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\begin{document}

\title{Reduced cue-induced reinstatement of cocaine-seeking behavior in
Plcb1+/- mice}



\author[1]{Judit Cabana-Dominguez}%
\author[2]{Elena Martin-Garcia}%
\author[3]{Ana Gallego-Roman}%
\author[3]{Rafael Maldonado}%
\author[1]{Noèlia Fernàndez-Castillo}%
\author[1]{Bru Cormand}%
\affil[1]{Universitat de Barcelona}%
\affil[2]{Universitat Pompeu Fabra}%
\affil[3]{Laboratori de Neurofarmacologia. Universitat Pompeu Fabra}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and Purpose: Cocaine addiction causes serious health problems
and no effective treatment is available yet. We previously identified a
genetic risk variant for cocaine addiction in the PLCB1 gene and found
this gene upregulated in postmortem brains of cocaine abusers and in
human dopaminergic neuron-like cells after an acute cocaine exposure.
Here, we functionally tested the contribution of PLCB1 gene to cocaine
addictive properties in mice. Experimental approach: We used
heterozygous Plcb1 knockout mice (Plcb1+/-) and characterized their
behavioral phenotype. Subsequently, mice were trained for operant
conditioning and self-administered cocaine for 10 days. Plcb1+/- mice
were assessed for cocaine motivation, followed by 26 days of extinction
and finally evaluated for cue-induced reinstatement of cocaine seeking.
Gene expression alterations after reinstatement were assessed in medial
prefrontal cortex (mPFC) and hippocampus (HPC) by RNAseq. Key Results:
Plcb1+/- mice showed normal behavior, although they had increased
anxiety and impaired short-term memory. Importantly, after cocaine
self-administration and extinction, we found a reduction in the
cue-induced reinstatement of cocaine-seeking behavior in Plcb1+/- mice.
After reinstatement, we identified transcriptomic alterations in the
medial prefrontal cortex of Plcb1+/- mice, mostly related to pathways
relevant to addiction like the dopaminergic synapse and long-term
potentiation. Conclusions and Implications: To conclude, we found that
heterozygous deletion of the Plcb1 gene decreases cue-induced
reinstatement of cocaine seeking, pointing at PLCB1 as a possible
therapeutic target for preventing relapse and treating cocaine
addiction.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Cocaine is the most used psychostimulant illicit drug worldwide (UNODC,
2019), causing severe health problems that include the development of
cocaine addiction in around 15-16\% of cocaine users (O'Brien and
Anthony, 2005). Cocaine addiction is a complex psychiatric disorder that
results from the interaction of genetic, epigenetic, and environmental
risk factors (Pierce et al., 2018). The heritability of cocaine
addiction is one of the highest among psychiatric disorders, estimated
around 65\% for women (Kendler et al., 2000) and 79\% for men (Kendler
and Prescott, 1998). However, the genetic factors and mechanisms that
underlie the transition from drug use to addiction and its establishment
remain unknown.

In a previous study, we identified a single nucleotide polymorphism in
the \emph{PLCB1} gene associated with drug dependence (rs1047383), and
especially with a subgroup of cocaine-addicted patients, that was
replicated in an independent clinical sample (Cabana-Dom\selectlanguage{ngerman}ínguez et al.,
2017). Also, genetic variants in this gene were found nominally
associated with an illegal substance and cocaine addiction in two GWAS
performed in European-American samples (Drgon et al., 2012). On the
other hand, we found that cocaine increased the expression
of\emph{PLCB1} both in human dopaminergic neuron-like cells
(differentiated SH-SY5Y cells) after acute cocaine exposure and in
postmortem samples of the nucleus accumbens (NAc) of cocaine abusers
(Cabana-Domínguez et al., 2017). Interestingly, this gene was also found
over-expressed in the same brain region in mice after cocaine
administration for 7 days and also during withdrawal (Eipper-Mains et
al., 2013). All this evidence suggest that \emph{PLCB1} may play a role
in cocaine addiction.

The \emph{PLCB1} gene encodes phospholipase C beta 1, and it is highly
expressed in the brain, mainly in the frontal cortex, basal ganglia
(caudate, putamen, and NAc) and hippocampus. These brain regions are
crucial for drug reward and the formation of drug-context associations,
both contributing to the development and maintenance of addiction
(Bisagno et al., 2016; Castilla-Ortega et al., 2016, 2017; Kutlu and
Gould, 2016; Pitts et al., 2016; Cooper et al., 2017). Several
neurotransmitters activate this protein, including dopamine through DRD1
and DRD2 (Lee et al., 2004; Rashid et al., 2007), serotonin by 5-HT2A
and 2C receptors (Hagberg et al., 1998; Chang et al., 2000) and
glutamate by mGluR1 (Conn and Pin, 1997; Hannan et al., 2001). Thus,
PLCB1 might be a point of convergence of neurotransmitter systems that
play an essential role in the development of addiction (recently
reviewed (Howell and Negus, 2014; Moretti et al., 2020)). The activation
of PLCB1 produces the cleavage of phosphatidylinositol 4,5-bisphosphate
(PIP2) into the second messengers diacylglycerol (DAG) and inositol
1,4,5-trisphosphate (IP3), responsible for intracellular signal
transduction. Alterations in PLCB1-mediated signaling in the brain have
been associated with other neuropsychiatric disorders such as epilepsy,
schizophrenia, and bipolar disorder (Yang et al., 2016).

Here we studied the contribution of the \emph{PLCB1} gene to cocaine
addiction and dissected its participation in the different aspects of
the addictive process. We used heterozygous knockout (KO) mice
(\emph{Plcb1+/-} ), as the homozygous KO (\emph{Plcb1} -/-) showed
seizure attacks and low viability after birth (Kim et al., 1997). In
addition, the use of a constitutive heterozygous KO mouse model allowed
us to assess \emph{Plcb1} haploinsufficiency during neurodevelopment in
mice similarly to humans, where inherited genetic risk or protective
variants can modulate the susceptibility to addiction (Cabana-Domínguez
et al., 2017). First, we performed a general phenotypic characterization
using a battery of behavioral tests, including memory, anxiety,
locomotor activity, coordination, food and water intake, and sucrose
preference. Then, we evaluated cocaine operant self-administration,
extinction, and cue-induced reinstatement. Finally, we studied
transcriptional alterations to further understand the molecular
mechanisms involved.

\textbf{RESULTS}

In the present work, we aimed to assess the contribution of
the\emph{PLCB1} gene in the different stages of cocaine addiction using
heterozygous knockout mice (\emph{Plcb1+/-} ). To do so, we performed a
general phenotypic characterization and, then we evaluated cocaine
operant self-administration, extinction, and cue-induced reinstatement
in those animals. Also, we studied transcriptional changes in mPFC and
HPC to understand the mechanisms involved.

\textbf{Behavioral tests for phenotype characterization}

We first evaluated the effects of the heterozygous deletion
of\emph{Plcb1} on general behavioral responses, including locomotor,
cognitive and emotional responses, food, and water
intake.\emph{Plcb1+/-} mice showed a lower discrimination index in the
novel object recognition compared to WT (\emph{t} -test=3.21,\emph{P}
\textless{}0.01, Figure 1B), suggesting an impairment in short-term
memory. This difference was not influenced by exploration time, as this
variable was equal between genotypes (Figure 1C). Furthermore, no
differences in locomotor activity were reported between genotypes,
discarding an involvement of the \emph{Plcb1} heterozygous deletion in
locomotion (Figure 1D). Mutant mice showed increased exploratory
activity with a higher number of rearings than the WT mice (\emph{U}
-Mann Whitney=67.5, \emph{P} \textless{}0.01, Figure 1E). An anxiogenic
profile was revealed in the elevated plus maze in mutants, as shown by
the reduced time spent in the open arms (\emph{t} -test=3.02,\emph{P}
\textless{}0.001, Figure 1F) and the percentage of time (\emph{t}
-test=3.16, \emph{P} \textless{}0.001, Figure 1G). Finally, the rota rod
test revealed that the heterozygous deletion of \emph{Plcb1}did not
affect motor coordination (Figure 1H-I).

Several consummatory and locomotor parameters were long-term monitored
in the PheComp boxes (Supplementary Figure 1A-H, n=6 per genotype). No
differences between genotypes were revealed during the whole
experimental period in body weight, food, and water intake, levels of
sucrose preference, stereotyped movements, horizontal and vertical
locomotor activity, indicating no altered behavior in the\emph{Plcb1+/-}
mice.

\textbf{Operant conditioning maintained by cocaine}

Then, we investigated the effects of the heterozygous deletion
of\emph{Plcb1} in cocaine behavioral responses related to its addictive
properties. For this purpose, \emph{Plcb1+/-} mice and their WT
littermates were trained for cocaine operant self-administration
(0.5mg/kg/infusion) during FR1 and FR3, progressive ratio, extinction
and cue-induced reinstatement (Figure 2A). Control mice trained with
saline were included for both genotypes. Results showed that the
percentage of mice reaching the criteria of operant conditioning was
100\% for both genotypes trained with cocaine and 33\% for mice trained
with saline: {[}\emph{Chi-square} test=9.50; \emph{P}
\textless{}0.01,\emph{Plcb1+/-} cocaine vs. \emph{Plcb1+/-} saline{]}
and {[}\emph{Chi-square} test=14.00; \emph{P} \textless{}0.001, WT
cocaine vs. WT saline{]}, as expected.

The primary reinforcing effects and the motivation for cocaine were
similar in both genotypes (Figure 2B-C). During FR1, both genotypes
similarly increased the number of cocaine infusions across sessions,
whereas mice trained with saline remained steady (repeated measures
ANOVA, interaction between genotype x drug x sessions,\emph{P}
\textless{}0.05, Figure 2B and Supplementary Table S1). Thus, the
evolution of operant responding was different in mice trained with
cocaine and saline, with an increased responding over nearly all
sessions in cocaine-trained mice . This enhancement in cocaine
responding was more pronounced in mutants than WT mice. Indeed, WT mice
showed a decrease in cocaine intake in the second session. The maximum
number of infusions was reached on session 5 in both genotypes and was
slightly superior in \emph{Plcb1+/-} mutants (36.35±1.47) than in WT
(29.61±2.23) but post hoc analyses demonstrated that this difference in
cocaine intake was not significant (Figure 2B). Similar findings were
observed for the total number of nose-pokes in which operant responding
increased in both genotypes with cocaine but remained stable with saline
(Supplementary Figure S2A). When the effort to obtain one dose of
cocaine increased to FR3, the number of infusions was stable across
sessions in WT and \emph{Plcb1} +/- mice. Similarly, operant responding
was higher for all groups than in FR1 with stable higher levels of
responding (Supplementary Figure S2A) and a higher number of infusions
(repeated measures ANOVA, the main effect of the drug,\emph{P}
\textless{}0.001, Figure 2B) in mice trained with cocaine than with
saline, independently of the genotype.

Motivation for cocaine was evaluated in a progressive ratio schedule,
and no significant differences were obtained between genotypes (Figure
2C). The levels of extinction of the operant behavior were similar
between genotypes and decreased progressively across sessions (repeated
measures ANOVA, interaction genotype x sessions,\emph{P}
\textless{}0.001, Figure 2D). The percentage of mice reaching
cocaine-seeking extinction criteria was similar in \emph{Plcb1+/-}
(73\%) and WT (78\%) mice.

Importantly, \emph{Plcb1+/-} mice showed significantly reduced
cue-induced reinstatement of cocaine-seeking compared to WT mice
(\emph{U} Mann-Whitney, \emph{P} \textless{}0.05, Figure 2E), with
27.59\% less active nose-pokes compared to WT mice trained with cocaine.
Furthermore, mice trained with saline from both genotypes exhibited
55.84\% reduction of active nose-pokes than WT mice trained with cocaine
and 39.20\% less than mutants trained with cocaine. No significant
differences were obtained between genotypes in inactive nose-pokes
during operant conditioning maintained by cocaine nor during extinction
(Supplementary Figure S2B-C). Both genotypes trained with cocaine
acquired the reinstatement criterion (double nose pokes in the active
hole than the number of nose pokes during the 3 consecutive days when
the mice acquired the extinction criteria) showing their capability to
maintain this conditioning learning task. These data showed
that\emph{Plcb1+/-} resulted in a phenotype of resistance to cue-induced
reinstatement with reduced cocaine-seeking (Figure 2E).

\textbf{Brain transcriptomic analysis after the reinstatement of
cocaine-seeking behavior}

To further understand the role of PLCB1 in the molecular mechanisms
involved in this cocaine relapse-related phenotype, we analyzed the
transcriptomic profiles of mPFC and HPC immediately after the
reinstatement of cocaine-seeking behavior in WT and \emph{Plcb1} +/-
mice trained with cocaine. We identified 2,115 protein-coding genes
differentially expressed (DEGs) in mPFC (1,231 downregulated and 943
upregulated) and only 12 in HPC, when comparing \emph{Plcb1+/-} and WT
animals (Supplementary Tables S2-3). In accordance, PCA analysis and
heatmap plots revealed that individuals with the same genotype plotted
together only in mPFC, but not in HPC (Supplementary Figure S3). This
suggested a more prominent role of mPFC, so further studies were carried
out only with DEGs of this brain area. Analysis of functional group
over-representation identified several processes previously related to
cocaine addiction, including dopaminergic synapse, learning, long-term
potentiation (LTP), neurotransmitter secretion, and axon guidance, as
well as other relevant signaling pathways such as MAPK, mTOR, and
neurotrophin (Figure 3A-B and Supplementary Tables S3-5). We focused on
the dopaminergic synapse pathway (enriched in the mPFCs DEGs;\emph{P}
\textsubscript{Adj}\textless{}0.05), in which phospholipase c (such as
Plcb1) is directly participating in signal transmission (Figure 4).
Interestingly, many genes coding for proteins in this pathway are
differentially expressed in \emph{Plcb1+/-} mice after cue-induced
reinstatement (Figure 4 and Supplementary Table S6). Furthermore, we
found that several TFBS were over\selectlanguage{english}-represented in the DEGs of mPFC,
including YY1, MYOD, NRF1, ERR1, FREAC2, NFY and E4F1 (complete list of
TFBS in Supplementary Table S7). Then, we filtered the DEGs on mPFC
based on fold-change (FC\textgreater{}\textbar{}1.2\textbar{}) and
obtained a list of 238 genes that we used for gene network construction.
This analysis showed a highly scored network (score=62, Figure 3C) that
includes 31 DEGs involved in ''cellular development, cellular growth and
proliferation, nervous system development and function''.

\textbf{DISCUSSION}

Here we investigated, for the first time, the contribution of
the\emph{PLCB1} gene to cocaine addictive properties using
\emph{Plcb1+/-}mice. We found that the heterozygous \emph{Plcb1}
genotype resulted in a phenotype of resistance to cue-induced
reinstatement of cocaine-seeking behavior. Furthermore, we found
relevant transcriptomic differences in\emph{Plcb1+/-} mice compared to
WT after cue-induced reinstatement of cocaine seeking in mPFC, a brain
area essential in relapse (Jasinska et al., 2015; Moorman et al., 2015).
This study supports a role for\emph{PLCB1} in cocaine addiction,
confirming previous findings in humans (Cabana-Dominguez et al., 2017),
and suggests it may be relevant in relapse to cocaine addiction.

The phenotype of \emph{Plcb1+/-} mice was characterized at different
levels to evaluate consummatory and general behavior. In general, mutant
mice presented normal levels of body weight, food and water intake,
locomotor activity, and motor coordination, demonstrating that this
animal model is valid to study the effects of the heterozygous deletion
of the \emph{Plcb1} gene in other behavioral responses.
\emph{Plcb1+/-}showed increased anxiety in the elevated plus maze,
reduced short-term memory in the novel object recognition paradigm,
without any sign of depressive-like behavior in the anhedonia sucrose
preference test. Thus, the single targeting of the \emph{Plcb1} gene has
an impact on selective emotional and cognitive responses.

The short-term memory impairment observed in the mutants did not affect
the acquisition of operant associative conditioning nor the instrumental
learning that drives the goal-directed action, as shown by similar
levels of operant cocaine self-administration and extinction learning
than WT. In agreement, both genotypes trained with cocaine accomplish
cue-induced reinstatement criterion with high enhancement of responding
during this test compared to extinction. Thus, different brain circuits
are involved in each kind of learning, with perirhinal-hippocampal
structures (Winters et al., 2004) participating in short-term memory in
the novel object recognition paradigm and mPFC-dorsal striatum pathway
in cue-associated seeking. Also differences in each paradigm such as the
acute retrieval or repetitive exposure involved respectively in each
task may have a crucial influence in cognition. Hence, cocaine seeking
can be multidimensional, involving different types of associative
learning that together lead to an extensive repertoire of conditioned
and instrumental responding.

The anxiogenic profile of \emph{Plcb1+/-} is in accordance with the
results previously reported in the \emph{Plcb4} -/- mice, which were
associated with alterations in the cholinergic activity of the medial
septum (Shin et al., 2009). However, the selective knock-down
of\emph{Plcb1} in the mPFC did not replicate this phenotype in a
previous study (Kim et al., 2015), suggesting that other areas may be
involved. The anxiogenic profile of \emph{Plcb1+/-} mice had no effect
on cocaine self-administration since the acquisition and extinction of
this operant behavior was not modified in the mutants. Besides, mutants
showed protection against cue-induced reinstatement of cocaine seeking,
instead of the expected cocaine seeking promoted by an anxiogenic
phenotype (Mantsch et al., 2016). Therefore, the association of an
anxiogenic profile with a phenotype resilient to cocaine seeking in
the\emph{Plcb1+/-} mice suggests modifications in specific brain areas
involved in cocaine relapse, such as the mPFC. Recently, a phenotype
resilient to develop food addiction has been associated with increased
strength of pyramidal glutamatergic synaptic transmission in the mPFC
related to decreased compulsivity in the face of negative consequences
(Domingo-Rodriguez et al., 2020) in mice with an anxiogenic profile
(Lutz et al., 2015). Concerning cue-induced cocaine seeking, the mPFC
has a crucial role and the network of glutamatergic projections from the
prelimbic mPFC to the dorsal striatum participates in this phenotype
(Luscher et al., 2020). In our model, \emph{Plcb1} haploinsufficiency is
linked to reduced cue-induced cocaine seeking possibly associated with
modifications in this top-down corticolimbic brain network. Furthermore,
these glutamatergic projections from mPFC to dorsal striatum receive
mesocortical dopaminergic inputs from the VTA that could be crucially
involved in the protective effects of \emph{Plcb1,} since this gene
plays an essential role in the dopaminergic signal transmission in the
mPFC and many of the genes encoding for proteins in this pathway are
differentially expressed in \emph{Plcb1+/-} mice after cue-induced
reinstatement (Figure 4).

Transcriptomic analyses performed after the cue-induced reinstatement of
cocaine-seeking behavior revealed some of the molecular underpinnings
that underlie the cocaine relapse-related phenotype observed in the
mutant mice. Interestingly, differences in gene expression
between\emph{Plcb1+/-} and WT were predominantly found in the mPFC,
whereas almost no differences were observed in the HPC. This evidence
highlights mPFC as a key region to explain these differences in
cocaine-seeking reinstatement observed in \emph{Plcb1+/-} mice.
Consistently, in DEGs in mPFC, we found enrichment on pathways that are
essential for the development of addiction, including the dopaminergic
neurotransmission (Figures 3-4) (Wise and Robble, 2020). \emph{PLCB1} is
part of the dopamine-DARPP-32 signaling pathway (Figure 4), which plays
a key role in cocaine reward (Borgkvist and Fisone, 2007; Nishi and
Shuto, 2017). In a previous study, we also found this pathway enriched
in DEGs in the frontal cortex and ventral striatum of mice that showed
frustrated expected reward, produced by the cue in the absence of
expected reward after a high level of effort in a progressive ratio
schedule of reinforcement with palatable food (Martin-Garcia et al.,
2015b). Notably, \emph{Plcb1} was upregulated in the frontal cortex of
those frustrated mice with increased responses to obtain the reward
(palatable food). These data are in line with our findings in which
decreased expression of \emph{Plcb1} (\emph{Plcb1} +/- mice) results in
decreased cue-induced responses to obtain cocaine after extinction. All
these results support the participation of \emph{Plcb1} in cue-induced
cocaine seeking revealed in the present study and the involvement of
mPFC .

Our transcriptomic analyses also found enrichment in genes related to
learning and memory processes, such as LTP in the mPFC. The transition
from drug use to drug addiction is a maladaptive process that directly
affects learning and memory (Torregrossa et al., 2011; Goodman and
Packard, 2016). LTP produces long-lasting activity-dependent synaptic
modifications that underlie memory, and drugs of abuse alter this
mechanism in several brain regions such as mPFC (Huang et al., 2007; Lu
et al., 2010), mesocorticolimbic system (Thomas et al., 2008), VTA
(Placzek et al., 2016) and hippocampus (Keralapurath et al., 2017;
Preston et al., 2019), among others. Importantly, genes related to the
glutamatergic system such as \emph{Gria2-4} (glutamate ionotropic
receptor AMPA2-4 (alpha 2-4)) and \emph{Grik2} (ionotropic glutamate
receptor kainate 2 (beta 2)) were found to be upregulated
in\emph{Plcb1+/-} mice. Meanwhile, \emph{Grin1, 2c, and 2d} (glutamate
ionotropic receptor NMDA1 (zeta 1, epsilon3 and epsilon4)) were
downregulated, suggesting an increase in AMPAR/NMDAR ratio in
cocaine-experienced and abstinent \emph{Plcb1 +/-} mice as compared to
WT, one of the key indicators of LTP induction and increased synaptic
strength (Luscher and Malenka, 2011). Furthermore, we found enrichment
in other neuroplasticity (synapse organization, neuron projection
development, and axon guidance) (Bahi and Dreyer, 2005; O'Brien, 2009;
Cooper et al., 2017; Dong et al., 2017) and signaling pathways (MAPK,
mTOR, and neurotrophin) (Li and Wolf, 2015; Garcia-Pardo et al., 2016)
related to addiction. The assessment of gene expression in the mPFC of
the \emph{Plcb1+/-} mice highlighted alterations in relevant pathways
for the addictive process, which could contribute to the results
observed in the cue-induced reinstatement.

Nowadays, there are few effective pharmacological treatments available
for cocaine use disorders, and frequently, psychosocial interventions in
combination with pharmacotherapy are needed (Kampman, 2019). Studies
performed in mice have pinpointed multiple potential therapeutic
approaches for the reinstatement of cocaine-seeking behavior, targeting
the reward circuit (Buchta et al., 2020). In humans, treatment with
bupropion, topiramate, or disulfiram has been widely used. Bupropion, a
non-tricyclic antidepressant that inhibits dopamine and norepinephrine
reuptake, is effective in reducing craving (Frishman, 2007). Topiramate,
a GABA/glutamatergic medication, has also been used to treat cocaine use
disorder as it reduces the activity of the mesocorticolimbic
dopaminergic system (Kampman, 2019). A completely different strategy is
the use of disulfiram which potentially inhibits the oxidoreductase
dopamine \selectlanguage{greek}β\selectlanguage{english}-\selectlanguage{english}hydroxylase (D\selectlanguage{greek}β\selectlanguage{english}H, encoded by the \emph{DBH} gene), which
converts dopamine to norepinephrine (Kampangkaew et al., 2019).
Significantly, a genetic variation in the \emph{SLC6A3} gene (encoding
DAT) has been associated with disulfiram treatment for cocaine
addiction, with patients with higher DAT levels having better treatment
outcomes than those with lower DAT levels (Kampangkaew et al., 2019).
Thus, further studies and new therapeutic targets are needed to obtain
effective treatment for cocaine addiction. The results obtained in the
present study underscore the relevance of the \emph{Plcb1} gene in the
cue-induced reinstatement of cocaine seeking after extinction. Together
with previous findings in humans (Drgon et al., 2012; Cabana-Dom\selectlanguage{ngerman}ínguez
et al., 2017) and mice (Eipper-Mains et al., 2013), \emph{PLCB1} merits
to be further evaluated as a promising novel therapeutic target for
preventing relapse and treating cocaine addiction .

The experimental approach used in our study, the \emph{Plcb1+/-} mouse
model, allowed us to reproduce better the molecular context observed in
humans in comparison to the use of a complete KO mouse (Hall et al.,
2013), as these animals preserved, at least, half of the expression
of\emph{Plcb1} . However, the haploinsufficiency of \emph{Plcb1} during
neurodevelopment in these animals could produce alterations that may
contribute to the phenotype observed in the present study. Nevertheless,
this may also be similar in humans with genetic risk variants that
decrease \emph{PLCB1} expression. Therefore, this approach is
appropriate to study a specific genetic alteration that confers
susceptibility to drug addiction and to delineate the precise
contribution of \emph{PLCB1.}

To sum up, we studied, for the first time, the contribution of
the\emph{PLCB1} gene to cocaine addictive properties using
\emph{Plcb1+/-}mice. Previous studies have revealed an up-regulation of
\emph{Plcb1} in brain areas related to the reward circuit after cocaine
exposure in animals (Eipper-Mains et al., 2013) and in human cocaine
abusers (Cabana-Domínguez et al., 2017). These changes, together with
our results, suggest that cocaine increases the expression of
\emph{Plcb1,}and this mechanism plays an essential role in cocaine
addiction, as revealed now by the resistance to cue-induced
reinstatement of cocaine-seeking behavior exhibited by \emph{Plcb1+/-}
mutant mice. These results highlight the importance of the \emph{Plcb1}
gene in the development of cocaine addiction and relapse and pinpoint
PLCB1 as a promising therapeutic target for cocaine addiction and
perhaps other types of addiction.

\textbf{METHODS}

\textbf{Animals}

Male mice, 8 weeks old, were housed individually at temperature- and
humidity-controlled laboratory conditions (21±1°C, 55±10\%) maintained
with food and water ad libitum. Mice were tested during the dark phase
of a reverse light cycle (lights off at 8.00h and on at
20.00h).\emph{Plcb1+/-} mice in a C57BL/6J background and their
wild-type (WT) littermates were used (Kim et al., 1997). All
experimental protocols were performed in accordance with the guidelines
of the European Communities Council Directive 2010/63/EU and approved by
the local ethics committee (Comitè Ètic d'Experimentació Animal-Parc de
Recerca Biomèdica de Barcelona, CEEA-PRBB, agreement N°9213). In
agreement, maximal efforts were made to reduce the suffering and the
number of mice used.

\textbf{Genotyping of transgenic mice}

\emph{Plcb1+/-} mice in a C57BL/6J background were kindly provided by
H.-S. Shin (Kim et al., 1997). Very briefly, the knockout animals were
generated by the replacement of exons that encode aminoacid residues
50--82 by a Neomycin cassette that was used both for gene disruption and
for positive selection.

To genotype the mice, genomic DNA from ear punches or tail tips was
extracted as previously described (Gómez-Grau et al., 2017). Genotyping
was carried out by PCR on extracted genomic DNA using three specific PCR
primers: a sense primer, 5\selectlanguage{english}'-GTTAAGTCCTCAGGCAAACACC-3', and two antisense
primers, 5\selectlanguage{english}'-ACCTTGGGAGCTTTGGCGTG3' and 5\selectlanguage{english}'-CTGACTAGGGGAGGAGTAGAAG-3',
that allowed us to amplify a 180bp \emph{Plcb1+/+} band and a
290bp\emph{Plcb1-/-} band (Filis et al., 2009). Fragments were separated
by 2.5\% agarose gel electrophoresis. Known WT and \emph{Plcb1+/-}
controls were always used to validate genotyping results. The genotype
of all animals used was confirmed after the cue-induced reinstatement.

\textbf{Behavioral tests for phenotype characterization}

A group of 25 WT and 12 \emph{Plcb1+/-} mice underwent different
behavioral tests for phenotype characterization for 10 days, as
described in Figure 1:

\emph{Short-term memory.} To measure short-term memory, the novel object
recognition (NOR) test was used. This test was performed in a V-maze
apparatus (40 cm per side, Panlab, Spain) as previously described by us
and others (Bura et al., 2010; Planaguma et al., 2015). On day 1 mice
were habituated for 9 minutes in the V-maze. On day 2, mice were put
back into the V-maze for 9 minutes; two identical objects were
presented, and the time the mice spent exploring each object was
recorded. After a retention phase of 3 hours, the mice were placed for
another 9 minutes into the V-maze; in each paradigm, one of the familiar
objects was replaced with a novel object and the total time spent
exploring each object (novel and familiar) was registered. Object
exploration was defined as the orientation of the nose to the object at
a distance of less than 2 cm. A discrimination index was calculated as
the difference in the time spent exploring the novel and the time spent
exploring the familiar object divided by the total time exploring both
objects. A higher discrimination index is considered to reflect more
excellent memory retention for the familiar object.

\emph{Locomotor activity.} Locomotor activity was assessed in locomotor
activity boxes (9x20x11 cm; Imetronic, Passac, France), equipped with 2
rows of photocell detectors, and placed in a low-luminosity environment
(20--25 lux), as previously described (Martin-Garcia et al., 2013). The
locomotor activity was recorded for 2 hours, and 2 variables were
measured: horizontal activity as horizontal displacement and vertical
activity as vertical exploration/elevation.

\emph{Elevated plus maze test.} This test was performed as previously
reported (Planaguma et al., 2015). The test measures the conflict
between the natural tendency of mice to avoid an illuminated and
elevated surface, and the natural tendency to explore new environments.
It consisted of a black plastic cross with arms 40 cm long and 6 cm wide
placed 50 cm above the floor. Two opposite arms were surrounded by walls
(15 cm high, closed arms, 10 lux), while the two other arms were devoid
of such walls (open arms, 200 lux). A central platform connected the
four arms. At the start of the session, the mouse was placed at the end
of a closed arm facing the wall. During the 5-minutes trial, the number
of entries and the time spent in each arm were recorded. Anxiety was
assessed as both the time spent avoiding the open arms and the number of
entries into them.

\emph{Motor coordination.} Rota rod apparatus was used to assess motor
coordination. Precisely, this test measures the ability of the mouse to
remain on a rotating rod in which the speed of rotation is gradually
increased and evaluate general motor coordination. The device is a round
drum (5 cm diameter) suspended between 2 plexiglass walls. The drum is
suspended at 24 cm from a soft mat covered tabletop. The speed of
rotation is controlled by an electric engine with a digital revolution
per min (rpm) display. The test consisted in five trial sessions. Each
trial started with the mouse being placed on the rotating rod at 4.0
rpm, and then every 3 s the speed increased by 1.0 rpm until 20.0 rpm.
The trial terminated when the mouse fell from the rod or after 90 s,
whichever occurred first. There was a 10 s interval between trials. The
average maximum rota rod speed and time to fall was calculated for each
mouse (Martin-Garcia et al., 2013).

\emph{Experimental procedure for food and drink monitoring.}

\emph{Habituation.} Six animals per genotype were taken from their home
cages and placed in experimental chambers named PHECOMP boxes (Panlab
and Harvard Apparatus) equipped with a food, drink and motor activity
monitoring system
(\emph{https://www.panlab.com/en/products/phecomp-system-panlab}) for
two weeks of habituation. Animals were monitored in these boxes 24 h a
day during the whole experimental period.

\emph{Sucrose preference test.} After a period of habituation, a sucrose
preference test was performed on the third week of PheComp cage housing
during one week, as previously described (Bura et al., 2010; Planaguma
et al., 2015). Two bottles of water, one with 2\% sucrose and the other
without, were placed in the cage. The position of bottles were exchanged
everyday, and the consumption was measured after a 24h interval. The
preference for sucrose was calculated as the relative amount of water
with sucrose versus total liquid (water with and without sucrose)
consumed by the mice (Bura et al., 2010).

\textbf{Operant conditioning maintained by cocaine}

\emph{Operant self-administration apparatus.} The self-administration
experiments were conducted in mouse operant chambers (Model ENV-307A-CT;
Med Associates Inc., Georgia, VT, USA) equipped with two holes, one
randomly selected as the active hole and the other as the inactive hole
as previously described (Gutierrez-Cuesta et al., 2014).

\emph{Operant conditioning maintained by cocaine.} Cocaine
self-administration experiments were performed as previously described
(Gutierrez-Cuesta et al., 2014; Martin-Garcia et al., 2015a). Mice were
deeply anesthetized by intraperitoneal injection of a mixture of
ketamine (75mg/kg; Imalgene; Merial Laboratorios S.A., Barcelona, Spain)
and medetomidine (1mg/kg; Domtor; Esteve, Barcelona, Spain). After
surgery, anesthesia was reversed by subcutaneous injection of the
synthetic \selectlanguage{greek}α\selectlanguage{english}2 adrenergic receptor antagonist, atipamezole (2.5mg/kg;
Revertor; Virbac, Barcelona, Spain) indicated for the reversal of the
sedative and analgesic effects of medetomidine (\selectlanguage{greek}α\selectlanguage{english}2 adrenergic receptor
agonist). In addition, mice received an intraperitoneal injection of
gentamicin (1 mg/kg; Genta-Gobens; Laboratorios Normon, S.A., Madrid,
Spain) along with subcutaneous administration of the analgesic meloxicam
(2 mg/kg; Metacam; Boehringer Ingelheim, Rhein, Germany). Each 2h daily
self-administration session started with a priming injection of the
drug. Cocaine (obtained from The Spanish Agency of Drugs, Madrid, Spain)
was intravenously infused in 23.5\selectlanguage{greek}μ\selectlanguage{english}l over 2s (0.5 mg/kg per injection).
Cue light, located above the active hole, was paired with the delivery
of the reinforcer. Mice (WT n=36; \emph{Plcb1+/-} n=26) were trained
under a fixed ratio 1 schedule of reinforcement (FR1; one nose-poke lead
to the delivery of one dose of cocaine) over 5 consecutive daily
sessions and under a fixed ratio 3 (FR3) over 5 consecutive daily
sessions. Control mice trained with saline were included for both
genotypes (WT n=6; \emph{Plcb1+/-} n=6). The timeout period after
infusion delivery was 10 s. Responses on the inactive hole and all
responses elicited during the 10-s timeout period were also recorded.
Responses during the 10-s timeout period were considered a measure of
impulsivity reflecting the inability to stop motor behavior once it is
initiated. The criteria for self-administration behavior were achieved
when all of the following conditions were met: (1) mice maintained
stable, responding with 20\% deviation from the mean of the total number
of reinforcers earned in three consecutive sessions (80\% of stability);
(2) at least 75\% of mice responding on the active hole; and (3) a
minimum of 10 reinforcers per session.

After the 10 FR sessions, animals were tested in a progressive ratio
schedule of 4h where the response requirement to earn the cocaine
escalated according to the following series:
1--2--3--5--12--18--27--40--60--90--135--200--300--450--675--1000. Then,
we proceed with the extinction phase, 2h daily sessions following the
same experimental conditions than the cocaine self-administration
sessions except that cocaine were not available, and cue-light was not
presented. Mice were given 2-h daily sessions until they achieved the
extinction criterion with a maximum of 26 sessions. The criterion for
extinction was achieved when, during 3 consecutive sessions, mice
completed a mean number of nose pokes in the active hole consisting of
30\% of the mean responses obtained during the 3 days to achieve the
acquisition criteria for cocaine self-administration training. On day
27, only mice that accomplished extinction criterion were tested in the
cue-induced reinstatement during a 2h session, to evaluate the
reinstatement of cocaine-seeking behavior. The test for cue-induced
reinstatement was conducted under the same conditions used in the
training phase except that cocaine was not available. The reinstatement
criterion was achieved when nose pokes in the active hole were double
the number of nose pokes in the active hole during the 3 consecutive
days when the mice acquired the extinction criteria.

The catheter was flushed daily with heparinized saline (30 USP
units/ml). The patency of intravenous catheters was evaluated after the
last cocaine self-administration session and whenever the behavior
appeared to deviate dramatically from that observed previously by
infusion of thiopental through the catheter. If prominent signs of
anesthesia were not apparent within 3s of the infusion, the mouse was
removed from the experiment. The success rate for maintaining patency of
the catheter (mean duration of 11 days) until the end of the cocaine
self-administration training was 90\%.

After the cue-induced reinstatement, animals were euthanized by
decapitation, brains were quickly removed, and the medial prefrontal
cortex (mPFC) and hippocampus (HPC) were dissected. Brain tissues were
then frozen by immersion in 2-methylbutane surrounded by dry ice and
stored at -80\selectlanguage{ngerman}°C for later RNA isolation and transcriptomic analyses.

\textbf{RNA extraction and RNA sequencing}

Total RNA of 12 WT and 11 \emph{Plcb1+/-} mice from mPFC and HPC were
isolated using the RNeasy Lipid Tissue Mini Kit (Qiagen Düsseldorf,
Germany) according to the manufacturer's protocol. RNA concentration was
determined using the NanoDrop ND\selectlanguage{english}-1000 spectrophotometer (NanoDrop
Technologies, Wilmington, DE, USA), and integrity was evaluated using
the Bioanalyzer2100 platform (Agilent Technologies, Santa Clara, CA,
USA). RNA samples were grouped in 4 pools consisting of 3 mice per pool
for each experimental group, except for 1 group in the
\emph{Plcb1+/-}mice were only 2 animals were pooled. The pools were
organized to homogenize the average number of nose pokes in the
different pools. The pooled individuals were the same for both mPFC and
HPC.\textbf{RNA sequencing}

RNA sequencing (RNAseq) was performed by the Centre de Regulacio
Genomica (CRG, Barcelona, Spain). Libraries were prepared using the
TruSeq Stranded mRNA Sample Prep Kit\_v2 (Illumina, San Diego, CA, USA)
according to the manufacturer's protocol and sequenced 2x75 on
Illumina's HiSeq3000 system for both mPFC and HPC. The Bioinformatics
service of CRG carried out the analysis of RNAseq. Briefly, FastQC
v0.11.5(Andrews S., 2010) was used to inspect the reads quality and
CutAdapt 1.7.1 (Martin, 2011) to clean the data of adapters and
low-quality reads. Then, reads were mapped to the \emph{Mus
musculus}genome of reference (GRCm38/mm10) with STAR 2.5.3a (Dobin et
al., 2013), and the differential expression analysis was done by DESeq2
(Love et al., 2014a) to compare WT and \emph{Plcb1+/-} . Corrections for
multiple testing were applied by adjusting the p-values with a 5\% False
Discovery Rate.

RNAseq data of mPFC and HPC were explored on a principal component
analysis (PCA) plot using the ''plotPCA'' method from the DESeq2 package
(Love et al., 2014b) and log2 gene expression data. The PCAs were
performed with the 500 genes showing the highest variance among the
samples to calculate the distance among them. The heatmaps were
performed using the ''heatmap'' function on R, and the hierarchical
clustering considered the euclidean distance between the samples
considering all the genes or only those with corrected p-value
\textless{} 1e-05.

\textbf{Functional annotation of RNAseq results}

We performed a functional group enrichment of differentially expressed
genes (DEGs) in mPFC using the DAVID Annotation Tool
(\emph{http://david.abcc.ncifcrf.gov})(Jiao et al., 2012) considering GO
(Gene Ontology) biological processes and KEGG pathways (Kyoto
Encyclopedia of Genes and Genomes). Then, we searched for
over\selectlanguage{english}-represented transcription factor\selectlanguage{english}-binding sites (TFBS) using the
information of MsigDB (\emph{https://www.gsea-msigdb.org/gsea/msigdb})
integrated on WebGestalt2019 (\emph{http://www.webgestalt.org/})(Liao et
al., 2019), and the default parameters, applying the weighted set cover
method to reduce redundancy. In both analyses, the Benjamin--Hochberg
procedure was performed for multiple testing. Finally, we investigated
the existence of gene networks with Ingenuity Pathway Analysis 8.8
software (IPA, http://www.ingenuity.com/products/ipa; Ingenuity Systems,
Redwood City, CA, USA) (Kramer et al., 2014) after selecting genes with
fold-change \textgreater{}\textbar{}1.2\textbar{}.

\textbf{Statistical analysis}

Three-way ANOVA with repeated measures was used to test the evolution
over sessions or days. Sessions or days were used as within-subject
factors and genotype (\emph{Plcb1+/-} or WT) and drug (cocaine or
saline) were used as between-subjects factors. Post-hoc analyses
(Newman-Keuls) were performed when required. Comparisons between two
groups were analyzed by Student \emph{t} -test or \emph{U} -Mann-Whitney
depending on the distribution defined by the Kolmogorov-Smirnov
normality test and the sample size. The chi-square analyses were
performed to compare the percentage of mice that acquired operant
learning criteria in the different experimental groups. Results are
expressed as mean +- SEM or individual values with the median and the
interquartile range specified in the figure legend. Differences were
considered significant at \emph{P} \textless{}0.05. The sample size was
calculated based on power analysis. The statistical analyses were
performed using the Statistical Package for Social Science tool
SPSS(r)25.0 (SPSS Inc, Chicago, USA).

See supplementary information for more details of genotyping of
transgenic mice, drugs, behavioral tests of phenotype characterization,
operant conditioning maintained by cocaine, RNA extraction and
sequencing.

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\emph{FIGURE LEGENDS}

\textbf{Figure 1. Behavioral tests for phenotype characterization.} (A)
Timeline of the experimental sequence. (B, C) Short-term memory measured
in the V-maze for novel object recognition task, (B) discrimination
index and (C) exploration. (D) Locomotor and (E) rearing activity
measured in actimetry boxes. (F) Anxiety-like behavior measured by the
time spent in the open arms and (G) the percentage of time in the open
arms in the elevated plus maze. Motor coordination measured in the rota
rod test by the (H) number of r.p.m. that the mice performed in the
average trials or the (I) mean time they remain in the apparatus. All
data are expressed in median and interquartile range and individual data
are shown (WT n=25; \emph{Plcb1+/-} n=12).

\textbf{Figure 2. Operant conditioning maintained by cocaine
in\emph{Plcb1+/-}, wild-type (WT) and corresponding control saline
mice.}(A) Timeline of the experimental sequence. (B) The number of
cocaine infusions (0.5 mg/kg/infusion) in both genotypes increased
progressively across sessions during FR1 (repeated measures ANOVA,
interaction between genotype x drug x sessions, \emph{P}
\textless{}0.05) and remained stable during FR3 and higher in mice
trained with cocaine (repeated measures ANOVA, main effect of drug,
***\emph{P} \textless{}0.001). The maximum number of infusions was
reached on session 5 in both genotypes and was slightly superior in
\emph{Plcb1+/-} (36.35\selectlanguage{ngerman}±1.47) than in WT (29.61±2.23) but post hoc
analyses demonstrated that this difference was not significant. (C) The
motivation for cocaine measured by the breaking point achieved in the
progressive ratio schedule of reinforcement (4h) was equivalent in both
genotypes trained with cocaine but higher than those trained with saline
independently of the genotype (individual data with interquartile range,
\emph{U} Mann-Whitney, \#\#\emph{P} \textless{}0.01 vs WT cocaine,
\$\$\emph{P} \textless{}0.01 vs \emph{Plcb1+/-} cocaine). (D) Both
genotypes also showed similar levels of extinction that decreased during
sessions, but the curve was more pronounced in WT mice than in mutants
(repeated measures ANOVA, interaction genotype x sessions,
\&\&\&\emph{P} \textless{}0.001). (E) Decreased cue-induced
reinstatement of cocaine seeking in\emph{Plcb1+/-} mice was obtained
compared to WT (individual data with interquartile range, \emph{U}
Mann-Whitney, \#\emph{P} \textless{}0.05, \#\#\emph{P} \textless{}0.01
vs WT cocaine). All data are expressed in mean ± SEM when sessions are
represented or median and interquartile range when individual data are
shown (WT cocaine n=28-36;\emph{Plcb1+/-} cocaine n=19-26; saline n=6
per genotype). Statistical details are included in Supplementary Table
S1.

\textbf{Figure 3. Gene expression changes in mPFC after cue-induced
reinstatement of cocaine seeking in \emph{Plcb1+/-} versus wild-type
(WT) mice.} (A) Selection of over-represented KEGG pathways (Kyoto
Encyclopedia of Genes and Genomes) and (B) GO (Gene Ontology) identified
by DAVID software among the differentially expressed genes. The number
of genes with altered expression included in each category is indicated
on the right side of the bar. In red, relevant pathways for the
addictive process. (C) Gene network involved in cellular development,
cellular growth and proliferation, nervous system development and
function (score = 62). The green nodes in the pathway indicate genes
with downregulated expression in \emph{Plcb1} +/- identified in RNAseq.

\textbf{Figure 4. Alterations in expression in the dopaminergic synapse
in mPFC of \emph{Plcb1}+/- mice after cue-induced reinstatement of
cocaine seeking.} Adapted from KEGG (Kyoto Encyclopedia of Genes and
Genomes) pathways (mmu04728). Enriched pathway in differentially
expressed genes in mPFC, 25 out of 131 genes in the pathway were
differently expressed (\emph{P\textsubscript{raw}}
=2e-03;\emph{P\textsubscript{ajd}} =0.02). In red, upregulated genes and
in green, downregulated genes in mPFC. *Protein complexes including
upregulated and downregulated genes. Correspondence between genes and
proteins can be found in Supplementary Table S6.\selectlanguage{english}
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