow per day) started and continued until the end of the experiment. Four to five mice were housed together, given access to food
pellets and water ad libitum, and maintained on a 12/12 h dark/light cycle (lights on at 7:00 AM). All animals were kept in a sound-
attenuated, isolated holding facility in the lab 1 week prior to surgery, post-surgery, and throughout the duration of the behavioral
assays to minimize stress.

METHOD DETAILS

Surgeries
All surgeries were performed under isoflurane (1.5/2 MAC) anesthesia under sterile conditions.
Intra cerebral injections
Rats were stereotaxically (World Precision Instruments, Sarasota, FL) injected with either norBNI (2 mg per side in 0.5 ml), HSV-
Dyn2.0-hM4di-IRES-mCherry (5x108 transducing units per ml - 0.5 mL per side), HSV-Dyn2.0-mCherry (5x108 transducing units
per ml - 0.5 mL per side) or implanted with guide cannula (Plastics One, Roanoke, VA) targeting the NAcShCS (stereotaxic coordinates
from Bregma: A/p = + 0.96mm, M/L = ± 0.8mm, D/V = – 6.5mm from the skull surface) (Castro and Berridge, 2014; Hipólito et al.,
2015). Animal’s skin was either sutured after bilateral injection using sterile nylon sutures or, when using guide cannula, the implants
were secured on the skull using two sterile bone screws and a dental cement head-cap (Lang Dental). Cannula were obstructed using
a dummy cannula and covered by a cannula cover until pharmacological injection.
Mice were anesthetized in an induction chamber (4 MAC Isoflurane) and placed into a stereotaxic frame (Kopf Instruments, Model
1900) where they were maintained at 1 MAC–2 MAC isoflurane. A craniotomy was performed and followed by a unilaterally
injection, using a blunt needle (86200, Hamilton Company), 300nl of AAV5-DIO-ChR2-eYFP or AAV5-DIO-eYFP controls (Hope
Center Viral Vector Core, viral titer 2 3 1013 vg/mL) into vNAcSh (stereotaxic coordinates from Bregma: A/P: + 1.30mm, M/L: ±
0.5mm, D/V: –4.75mm) (Al-Hasani et al., 2015). Two weeks after this injection mice underwent a second surgery where norBNI
(2 mg or 4 mg /0.5 ml) was locally injected and a fiber optic was placed in the vNAcSh (Al-Hasani et al., 2015; Siuda et al., 2015).
The implants were secured using two bone screws and a dental cement head-cap (Lang Dental). Mice were allowed to recover
one week before running any behavioral experiment, well within the limits of norBNI antagonism. Furthermore, the experiments
started 3 weeks after AAV5-DIO-ChR2-eYFP injection, permitting optimal expression of ChR2 in the dyn-Cre cell bodies.
A similar surgical procedure was used for pharmacological microinjection of norBNI (Figures 1E–1H). Briefly, following craniotomy
norBNI (2 mg or 4 mg /0.5 ml) was bilaterally injected into vNAcSh (stereotaxic coordinates from Bregma: A/P: + 1.30mm, M/L: ±
0.5mm, D/V: –4.75mm)5. Mice’s skin was sutured after bilateral injection using sterile nylon sutures and this animal was allowed
to recovery for a week before running any behavioral experiment.
CFA administration
After confirming sedation by the absence of reflex during a toe-pinch, rats and mice were injected with in the right hindpaw with
properly resuspended 150ul or 50ul of CFA solution (Thermo Fisher), respectively. Animal’ recovery and general behavior (feeding,
drinking, mobility) were monitored through the rest of the experiment.

Chemogenetics, Optogenetics and Behavioral Assays

Sucrose Self-administration
Rat operant-conditioning chambers (Med Associates, Fairfax, VT) were equipped with two retractable levers positioned on the right-
hand wall 12.5 cm apart and 5 cm above the floor, a food magazine connected to a food pellet dispenser, two cue lights positioned
2 cm above the levers, and one house light positioned on the top left-hand wall. At the beginning of the session, both levers (active
and inactive) were presented and a white light was on above the active lever. Pressing the active lever resulted in sucrose pellet
dispense and a 20 s’ retraction of both active and inactive levers, along with the turning off of the light cue above the active lever
after the retraction of the levers. Pressing the inactive lever had no effect. Animals were gently placed in the self-administration boxes
for 2 h sessions where a press on the active lever results in sucrose pellet dispense - fixed-ratio (FR) 1 schedule. Once rats acquired
the self-administration behavior (by obtaining 60 pellets/session on 5 consecutive sessions), the schedule of reinforcement was
changed to FR2 for the other 3 sessions, and then FR5 for another 3 sessions. Once they completed the training, the rat’s motivation
for sucrose self-administration was tested using a Progressive Ratio (PR) schedule of reinforcement (PR1). In the PR session, the
number of responses on the active lever to obtain the reward increased with the dose. The increase in the number of correct re-
sponses followed the equation response ratio = (5 3 e(0.2 3 infusion number) – 5 rounded to the nearest integer resulting in the following
PR steps: 1, 2, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95. (Hipólito et al., 2015; Roberts and Bennett, 1993; Schwartz et al., 2014).
For the studies on the necessity of KOR on pain effects on motivated behavior, rats were injected with either norBNI (2 mg per side)
or aCSF in the NAcShCS (A/P: + 0.96mm; M/L: +/– 0.8mm; D/V: - 6.5mm) (Castro and Berridge, 2014). A week after, to allow full
recovery, animals were tested on a second PR schedule of reinforcement (PR2) to assess any effect of KOR antagonism on

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 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

motivation. Right after, rats were injected with 150 mL of saline or CFA solution in the hindpaw; and placed again in the self-
administration chambers 48 h after injection to undergo an additional PR session.
For the studies on the sufficiency of KOR on pain effects on motivated behavior, after training and a first PR ratio (PR1) as a baseline
measurement, rats were deeply anesthetized and guide cannula were placed bilaterally 1mm above the NAcShCS (A/P: - 0.96mm;
M/L: +/– 0.8mm; D/V: - 5.5mm) (Castro and Berridge, 2014). One week after surgery, to allow full recovery, animals were injected in
the NAcShCS with KOR agonist (U50,488 – 1 mg per side) or aCSF using a microinjection pump (rate 0.25 mg per minute) and the
injector, projecting 1mm below the cannula, was left in place for an additional 5 min to allow compound diffusion. 30 min after
U50,488 injection rats were tested for thermal hyperalgesia using Hargreaves test. 30 min later these animals were gently placed
in the self-administration boxes for a second PR test (PR2), thus assessing the role of KOR stimulation on sucrose self-administration.
Lastly, and because U50,488 is a reversible KOR agonist, a third PR test was performed 24 h later to assess if the effects of KOR
activation were transient or sustainable.
To determine the involvement of KOR stimulation during a FR schedule of reinforcement, animals were trained to self-administer
sucrose as pellets as mentioned above. After completion of three successive FR5 sessions, rats were deeply anesthetized and guide
cannula were placed bilaterally 1mm above the NAcShCS (A/P: - 0.96mm; M/L: +/– 0.8mm; D/V: - 5.5mm) (Castro and Berridge,
2014). A week after surgery, to allow full recovery, animals were injected in the NAcShCS with KOR agonist (U50,488 – 1 mg per
side) or aCSF using a microinjection pump (rate 0.25 mg per minute) and the injector was left in place for an additional 5 min to allow
compound diffusion. 1 h after U50,488 injection rats were gently placed in the self-administration boxes for new FR5 session, thus
assessing the role of KOR stimulation on a fixed ratio schedule of reinforcement for sucrose self-administration.
For the studies on the necessity of dynorphin containing neurons activity on pain effects on motivated behavior rats were injected
with either HSV-Dyn2.0-hM4Di-IRES-mCherry, HSV-Dyn2.0- mCherry (0.5 mL per side, viral titer: 5 3 108 transducing units per ml,
provided by Rachael Neve, MIT, Boston, Massachusetts) or aCSF in the NAcShCS (A/P: +0.96mm; M/L: +/–0.8mm; D/V: –6.5mm).
Three days after, animals were injected with 150 mL of saline or CFA solution in the hindpaw; and 48 h later, when inflammation was
stable, the rats were injected i.p with CNO (1 mg.kg-1) or saline as a control and placed again in the self-administration chambers to
undergo a second PR session 15 min after i.p. treatment.
For assessment of motivation in mice a PR schedule of reinforcement for sucrose pellet self-administration was also used and per-
formed as described above with slight modifications. Mice operant-conditioning chambers (Med Associates, Fairfax, VT) were equip-
ped with nose poke holes, both presenting a cue light, accessible to animals. An active nose poke resulted in a sucrose delivery in
the food magazine together with house light cue for 20 s (FR1). During this 20 s period, no further action had consequence (time out
period). Poking in the inactive hole had no consequence. Mice were trained to discriminate in between active and inactive lever
during 1 h long FR1 sessions. Once discrimination was acquired (less than 30% of total poking in the inactive hole), mice
underwent 3 consecutive sessions of FR2 and 3 consecutive sessions of FR5. Mice’s motivation was then assessed using a 1 h
session of progressive ratio schedule of reinforcement. To test the necessity of KOR to drive pain-induced negative affect, intra ac-
cumbal (AP: +1.3mm; L: +/– 0.5mm; DV: –4.75mm) injection of 0.5 mL of either aCSF or norBNI (2 mg per side) were then
performed. After surgery recovery, a second progressive ratio testing was performed to assess the consequences of kappa
opioid receptors antagonism on mice’s motivation (PR2). Following this, 50nl of either sterile saline or CFA solution was injected
the mice’ right hindpaw. 48 h later, when inflammation is stable, animals underwent a third progressive ratio session. To test
the sufficiency of dynorphin-containing neurons photostimulation to drive negative affective states, dynorphin-Cre mice received
an injection of AAV cre-dependent channelrhodopsin in the vNAcSh (AP: +1.3mm; L: +/– 0.5mm; DV: –4.75mm). After two weeks
recovery, using the same stereotaxic coordinates, the animals were implanted with fiber implant in the vNAcSh. Training period
was then performed as described above a week after surgery to allow full recovery. Following training the animals were exposed
to a PR session in which fiber implant was connected to a 473nm laser emitting constant photo-stimulation (20Hz, 10ms width) during
the test.
Plantar test for thermal sensitivity – Hargreaves test
The hyperalgesic effects induced by CFA injection in the rat’s hindpaw were examined using the thermal plantar test (Hargreaves
method, IITC Life Science). Animals were placed in Plexiglas boxes on top of a glass surface. After 30 min of habituation, a radiant
heat source was applied on the plantar surface of the right hindpaw, and the latency of paw withdrawal from the radiant heat stimulus
was recorded. Four measurements with at least a 5-min interval between trials were obtained for each session. The intensity of the
light beam was adjusted so that baseline latencies were 15 s in naive rats. A cutoff time of 30 s was imposed to prevent tissue
damage. Paw withdrawal thresholds (4 measurements with 5 min of resting period) were measured for 5 consecutive days during
the four last sucrose self-administration training days. These thermal sensitivity recordings were repeated 30 min before any progres-
sive ratio schedule of reinforcement test to confirm inflammation-induced hyperalgesia.
Real-Time Place Testing (RTPT)
All behaviors were performed within a sound-attenuated room maintained at 23 C at least 1 week after habituation to the holding
room and the final surgery. Lighting was stabilized at 1,500 lux for aversion behaviors, 250 lux for anxiety-like behaviors. Move-
ments were video recorded and analyzed using Ethovision XT 10.0 (Noldus Information Technologies).
For real-time place testing, after recovery from surgery (please refer to the surgeries section for further information on procedure)
mice were gently placed in a custom-made unbiased, balanced two-compartment conditioning apparatus (52.5 3 25.5 3 25.5 cm) as
described previously (Al-Hasani et al., 2015; McCall et al., 2015; Siuda et al., 2015). During a 20-min trial, entry into one compartment

Neuron 102, 1–10.e1–e6, May 8, 2019 e3

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

triggered photo-stimulation (20Hz, 10ms pulse width) while the animal remained in the light-paired chamber and entry into the other
chamber ended photo-stimulation.
Open Field Test
Open field testing was performed in a square enclosure (55 3 55 cm) within a sound attenuated room maintained at 23 C. Lighting
was measured and stabilized at 25 lux. Mice were placed in the center of the open field and allowed to roam freely for 21 min.
Throughout the 21 min mice received constant photo-stimulation (20Hz, 10ms pulse width). The open field was cleaned with 70%
ethanol between each trial. Movements were video recorded and analyzed using Ethovision XT 10.0 (Noldus Information Technol-
ogies, Leesburg, VA). The center was defined as a square comprised of 50% the total area of the open field chamber. Time in the
center was the primary measure of anxiety-like behaviors.

Receptor Function Assessment

Incorporation of GTPgS in membranes
NAc tissue was micro-dissected from saline and CFA-injected rat and tested in GTPyS coupling assay as described previously (Kiss-
ler et al., 2014), with slight modifications. Briefly, tissue was homogenized (40-45 strokes; glass homogenizer with Teflon plunger; on
ice) in 1.5 mL of membrane buffer (pH 7.4, 50.0 mM Tris–HCl, 3.0 mM MgCl2, and 1.0 mM EGTA). Homogenates were centrifuged
(15000rpm, 4 C for 30 min), re-suspended in 1.5 mL membrane buffer, homogenized (12-15 strokes; on ice) again and finally centri-
fuged. Pellet was homogenized (12-15 strokes; on ice) in 1.5 mL assay buffer (pH 7.4, 50.0 mM Tris–HCl, 3.0 mM MgCl2, 0.2 mM
EGTA, 100.0 mM NaCl). Protein estimation was conducted using BCA protein assay (Pierce). Protein (5.0 mg) was homogenized
(12-15 strokes; on ice) and incubated with Dynorphin A (0.1-1.0 uM), at least in duplicate, in assay buffer for 90 min at 25 C, with
10 mM GDP and 0.4 nM [35S] GTPgS in a total volume of 250.0 ml. Unlabeled GTPgS (10.0 mM) was used to assess nonspecific bind-
ing. Specific binding was obtained by subtracting nonspecific binding from total binding. The reaction was quickly terminated by
filtration through UniFilter-96 GF/B filter plates using a cell harvester (Brandel, Gaithersburg, MD), followed by 8-10 washes with
wash buffer (ice-cold phosphate buffer (pH 7.4), 5.0 mM MgCl2). Bound radioactivity on the filters was counted by Microbeta liquid
scintillation counter (PerkinElmer Life Sciences) on the following day. GTPyS coupling data were analyzed using a mixed-model
two-way ANOVA to compare changes in GTPgS signaling for the controls and CFA-treated animals. The within-subject variable
was DYN concentration and the between-groups variable was treatment condition.
Incorporation of GTPgS in slices (autoradiography)
Brains were collected from saline or CFA injected rats 48h post-injection and were snap-frozen with isopentane at –30 C, and stored
in –80 C until further processing. The brains were then coronal-sectioned via cryostat (20 mm thick) at –20 C, and thaw-mounted on
SuperFrost charged slides. Sections were pre-incubated in assay buffer (50mM Tris-HCl, 3mM MgCl2, 0.2mM EGTA, 100 mM NaCl,
2 mM GDP, 1 mM DPCPX, pH = 7.4) for 15 min. Agonist-stimulated KOR activity was determined by incubating brain sections in [35S]
GTPgS (40 pM) with U69,593 (10 mM) +/– JdTic (10 mM) for 1 h at RT. After incubation, slides were washed 2x in ice-cold wash buffer
(50 mm Tris-HCl, pH 7.4) followed by a brief wash in ice-cold deionized water (30 s). Slides were air-dried and exposed to Kodak
Biomax film together with [14C] standards for 2 days. Films were developed using Kodak GBX Developer and RapidFix solutions.
Films were digitally analyzed and quantified using MicroComputer Image Device (MCID) normalized to the [14C] standard curve,
measured in dpm/mg (MCID Imaging Research, St. Catherine, Ontario, Canada). The resulting agonist-stimulated samples were
compared to non-agonist-treated brain samples to determine the percent activation of KOR above basal.
Slices radiography for KOR occupancy
Brains were dissected and frozen for cryostat sectioning. Coronal, 50mm thick sections were thaw-mounted in pairs on poly-L-lysine
microscope slides and allowed to air-dry. The slides were then stored at –80 until used. Slides were removed from the –80 and
allowed to warm to room temperature. Paired sections were traced around using a hydrophobic pen. Each paired section was filled
with 1.0ml of either 50mci/mL of [11C]-LY2795050 and allowed to incubate for the allotted time (10-40 min). Once the incubation time is
complete, the radiopharmaceutical was removed and MilliQ water was replaced in the barrier area for approximately 10 s. The MilliQ
wash was repeated 3 times. Slides were allowed to air dry. The slides were then placed in a cassette and exposed to the phosphor
imaging screen overnight. The screen was then imaged using the typhoon imager.

Electrophysiology
In vitro electrophysiological experiments on brain slices were conducted 48 h after saline or CFA injection in the hindpaw.
To measure dynorphin neurons excitability, male and female Ai14-Dynorphin-Cre mice (5-10 weeks old) were deeply anesthetized
with isoflurane and perfused transcardially with 34 C aCSF (in mM as follows: 0.001 MK-801, 126 NaCl, 2.5 KCl, 1.4 NaH2PO4,
1.2 MgCl2, 2.4CaCl2, 11 glucose, and 25 NaHCO3) before being decapitated. Brains were quickly removed and submerged in
warm MK-801-buffered aCSF. Coronal forebrain sections (230–250 mm) that contained the NAc were cut using a vibratome
(VT1200S, Leica Microsystems) in warm aCSF containing 1 mM MK-801. NAc slices were submerged in aCSF (in mM as follows:
0.01 MK-801 126 NaCl, 2.5 KCl, 1.4 NaH2PO4, 1.2 MgCl2, 2.4CaCl2, 11 glucose, and 25 NaHCO3) at 34 C for 30 m, and equilibrated
with 95% O2 and 5% CO2 before maintenance at room temperature in aCSF without MK-801. Slices were then individually
transferred to the recording chamber (volume 0.8 ml) and superfused continuously (2.2 mL/min) with 34 C aCSF. NAc neurons
were visualized using differential interference contrast optics on an upright microscope (BX50WI, Olympus). Ai14 reporter
animals contain a cre-dependent tdTomato, which was visualized using a 530 nM light shone through the 40X objective.

e4 Neuron 102, 1–10.e1–e6, May 8, 2019

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

Whole-cell current-clamp recordings of NAc neurons were obtained using an Axopatch 200B amplifier (Molecular Devices).
Electrodes (3–4.5 MU) were filled with pipette solution containing the following (in mM): 120 K-gluconate, 5 NaCl, 2 MgCl2, 0.1
CaCl2, 10 HEPES, 1.1 EGTA, 4 Na2ATP, 0.4 Na2GTP, 10 Na2Phosphocreatine, and 0.05% biocytin. The internal solution had
pH 7.3 and osmolality 288 mOsm/l. Membrane voltage and cellular responses to current steps (–50 pA, D5 pA, 200ms) were
recorded in the presence of DNQX (5 mM), strychnine (5 mM), CGP 55845 (1 mM), and APV (50 mM). All recordings were filtered
(2 kHz low-pass filter) and sampled (10 kHz) for online and later offline analysis (Axograph X; Axograph Scientific Software).
To assess sIPSCs, whole-cell voltage clamp experiments on brain slices were conducted 48 h after saline or CFA injection in the
hindpaw. Male and female Ai9-Dynorphin-Cre mice (8-10 weeks old) were deeply anesthetized with a cocktail of ketamine/xylazine/
acepromazine and perfused transcardially with NMDG-substituted aCSF containing (in mM): 93 NMDG, 2.5 KCl, 1.25 NaH2PO4,
30 NaHCO3, 20 HEPES, 25 glucose, 5 ascorbic acid, 2 thiourea, 3 Na-pyruvate, 12 N-acetyl-L-cysteine, 10 MgSO4, 0.5 CaCl2,
pH = 7.3. After decapitation, 300 mm thick coronal sections containing the NAc were cut using a Vibratome VT1000s (Leica) and
transferred to an oxygenated recovery chamber containing NMDG aCSF for 10–12 min at 32–34 C, before being transferred to a
holding chamber filled with aCSF containing (in mM): oxygenated aCSF containing (in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 24
NaHCO3, 5 HEPES, 12.5 glucose, 2 CaCl2, 1 MgCl2; pH = 7.3, 300-315 mOsm and were incubated in the dark at room temperature
for >1 hr before recording. Slices were transferred to a heated (32-34 C) recording chamber in the dark and perfused (2 mL/min)
with oxygenated aCSF containing. Dynorphinergic neurons in the NAc were visualized through a 40x objective using IR-DIC micro-
scopy on an Olympus BX51 microscope, and tdT+ neurons were identified using epifluorescent illumination. Electrodes (3–4.5 MU)
were filled with pipette solution containing the following (in mM): 110 D-gluconic acid, 110 CsOH, 0.1 CaCL2, 4 MG2ATP, 0.4
Na2GTP, 10 Na2Phosphocreatine, 10 HEPES, 1.1 EGTA, 8 TEA+CL-, 3 QX314+Br-, and 0.05% neurobiotin. The internal solution
had pH 7.3 and osmolality 290 mOsm/l. Recordings were made with Patchmaster software controlling a HEKA EPC10 and
EPC10 Double amplifiers. Following gigaseal formation and stable whole-cell access, spontaneous inhibitory postsynaptic
currents were isolated by blocking AMPA/KARs (10 mM NBQX, Abcam) and NMDARs (50 mM D-APV, Abcam). Neurons were voltage
clamped at 0 mV and whole-cell currents were recorded using a gap-free protocol. In a subset of cells, GABAARs were then
blocked using 100 mM picrotoxin (Abcam) and 10 mM biccuculine to confirm as GABAAR IPSCs. Only cells with a stable Rs
(+/– 20%) less than 15 MOhm were included in our analysis. For sIPSC recordings, amplitude, frequency, and the kinetics of currents
were extracted offline using the Mini Analysis software (Synaptosoft).
Ex vivo confirmation of Gi-DREADD functionality was performed using whole cell-patch clamp electrophysiology (recording details
below). Rats were injected with either HSV-Dyn2.0-hM4Di-IRES-mCherry or HSV-Dyn2.0- mCherry in the NAcShCS. Three to five
days later, animals were sacrificed and brains were extracted for current-clamp recordings of NAc neurons. Membrane voltage
and cellular responses to current steps were recorded in the presence of DNQX (5 mm), strychnine (5 mm), CGP 55845 (1 mm), and
APV (50 mm) before and during the bath application of 10 mM CNO. After the determination of baseline rheobase, current steps of
1, 2, 3 or 4 times the rheobase were applied to examine the magnitude of neuronal inhibition by CNO.

Immunohistochemistry
Dynorphin HRP staining
Rats were pretreated with 20 mg colchicine (Sigma-Aldrich) through i.c.v. injection (stereotaxic coordinates: A/P: – 1.5 mm; M/L:
–0.8 mm; D/V: –4.5 mm) under isoflurane (1.5%–2% MAC) anesthesia. Animals were injected with saline or CFA into the plantar
surface of the hindpaw as previously described. 48 h after, rats were trans-cardially perfused with phosphate buffered saline
(PBS) followed by 4% paraformaldehyde (PFA) diluted in PBS. Brains were removed and post-fixed 20h in 4% PFA, then transferred
in 30% sucrose in PBS. After flash freezing, brains were cut in 40 mm coronal slices using a cryostat (Leica CM 1950). Free-floating
sections were then washed in Tris-based saline buffer (TBS) before and after every incubation. First, slices were incubated for
30 min with 1% hydrogen peroxide in TBS to quench peroxidase activity. Then they were incubated with rabbit-anti-dynorphin A
(1:1000, T-4268, Peninsula Laboratories International Inc.) followed by biotinylated anti-Rabbit antibody (1:1000, BioRad) diluted
in TBS - 0.3% Triton X-100 - 5% normal goat serum. Biotinylated anti-rabbit antibodies were revealed using avidin-streptavidin
complex (ABC-Kit, Vector) followed by the reaction with DAB (Sigmafast, Sigma). Slices were then mounted and coversliped with
Eukitt (Microptic). All pictures were taken at 10x with a Leica DMR microscope using constant set up and lighting within the same
day. ImageJ software was used to measure I.O.D. Pictures were first converted into 8-bit (grayscale) and the background (measured
as staining in the anterior commissure) was subtracted. A ROI was selected based on kappa NAcShCS6 to measure I.O.D. in the
inverted picture.
HSV/dynorphin A co-staining
Rats were injected with 20 mg colchicine (Sigma-Aldrich) through i.c.v. injection (stereotaxic coordinates: A/P: – 1.5 mm; M/L:
–0.8 mm; D/V: –4.5 mm) and HSV-dyn2.0-hM4Di-IRES-mCherry in the NAc (stereotaxic coordinates from Bregma: A/p = +
0.96mm, M/L = ± 0.8mm, D/V = – 6.5mm from the skull surface) under isoflurane (1.5%–2% MAC) anesthesia. Animals were trans-
cardially perfused 24hours after injections with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) diluted in
PBS. Brains were removed and post-fixed 20h in 4% PFA, then transferred in 30% sucrose in PBS. After flash freezing, brains
were cut in 40 mm coronal slices using a cryostat (Leica CM 1950). Free-floating sections were then washed in phosphate based saline
buffer (PBS) before and after every incubation. First, slices were incubated endogenous biotin blocking kit (ThermoFisher Scientific,
USA). After PBS washes, slices were bathed with blocking solution composed of normal goat serum (5%) and 0.01% Tween 20 diluted

Neuron 102, 1–10.e1–e6, May 8, 2019 e5

 Please cite this article in press as: Massaly et al., Pain-Induced Negative Affect Is Mediated via Recruitment of The Nucleus Accumbens Kappa Opioid
System, Neuron (2019), https://doi.org/10.1016/j.neuron.2019.02.029

in PBS, before being incubated with both rabbit anti-DynorphinA (1/1000, Peninsula Labs, CA) and mouse anti-mCherry (1/1000,
DHBS, IO) antibodies diluted in blocking buffer overnight at 4C. Slices were then exposed to a goat anti-mouse Alexa 568 (1/500,
ThermoFischer) and biotin conjugated anti-rabbit (1/500, ThermoFischer Scientific) for 2 h at room temperature. Lastly, slices were
incubated with Alexa 488 conjugated streptavidin (10 mg per ml, ThermoFischer Scientific) for 2 h at room temperature to amplify dy-
norphin A signal. Slices were mounted and coverslipped with HardSet DAPI staining and analyzed using a Leica confocal microscope.

Positron Emission Tomography (Pet) Imaging

Animals were transported to the preclinical PET imaging facility 1 h before starting the experiment to allow room habituation. Preclin-
ical PET imaging was performed on either the Siemens Inveon PET/CT or the microPET Focus F220. A 30min dynamic PET image
acquisition followed a bolus injection of [11C]-LY2795050 (1mCi) via the tail-vain. [11C]-LY2795050 is an antagonist of the kappa
opioid receptor (KOR) and has demonstrated invaluable utility in quantifying the density and occupancy of kappa opioid receptors
in numerous applications (Shoghi and Welch, 2007; Zheng et al., 2013). All PET images were reconstructed using the OSEM algo-
rithm. Regions of interest (ROIs) were drawn on the whole brain. In the absence of a reference region in the brain, we used muscle
as a reference region to quantify the distribution volume. To calculate the distribution volume (DV), we performed Logan graphical
analysis41 using whole-brain kinetics of [11C]-LY2795050 with muscle kinetics as a reference region. The linear segment of the
linearized plot of the Logan plot (Logan, 2000) was used to derive the distribution volume of the tracer as a measure of apparent
receptor density/availability.

In Situ Hibridization
Following rapid decapitation of C57/BL6 mice, brains were rapidly frozen in 100 mL –50 C isopentane and stored at –80 C. Coronal
sections containing the VTA and NAc were cut at 20 mM at –20 C and thaw-mounted onto Super Frost Plus slides (Fisher, Waltham,
MA). Slides were stored at –80 C until further processing. Fluorescent in situ hybridization was performed according to the
RNAScope 2.0 Fluorescent Multiple Kit User Manual for Fresh Frozen Tissue (Advanced Cell Diagnostics, Inc.) as described previ-
ously (Wang et al., 2012). Briefly, sections were fixed in 4% PFA, dehydrated, and treated with pretreatment 4 protease solution.
Sections were then incubated for target probes for mouse Oprk1 (Tejeda et al., 2017). Probe consisted of 20 ZZ oligonucleotides
and was obtained from Advanced Cell Diagnostics. Following probe hybridization, sections underwent a series of probe signal
amplification steps followed by incubation of fluorescently labeled probes designed to target the specified channel associated
with Oprk1. Slides were counterstained with DAPI, and coverslips were mounted with Vectashield Hard Set mounting medium
(Vector Laboratories). Images were obtained on a Leica TCS SPE confocal microscope and analyzed with Application Suite
Advanced Fluorescence (Leica).

Histology
Please refer to Figure S4. After perfusion brain were kept in 4% paraformaldehyde at 4 C for 24 h. They were then transferred in 30%
sucrose solution for two to five days (until they sank in the vial) at 4C degrees. Brain were frozen in isopentane (–80 C) for 1minute
and kept in –20 C freezer until sliced using cryostat. Tissue was 40 mm thick and mounted on slides. An experimenter blinded to
treatments assessed guide-cannulae, fiber optic and injections placement. Only animals confirmed to have correct cannula place-
ment were included in the final data and statistical analyses.

QUANTIFICATION AND STATISTICAL ANALYSIS

All experiments were performed at least twice, including all treatment groups in each round, to avoid any unspecific day/condition
effect. Treatments (i.e., intra-NAc, inflammation and subcutaneous) were randomly assigned to animals before testing. Statistical
significance was taken as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined by a one-way ANOVA or a two-
way repeated-measures ANOVA followed by a Sidak post hoc tests, two-tailed unpaired or paired t test, one sample t test compared
to hypothetical value (50%) or Mann-Whitney for unpaired values as appropriate. All data samples were tested for normality of
distribution using Shapiro-Wilk test before being assigned to ANOVAs, or t test analysis. All data are expressed as mean ± SEM.
Sample size (n number) always refers to the value obtained from an individual animal when referring to behavioral experiments (Fig-
ures 1A–1L, Figures 2A–2H, Figures 4A–4E, and Figures S1A–S1I) and anatomical slice analysis (Figures 3A–3F and Figures S2A
and S2B), the total number of mCherry positive neurons (Figure S3B) or an individual cell recording (Figures 3J–3P and Figures
S2C–S2J). For experiments that include samples from model organisms, the sample size (n value) was reported as ncells/animals
(see Figures 2G–2P and Figure S3). For each experiment detailed statistical analysis and sample size (n number) are carefully
reported in figure legends. Statistical analyses were performed in GraphPad Prism 6.0 and SPSS.

DATA AND SOFTWARE AVAILABILITY

Authors can confirm that all relevant data are included in the paper and its supplementary information files.

e6 Neuron 102, 1–10.e1–e6, May 8, 2019

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—¿No tiene usted valimiento con los ministros liberales?
—Ninguno. Mi nombre solo les sonará a proclama realista.
—Entonces....
—Cuento con la protección de los jefes del ejército francés.
—Y con los servicios de un leal amigo... El objeto principal es
detener al ladrón.
—¡Detenerle y amarrarle y arrastrarle! —exclamé con furor—. Pero
deseo hacer mi justicia a espaldas de la curia, porque aborrezco los
pleitos, aun cuando los gane.
—¡Oh!, eso es muy español. Se trata, pues, de cazar a un hombre
¿por ventura eso es fácil todavía?
—Fácil no.
—Y para una dama...
—Pero yo no estoy sola. Tengo servidores leales que solo esperan
una orden mía para...
—Para matar...
—No tanto —dije riendo—. Esto le parecerá a usted leyenda
novela, romance o lo que quiera; pero no, mis propósitos no son tan
trágicos.
—Lo supongo... pero siempre serán interesantes... ¿Ha dejado
usted criados en Sevilla?
—Uno tengo a mis órdenes. Le mandé por delante, y en Cádiz está
ya.
—¿Vigilando...?
—Acechando.
—Bien: le seguirá de noche embozado hasta las cejas, espiará sus
acciones, se informará de su método de vida. ¿Y ese criado es fiel?
—Como un perro... Examinemos bien mi situación, señor conde
¿Se puede entrar en Cádiz?
—Es muy difícil, señora, sobre todo para los que son sospechosos
al gobierno liberal.
—¿Y por mar?
—Ya sabe usted que en la bahía tenemos nuestra escuadra.
—¿Cuándo tomarán ustedes la plaza?
 —Pronto. Esperamos a que venga Su Alteza para forzar el sitio.
—¿Y podrán escaparse los milicianos y el gobierno?
—Es difícil saberlo. Ignorarnos si habrá capitulación; no sabemos e
grado de resistencia que presentarán los insurgentes.
—¡Oh! —exclamé sin saber lo que decía, obcecada por mis
pasiones—. Ustedes los realistas no sirven para esto. Si Napoleón
estuviera aquí, amigo mío, mañana, mañana mismo, sí señor, mañana
sería tomada por asalto esa ciudad rebelde y pasados a cuchillo los
insensatos que la defienden.
—Me parece demasiado pronto —dijo Montguyon sonriendo—. En
fin, comprendo la impaciencia de usted.
—Sí, quien ha sido robada, vilmente estafada, no puede aproba
estas dilaciones que dan fuerza al enemigo. Señor conde, es preciso
entrar en Cádiz.
—Si de mí dependiera, señora, esta tarde mandaba dar el asalto —
repuso con entusiasmo—. Sorprendería a la guarnición, encarcelaría a
los diputados y a las Cortes, y pondría en libertad al rey.
—Ya eso no me importa tanto —dije en tono de conquistador—. Yo
entraría al asalto sorprendiendo la guarnición. Dejaría, a los diputados
que hicieran lo que les acomodase, mandaría al rey a paseo...
—¡Señora!...
—Buscaría a mi hombre, revolvería todos los rincones, todos los
escondrijos de Cádiz hasta encontrarle... y después que le hallara...
—Después...
—Después, señor conde... ¡Oh!, mi sangre se abrasa...
—En los divinos ojos de usted, Jenara —me dijo—, brilla el fuego
de la venganza. Parece usted una Medea.
—No me impulsan los celos —dije serenándome.
—Una Judith.
—Ni la idea política.
—Una...
—Parezca lo que parezca, señor conde, es preciso entrar en Cádiz.
—Entraremos.
—¿No sirve usted ahora en el Estado Mayor del general Bourmont?
—En él estoy a las órdenes de la que es imán de mi vida —repuso
poniendo los ojos en blanco.
—¿Será Bourmont nombrado comandante general de Cádiz, luego
que la plaza se rinda?
 —Así se dice.
—¿Hará usted prender a mi mayordomo?...
—Le haré fusilar...
—¿Me lo entregará atado de pies y manos?
—Siempre que no huya antes, sí, señora.
—¡Huir! Pues qué, ¿tendrá ese hombre la vileza de huir, de no
esperar?...
—El criminal, amiga mía de mi corazón, pone su seguridad ante
todo.
—¿No dice usted que hay una especie de escuadra?
—Una escuadra en toda regla.
—¿Pues de qué sirven esos barcos, señor mío —dije de muy ma
talante—, si permiten que se escape... ese?
—Quizás no se escape.
—¿De qué sirve la escuadra? —añadí con la más viva inquietud—
¿Quién es el almirante que la manda? Yo quiero ver a ese almirante
quiero hablar con él...
—Nada más fácil; pero dudo...
—Me ocurre que si hay capitulación, será más fácil atraparle...
—¿Al almirante?
—No; a... a ese.
—Sin duda. En tal caso se quedaría tranquilo en Cádiz, al menos
por unos días.
—Bien, muy bien. Si hay capitulación, arreglo, perdón de vidas y
libertad para todos... Señor conde, aconsejaremos al príncipe que
capitule... ¡Pero qué tonterías digo!
—Está patente en su espíritu de usted la obsesión de ese asunto.
—¡Oh!, sí. No puedo pensar en otra cosa. El caso es grave. Si no
consigo apoderarme de ese hombre... no sé... creo que me costará la
vida.
—Yo también le aborrezco... ¡Hombre maldito!... Pero le cogeremos
señora. Me pongo al servicio de este gran propósito con la sumisión de
un esclavo. ¿Acepta usted mi cooperación?
Al decir esto, me besaba la mano.
—La acepto, sí, hombre generoso y leal, la acepto con gratitud y
profundo cariño.
Al decir esto, yo ponía en mi semblante una sensibilidad capaz de
conmover a las piedras, y en mis pestañas temblaba una lágrima.
 —Y entonces —añadió Montguyon con voz turbada—, cuando
nuestro triunfo sea seguro, ¿podré esperar que el hueco que se me
destina en ese corazón no sea tan pequeño?
—¿Pequeño?
—Si es evidente, por confesión de él mismo, que ya tengo una
parte en sus sublimes afectos, ¿no puedo esperar...?
—¿Una parte? ¡Oh! no. Todo, todo.
El inflamado galán abrió sus brazos para estrecharme en ellos; pero
evadí prontamente aquella prueba de su insensato ardor, y
poniéndome primero seria y después amable, con una especie de
enojo gracioso y virtud tolerante, le dije que ni Zamora ni yo podíamos
ser ganadas en una hora. Al decir esto, violentos cañonazos me
hicieron estremecer y corrí al balcón.
—Son los primeros tiros de las baterías que se han armado para
atacar el Trocadero —me dijo el conde.
—¿Y esas bombas van a Cádiz?—pregunté poniendo inmenso
interés en aquel asunto.
—Van al Trocadero.
—¿Y qué es eso?
—Un fuerte que está en medio de las marismas.
—¿Y allí están...?
—Los liberales.
—¿Muchos?
— Mil y quinientos hombres.
—¿Paisanos?
—Hay muchos paisanos y milicianos.
—¡Oh!, morirá mucha gente.
—Eso es lo que deseamos. Parece que siente usted gran pena po
ello.
—La verdad —repuse, ocultando los sentimientos que bruscamente
me asaltaban—, no me gusta que muera gente.
—A excepción de su enemigo.
—Ese..., pero ¿estará en el Trocadero?
—¡Quién sabe!... Está usted aterrada.
—¡Oh!, yo quiero ir al Trocadero.
—Señora...
—Quiero ir al Trocadero.
 —Eso mismo deseamos nosotros —me dijo riendo—, y para
conseguirlo enviaremos por delante algunos centenares de bombas.
—¿Dónde está el Trocadero? —pregunté corriendo otra vez a la
ventana.
—Allí —dijo Montguyon asomándose y alargando el brazo.
Hízome explicaciones y descripciones muy prolijas de la bahía y de
los fuertes; pero bien comprendí que antes que mostrar sus
conocimientos deseaba estar cerca de mí, aproximando bastante su
cabeza a la mía, y embriagándose con el calor de mi rostro y con e
roce de mis cabellos.
 XXXIII

¡Qué aparato desplegaron contra aquellas fortalezas que se alzan

entre charcos salubres y que llevan por nombre el Trocadero! Desde
que llegó Su Alteza a mediados de agosto, no hacían más que
disparar bombas y balas contra los fuertes, esperando abrir brecha en
sus gloriosos muros. ¡Figúrese el buen lector mi aburrimiento
Considere con cuánta tristeza y tedio vería yo pasar día tras día sin
más distracción que oír los disparos y ver por las noches las
majestuosas curvas de los proyectiles. Me consumía en mi casa de
Puerto sin tener noticias del interior de Cádiz, ni esperanza de pode
penetrar en la plaza. Ni parecía aquello guerra formal y heroica como
creía yo que debían de ser las guerras, y como las que vi en mi niñez y
en tiempo del Imperio. Casi todo el ejército sitiador estaba con los
brazos cruzados: los oficiales paseaban fumando; los soldados hacían
menos pesado el tiempo con bailoteo y cantos.
No debo pasar en silencio que el duque del Infantado, que llegó de
Madrid en aquellos días, me llevó a visitar a Su Alteza, nuestro
salvador y el ángel tutelar de la moribunda España por aquellos días
Luis Antonio era un rubio desabrido, cuyo semblante respiraba
honradez y buena fe; pero la aureola del genio no circundaba su
frente. Fuera de aquel sitio, lejos de aquella deslumbradora posición y
con otro nombre, el hijo del conde de Artois habría sido un joven de
buen ver; mas no en tal manera que por su aspecto descollase entre la
muchedumbre. Para hallar en él lo que realmente le distinguía era
preciso que un trato frecuente hiciese resaltar las perfecciones
morales de su alma privilegiada, su lealtad sin tacha y aquel levantado
espíritu caballeresco sin quijotismo que le hacía estimable en la corte
de Francia. Era valiente, humanitario, cortés, puntual y riguroso en e
cumplimiento del deber. Si estas cualidades no eran suficientes a
formar un gran guerrero, ¿qué importaba? La pericia militar diéronsela
sus prácticos generales y nuestros desaciertos, que fueron el principa
estro marcial de la segunda invasión.
Recibiome Angulema con la más fina delicadeza y urbanidad; pero
de todas sus cortesanías la que más me agradó fue la de disponer e
asalto del Trocadero. «¡Al fin, al fin —exclamaba yo—, será nuestro e
horrible fuerte que nos abrirá las puertas de Cádiz!»
El 19 abrieron brecha; pero hasta la noche del 30 no se dio e
asalto, habiéndose guardado secreto sobre esto en los días anteriores
aunque yo lo supe por el conde de Montguyon, que no me ocultaba
nada referente a las operaciones. ¡Noche terrible la del 30 al 31 de
agosto! Noche que me pareció día por lo clara y hermosa, así como
por el estrépito guerrero que en ella resonara y las acciones heroicas
dignas de ser alumbradas por el sol... Apretado fue el lance del asalto
según oí contar, y Su Alteza y el príncipe de Carignan se portaron
bravamente, combatiendo como soldados en los sitios más peligrosos
No fue el hecho del Trocadero una de aquellas páginas de epopeya
que ilustraron el Imperio: fue más bien lo que los dramaturgos
franceses llaman succès d’estime, un éxito que no tiene envidiosos
Pero a la Restauración le convenía cacarearlo mucho, ciñendo a la
inofensiva frente del duque los laureles napoleónicos; y se tocó la
trompa sobre este tema hasta reventar, resultando del entusiasmo
oficial que no hubo en Francia calle ni plaza que no llevase el nombre
del Trocadero, y hasta el famoso arco de la Estrella, en cuyas piedras
se habían grabado los nombres de Austerlitz y Wagram, fue durante
algún tiempo Arco del Trocadero.
Yo me había trasladado a Puerto Real para estar más cerca. En la
mañana del 31, cuando vi pasar a los prisioneros hechos en los
fuertes, me sentí morir de zozobra. Entre aquellas caras atezadas a
cada instante creía ver la suya. Largo rato tardaron en pasar, porque
eran más de mil entre paisanos y militares. Creo que los miré uno po
uno; y al fin, cuando ya quedaban pocos, redoblé mi atención. ¡Oh
misericordioso Dios, qué estupendas cosas permites! En la última fila
casi solo, más abatido, más quemado del sol, más demacrado, con los
vestidos más rotos que los demás, pasó él, él mismo... no podía
dudarlo, porque le estaba viendo, viendo, sí, con mis propios ojos
arrasados de lágrimas. Llevaba la mano izquierda en cabestrillo, hecho
con un andrajo, y su paso era inseguro y como dolorido, sin duda po
tener lleno de contusiones el cuerpo. Al verle extendí los brazos y grité
con toda la fuerza de mi voz. Mi enamorada exclamación hizo volver la
cabeza a todos los que iban delante y a los curiosos que le rodeaban
Él, alzando los amortiguados ojos, me miró con expresión tan triste
que sentí partido mi corazón y estuve a punto de desmayarme. Creo
que pronunció algunas palabras; pero no oí sino un adiós tan lúgubre
como campanada funeral, y movió la mano en ademán de cariñoso
saludo, y pasó, desapareciendo con los demás en una vuelta de
camino.
Mi primera intención fue correr tras él: pero en la casa me
detuvieron. Cuando serenamente me hice cargo de la situación, formé
diversos planes; pero todos los desechaba al punto por descabellados
Pensándolo bien, comprendí que no era tan difícil conseguir su
libertad. Me congratulaba de que al cabo de tantas fatigas el destino
me le presentara prisionero, para poder decir con más calor que
nunca: «Ahora sí que no se me puede escapar.»
 XXXIV

Envié recados al conde de Montguyon; pero no se le podía

encontrar por ninguna parte. Unos decían que estaba en el Trocadero
otros que en el Puerto, otros que había ido a las fragatas con una
comisión. Por último, averigüé con certeza su paradero, y le escrib
una carta muy cariñosa. Mas pasó un día, pasaron dos, y yo me moría
de impaciencia, sin poder ver al prisionero, ni aun saber dónde le
habían llevado. El conde, robando al fin un rato a sus quehaceres, vino
a verme el día 4. Yo estaba otra vez medio loca; no tenía humor para
hacer papeles, y espontáneamente dejaba que se desbordasen los
sentimientos de mi corazón.
—¡Oh, cuánto me alegro de ver a usted! —le dije—. Si usted no
viene pronto, señor conde, me hubiera muerto de pena.
Con estas palabras, que creyó dictadas por un vivo interés hacia él
se puso el noble francés un poco chispo, que así denomino yo a
embobamiento de los hombres enamorados. Se deshizo en
galanterías, a las cuales daba cierto tono de intimidad cargante, y
después me dijo:
—Pronto, muy pronto, libertaremos a Su Majestad el rey de España
y entraremos en Cádiz. El sol de ese día, señora, ¡cuán alegremente
brillará sobre toda España, y especialmente sobre nuestros corazones
—Mi estimado amigo —indiqué riendo—, no diga usted tonterías.
Montguyon se quedó cortado.
—Basta de tonterías —añadí— y óigame usted lo que voy a decirle
Ya he encontrado al hombre que buscaba...
—¿Dónde... cómo... ese malvado?
—No es malvado.
—¿Cómo no? Me dijo usted que le había robado sus alhajas.
 —¡No es ese... por Dios! ¿Cuándo entenderá usted las cosas a
derecho?
—Siempre que no se me expliquen al revés.
—He encontrado a ese hombre... Pero entendamos. ¿No dije a
usted que había venido delante de mí un fiel criado de mi casa, el cua
entró en Cádiz?...
—¡Ah! sí... entró para observar los pasos del ladrón.
—Pues ese fiel criado tiene el defecto de ser algo patriota..
¡debilidades humanas! y como es algo patriota, se puso a pelear en e
Trocadero por una causa que no le importaba.
—Ya comprendo: y ha caído prisionero. ¿Le ha visto usted?
—Le vi cuando los prisioneros pasaron por aquí, pero no le he visto
más; y ahora, señor conde, quiero que usted me le ponga en libertad.
—Señora, si Cádiz se rinde pronto, como creo, y todo se arregla
espero conseguir lo que usted me pide.
—¡Qué gracia! Para eso no necesito yo de la amistad de un jefe de
brigada —dije con enfado—. Ha de ser antes, mañana mismo.
—¡Oh! Señora, usted somete mi amor a pruebas demasiado
fuertes.
—¿Quiere usted que dejemos a un lado el amor —le dije
poniéndome muy seria— y que hablemos como amigos?
Montguyon palideció.
—¿Esa persona —me dijo— interesa a usted tanto que no puede
esperar a que concluya la guerra, dando yo mi palabra de que e
prisionero será bien atendido?
—No basta que sea atendido —afirmé con resolución—. No basta
eso: quiero su libertad; quiero atenderle yo misma, cuidarle, curar sus
heridas, tenerle a mi lado, llevarle a sitio seguro...
Me expresé, al decir esto, con vehemencia suma, porque me era ya
muy difícil contener mi corazón, que iba al galope en busca de las
anheladas soluciones. El conde me oía con cierto terror.
—¿Tanto interesa a usted —repitió—, tanto interesa a usted... un
criado?
—No es criado.
—¿Tal vez un anciano servidor de la casa?
—No es anciano.
—¿Un joven?... Supongo que no será el ladrón.
—¿Qué ladrón?
 —El ladrón de quien usted me habló...
—¡Ah! No me acordaba... Ya no me ocupo de eso.
—¿Abandona usted la empresa de detener y castigar a ese
miserable?
—La abandono.
—¡Qué inconstancia!
—Yo soy así.
—Pero ese, ese otro... ¿interesa a usted tanto...?
—Muchísimo.
—¿Es pariente de usted?
—No. Es compañero de la infancia.
—¿Es militar?
—Paisano, señor conde —dije con el tono de severa autoridad que
sé emplear cuando me conviene—. Si se empeña usted en se
catecismo, buscaré otra persona más galante y más generosa que
sepa prestar un servicio, economizando las preguntas.
—Creo tener algún derecho a ello —repuso con gravedad.
—No tiene usted ninguno —afirmé con desenfado—, porque este
derecho yo sola podría darlo, y yo lo niego.
—Entonces, señora —objetó, encubriendo su ira bajo formas
urbanas—, he padecido una equivocación.
—Si cree usted que le amo, sí. La equivocación no puede ser más
completa.
Montguyon se levantó. Sus ojos, en los cuales se leía el furo
mezclado con la dignidad, me dirigieron una mirada que debía ser la
última. Yo corrí a él, y tomándole la mano le rogué que se sentase a m
lado.
—Es usted un caballero —le dije—. Ningún otro ha merecido más
que usted mi estimación, lo juro. Dios sabe que al decir esto hablo con
el corazón.
— Dios lo sabrá —repuso Montguyon muy afligido—; mas para mí
y de aquí en adelante, las palabras de usted están escritas en el agua.
—Considere las que le diga hoy como si estuvieran grabadas en
bronce. La que confiesa hechos que no le favorecen, ¿no tiene
derecho a ser creída?
—A veces sí. Confiéseme usted que su conducta conmigo no ha
sido leal.
 —Lo confieso —repliqué bajando los ojos, y realmente
avergonzada.
—Confiese usted que yo no merecía servir de juguete a una muje
voluntariosa.
—También es cierto.
—Declare usted que ama a otro.
—¡Oh!, sí, lo declaro con todo mi corazón, y si cien bocas tuviera
con todas lo diría.
El leal caballero se quedó atónito y espantado. Estaba, como ellos
dicen, foudroyé. Durante breve rato no me dijo nada; pero yo
comprendí su martirio y le tenía lástima. ¡Oh, qué mala he sido
siempre!
—Ese hombre... —murmuró Montguyon—, ese hombre...
—Ahora, reconociéndome culpable, reconociéndome inferior a
usted —dije—, le autorizo para que me abrume a preguntas, si gusta
y aun para que me eche en cara mi ligereza.
—Ese hombre... —prosiguió el francés—. Perdone usted; pero nada
es más curioso que la desgracia. El amor desairado quiere tener miles
de ojos para sondear las causas de su desdicha. Ese hombre... ¿quién
es?
—Un hombre.
—¿De familia ilustre?
—No, señor: de origen muy humilde.
—¿Le ama usted hace tiempo?
—Hace mucho tiempo.
—Él... ¿la ama a usted?
—No estoy muy segura de ello.
—¡Oh! ¡Qué iniquidad! Es un miserable.
—Un ingrato, y es bastante.
—¿Y a pesar de su ingratitud le ama usted?
—Tengo esa debilidad, que no puedo dominar.
—Aborrézcale usted.
—Si fuera fácil... Difícil cosa es esa.
—¡Es verdad, difícil cosa! —exclamó Montguyon con tristeza—. ¿Y
ese hombre...?
—¿Pero hay más preguntas todavía?
—No, ya no más. Me basta lo que sé, y me retiro.
 —Se conduce usted como un cualquiera —le dije afectuosa
deteniéndole—. Me abandona, precisamente cuando mi sinceridad
merece alguna recompensa. ¿Será posible que cuando yo empiezo a
tener franqueza, deje usted de tener generosidad?
—¡Oh! señora, toca usted una fibra de mi corazón que siempre
responde, aun cuando la hieran con un puñal.
—Sí, sí, amigo mío. Es usted generoso y noble en gran manera
Para que la diferencia entre los dos sea siempre grande, para que
usted sea siempre un caballero y yo una miserable, págueme usted
como pagan en todas ocasiones las almas elevadas. Pues yo me he
portado mal, pórtese usted bien conmigo. Haga cada cual su papel
Cumpla usted el precepto que manda volver bien por mal. Así crecerá
más a mis ojos; así me abatiré yo más a los suyos; así su generosidad
será mayor y mi culpa también, y usted tendrá en su vida una página
más gloriosa que la victoria que acaba de alcanzar frente al enemigo.
—Comprendo lo que usted me dice —murmuró el francés
descansando por breve rato su frente en la palma de la mano—. Yo
seré siempre digno de mi nombre.
—¡Caballero leal antes, ahora y siempre!
—Bien, señora —dijo levantándose y alargándome la mano, que
estreché cordialmente—. Lo que usted desea de mí es bastante claro.
—Sí.
—Y yo —añadió con manifiesta emoción— empeño mi palabra de
honor...
—¡Oh! Lo esperaba, lo esperaba.
—Bajo mi palabra de honor, haré cuanto esté en mi mano para
devolver a usted la felicidad, entregándole a su amante.
—Gracias, gracias —exclamé derramando lágrimas de admiración y
agradecimiento.
Saludándome ceremoniosamente, el conde se retiró. De buena
gana le habría dado un abrazo.
 XXXV

¡Cuántos días pasaron! Yo contaba las horas, los minutos, como s

de la duración de ellos dependiese mi vida. Entre españoles y
franceses era opinión corriente que la guerra acabaría pronto, que
Cádiz expiraba, que las Cortes se morían por momentos. Sin embargo
aún resistía el gobierno liberal y sus secuaces, como la bestia herida
que no quiere soltar su presa mientras tenga un hálito de existencia
Esta constancia no carecía de mérito, y lo tendría mayor si se
empleara en causa menos perdida. ¡Inútil sacrificio! No tenían
hombres, porque los alistamientos no producían efecto. No tenían
dinero, porque el empréstito que levantaron en Londres produjo... una
libra esterlina. Yo creo que si mi espíritu hubiera estado en disposición
de admirar algo, habría admirado la perseverancia de aquel gobierno
que no pudo encontrar en toda Europa quien le prestase más de cinco
duros.
Mi deseo era que se rindiese todo el mundo, que el rey y la nación
arreglasen pronto sus diferencias, aunque las arreglaran devorándose
mutuamente. Yo quería tener el campo libre para el desenlace de m
campaña amorosa, que veía ya seguro y feliz.
Casi todo septiembre lo pasaron Angulema y las Cortes en dimes y
diretes. Mil recados atravesaban la bahía en un bote; callaban los
cañones para que hablaran los parlamentarios. Tales comedias me
ponían furiosa, porque no se decidía la suerte de los infelices
prisioneros del Trocadero, que habían sido repartidos entre los
Dominicos del Puerto y la Cartuja de Jerez.
Montguyon me visitó el 12 para informarme de que había visto a
prisionero, cuyo nombre y señas le había dado yo oportunamente.
—Está sumamente abatido y melancólico —me dijo—. Se ha
negado a recibir los auxilios pecuniarios que le ofrecí de parte de
usted; pero se ha mostrado muy agradecido. Al oír que Jenara tenía
gran empeño en conseguir su libertad, pareció muy turbado
pronunciando palabras sueltas cuyo sentido no pude comprender.
—¿Y no desea verme?
—Parece que lo desea ardientemente.
—¡Oh! ¡Estas dilaciones son horribles! ¿Y qué más dijo?
—Cosas tristes y peregrinas. Afirma que desea la libertad para
conseguir por ella el destierro.
—¡El destierro!
—Dice que aborrece a su país, y que la idea de emigración le
consuela.
—Le conozco, sí... Esa idea es suya.
Otras cosas me dijo el conde; pero se referían al trato que se daba
a los prisioneros y a las excepciones ventajosas que él estableciera en
beneficio de mi amado. ¡Cuánto le agradecí sus delicadezas! Mientras
viva tendré buenos recuerdos de hombre tan caballeroso y
humanitario.
Interrumpidos los tratos por la terquedad de las Cortes, tomó de
nuevo la palabra el cañón, y el día 20 fue ganado por los franceses
con otro brioso asalto, el castillo de Sancti-Petri. Después de este
hecho de armas Angulema habló fuerte a los tenaces liberales
pegados como lapas a la roca constitucional, y les amenazó con pasa
a cuchillo a toda la guarnición de Cádiz si Fernando VII no era puerto
inmediatamente en libertad. El 26 se sublevó contra la Constitución e
batallón de San Marcial, que guarnecía la batería de Urrutia en la
costa; y la armada francesa, secundando el fuego de las baterías de
Trocadero, arrojaba bombas sobre Cádiz. No era posible mayo
resistencia. Era una tenacidad que empezaba a confundirse con e
heroísmo, y la Constitución moría como había nacido, entre espantosa
lluvia de balas, saludada en su triste ocaso, como en su dramático
oriente, por las salvas del ejército francés.
Por fin llegaba el anhelado día.
—Habrá perdón general —decía yo para mí—. Todos los
prisioneros serán puestos en libertad. Huiremos. ¡Cuán grato es e
destierro! Comeremos los dos el dulce pan de la emigración, lejos de
indiscretas miradas, libres y felices fuera de esta loca patria
perturbada, donde ni aun los corazones pueden latir en paz.
Montguyon me trajo el 29 malas noticias.
 —El duque ha resuelto poner en libertad a todos los prisioneros de
guerra. Pero...
—¿Pero qué?
—Ha dispuesto que sean entregados a las autoridades españolas
los individuos que en Cádiz desempeñaban comisiones políticas.
—¿Él está comprendido?
—Sí, señora. Desgraciadamente, se tienen de él las peores
noticias. Había recorrido los pueblos alistando gente por orden de
Calatrava; había venido desde Cataluña con órdenes de Mina para
realizar asesinatos de franceses. Había organizado las partidas de
gente soez que en el tránsito de Sevilla a Cádiz insultaron a Su
Majestad.
—¡Oh, eso es falso, falso, mil veces falso! —grité sin pode
contener mi indignación.
Y en efecto, tales suposiciones eran infames calumnias.
—Ha llegado al Puerto de Santa María —añadió Montguyon— e
señor don Víctor Sáez, Secretario de Estado. ¿Por qué no le ve usted?
—No quiero nada con hombres de ese jaez —repuse con enojo—
Usted me ha dado su palabra de honor; usted ha empeñado su
nombre de caballero, y con usted solo debo contar. ¡Oh, señor conde!
si mi prisionero es entregado a la brutalidad de las autoridades
españolas, sedientas hoy de sangre y de venganza, sospecharé que
usted me hace traición.
Palideció el caballero francés. Dirigiéndome una mirada desdeñosa
me dijo al despedirse:
—Todavía, señora, no sabe usted quién soy yo.
A pesar de mis propósitos, determiné visitar a Sáez, porque bueno
es tener amigos aunque sea en el infierno. Vencí mis recientes
antipatías, y tomando un coche me encaminé al Puerto de Santa
María. Era el 1.º de octubre, día solemne en los fastos españoles.
Hallé al buen canónigo más soplado y presuntuoso que nunca
como todo aquel que se ve en altura a donde nunca debió llegar; pero
contra lo que yo esperaba, recibiome afablemente, y no me dijo una
sola palabra acerca de mi conversión al absolutismo. Parecía no da
valor a estas pequeñeces, y ocuparse tan solo, como Jiménez de
Cisneros, en los negocios públicos de ambos mundos.
—Hoy es día placentero, señora, día feliz entre todos los días
felices de la tierra —me dijo—. Su Majestad don Fernando, ese ilustre
mártir de los excesos revolucionarios, es ya libre.
—¿Ya?
—Hoy nos le entregan. Al fin han comprendido esos locos que su
resistencia les podría costar muy cara, pero muy cara. El duque tiene
malas moscas.
—Felicitémonos, señor don Víctor —dije con afectado entusiasmo
—, de esta solución lisonjera. España y el mundo están de
enhorabuena. Mas para que se completara la dicha, convendría que
tantas y tan graves heridas no se ensañasen con la venganza y la
crueldad del partido vencedor, y que un generoso olvido de los errores
pasados inaugurase la venturosa era que empieza hoy.
—Así será, señora —repuso sonriendo de un modo que me pareció
algo hipócrita—. Su Majestad ha dado ayer en Cádiz un manifiesto en
que ofrece perdonar a todo el mundo y no acordarse para nada de los
que le han ofendido. ¡Cuánta magnanimidad! ¡Cuánta nobleza!
—¡Oh, sí, conducta digna de un descendiente de cien reyes, digna
de quien da el perdón y del pueblo que la recibe! Si Fernando cumple
lo que promete, será grande entre todos los reyes de España.
—Lo cumplirá, señora, lo cumplirá.
Aunque no tenía gran confianza en las afirmaciones de Sáez, d
crédito a estos propósitos por creerlos inspiración del duque de
Angulema.
Invitome luego a presenciar el desembarco de Su Majestad, a lo
que accedí muy gustosa. Nos trasladamos al muelle, y habiendo sido
colocada por un oficial francés en sitio muy conveniente para ver todo
presencié aquel acto, que debía ser uno de los más notables recodos
uno de los más bruscos ángulos de la historia de España en e
tortuoso siglo presente.
¡Espectáculo conmovedor! La regia falúa, cuyo timón gobernaba e
almirante Valdés, glorioso marino de Trafalgar, se acercaba al muelle
En ella venía toda la familia real, la monarquía histórica secuestrada
por el liberalismo. La conciliación ideada por cabezas insensatas era
imposible, y aquellos regios rehenes que la nación había tomado, eran
devueltos al absolutismo, contra el cual no podían prevalecer aún los
infiernos de la demagogia. En una lancha volvían del purgatorio
constitucional las ánimas angustiadas del rey y los príncipes.
Mientras el victorioso despotismo recobraba sus personas
sagradas, allá lejos, sobre la gloriosa peña inundada de luz y ceñida
por coronas de blancas olas, los pobres pensadores desesperados, los
utopistas sin ilusiones, los desengañados patricios lloraban sus
errores, y buscando hospitalidad en naves extranjeras, se disponían a
huir para siempre de la patria a quien no habían podido convencer.
Así acaban los esfuerzos superiores a la energía humana, las
luchas imposibles con monstruos potentes de terribles lazos, y que
hunden en el suelo sus patas para estar más seguros, como hunde
sus raíces el árbol. Tal era la contienda con el absolutismo. Querían
vencerle cortándole las ramas, y él retoñaba con más fuerza. Querían
ahogarle, y regándole daban jugo a sus raíces. ¡A vosotros, oh
venideros días del siglo, tocaba atacarlo en lo hondo, arrancándolo de
cuajo!... Pero advierto que estoy hablando la jerga liberal. ¡Qué horror
Verdad es que escribo veinte años después de aquellos sucesos; que
ya soy vieja, y que a los viejos, como a los sabios, se les permite
mudar de parecer.
Fernando puso el pie en tierra. Dicen que al verse en suelo firme
dirigió a Valdés una mirada terrible, una mirada que era un programa
político: el programa de la venganza. Yo no lo vi, pero debió de se
cierto, porque me lo dijo quien estaba muy cerca. Lo que sí puedo
asegurar es que Angulema, hincando en tierra la rodilla, besó la mano
al rey; que luego se abrazaron todos; que don Víctor Sáez lloraba
como un simple, y que los vivas y las exclamaciones de entusiasmo
me volvieron loca. Los franceses gritaban, los españoles gritaban
también, celebrando la feliz resurrección de la monarquía tradicional y
la miserable muerte del impío constitucionalismo. El glorioso imperio
de las caenas había empezado. Ya se podía decir con toda el alma
«¡Viva el rey absoluto! ¡Muera la nación!»
 XXXVI

Faltaba la solución mía. Mi corazón estaba como el reo cuya

sentencia no se ha escrito aún. El 1.º de octubre por la tarde y el día 2
hice diligencias sin fruto, no siéndome posible ver a Sáez ni a
Montguyon, a quien envié frecuentes y apremiantes recados. Ninguna
noticia pude adquirir tampoco de los prisioneros. Creo que me hubiera
repetido el ataque cerebral que padecí en Sevilla, si en el momento de
mi mayor desesperación no apareciese mi generoso galán francés a
devolverme la vida. Estaba pálido y parecía muy agitado.
—Vengo de Cádiz —me dijo—. Dispénseme usted si no he podido
servirla más pronto.
—¿Y qué hay? —pregunté con la vida toda en suspenso.
—Deme usted su mano —dijo Montguyon ceremoniosamente.
Se la di y la besó con amor.
—Ahora, señora, todo ha acabado entre nosotros. Mi deber está
cumplido, y mi deber es perdonar, pagando las ofensas con beneficios.
Yo me sentía muy conmovida y no pude decirle nada.
—Ni un momento he dudado de su hidalguía —indiqué con acento
de pura verdad—. A veces tropezamos en la vida con el bien y
pasamos sin verlo. Señor conde, mi gratitud será eterna.
—No quiero gratitud —díjome con honda tristeza—. Es un
sentimiento que no me gusta recibido, sino dado. Deseo tan solo un
recuerdo bueno y constante.
—¡Y una amistad entrañable, una estimación profunda! —exclamé
derramando lágrimas.
—Todo está hecho.
—¿Conforme a mi deseo...? ¡Bendito sea el momento en que nos
conocimos!
—Señora, su prisionero de usted está sano y salvo a bordo de la
corbeta Tisbe, que parte esta tarde para Gibraltar.
 —¿Y cómo?
—Por sus antecedentes debía ser condenado a muerte. Otros
menos criminales subirán al cadalso, si no se escapan a tiempo. Yo le
saqué anoche furtivamente de los Dominicos y le embarqué esta
mañana. Ya no corre peligro alguno. Está bajo la salvaguardia de
noble pabellón inglés.
—¡Oh, gracias, gracias!
—Además del servicio que a usted presto, creo cumplir un deber de
conciencia arrancando una víctima a los feroces ministros del rey de
España.
—¿Pues qué —pregunté con asombro—, Su Majestad no ha
ofrecido en su manifiesto de Cádiz perdonar a todo el mundo?
—¡Palabras de rey prisionero! Las palabras del déspota libre son las
que rigen ahora. Su Majestad ha promulgado otro decreto que es la
negra bandera de las proscripciones, un programa de sangre y
exterminio. Innumerables personas han sido condenadas a muerte.
—Esto es una infamia... Pero, en fin, ¿está él en salvo?...
—En salvo.
—¿Y sabe que me lo debe a mí..., sabe que yo...? ¡Oh, seño
conde!, no extrañe usted mi egoísmo. Estoy loca de alegría, y puedo
repetir con toda mi alma: «Ahora sí que no se me puede escapar.»
—Sabe que a usted lo debe todo, y espera abrazarla pronto.
—¿Cómo?
—Muy fácilmente. Comprendiendo que usted desea ir en su
compañía, he pedido otro pasaporte para doña Jenara de Baraona.
—¿De modo que yo...?
—Puede embarcarse usted esta tarde antes de las cuatro a bordo
de la Tisbe.
—¿Es verdad lo que oigo?
—Aquí está la orden firmada por el almirante inglés. Me la ha dado
con las que ponen en salvo a los exregentes Císcar y Valdés
impíamente condenados a muerte por el rey.
—¡Oh..., soy feliz, y todo lo debo a usted!... ¡Qué admirable
conducta!
Sin poder contenerme, caí de rodillas, y con mis lágrimas bañé las
generosas manos de aquel hombre.
—Así castigo yo —me dijo, levantándome—. Prepárese usted. A las
tres y media vengo a buscarla para conducirla a bordo del bote francés