DISCUSSION
Here we investigated, for the first time, the contribution of thePLCB1 gene to cocaine addictive properties using Plcb1+/-mice. We found that the heterozygous Plcb1 genotype resulted in a
phenotype of resistance to cue-induced reinstatement of cocaine-seeking
behavior. Furthermore, we found relevant transcriptomic differences inPlcb1+/- 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 forPLCB1 in cocaine addiction, confirming previous findings in
humans (Cabana-Domínguez et al., 2017), and suggests it may be relevant
in relapse to cocaine addiction.
The phenotype of 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 Plcb1 gene in other behavioral responses. 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 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 Plcb1+/- is in accordance with the
results previously reported in the Plcb4 -/- mice, which were
associated with alterations in the cholinergic activity of the medial
septum (Shin et al., 2009). However, the selective knock-down ofPlcb1 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 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 thePlcb1+/- 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
(Lüscher et al., 2020). In our model, 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 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 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 betweenPlcb1+/- 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 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). 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 (Martín-García et al., 2015b).
Notably, 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 Plcb1 (Plcb1 +/- mice) results in decreased
cue-induced responses to obtain cocaine after extinction. All these
results support the participation of 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 Gria2-4 (glutamate ionotropic
receptor AMPA2-4 (alpha 2-4)) and Grik2 (ionotropic glutamate
receptor kainate 2 (beta 2)) were found to be upregulated inPlcb1+/- mice. Meanwhile, 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 Plcb1 +/- mice as compared to
WT, one of the key indicators of LTP induction and increased synaptic
strength (Lüscher 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; García-Pardo et al., 2016)
related to addiction. The assessment of gene expression in the mPFC of
the 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 β‐hydroxylase (DβH, encoded by the DBH gene), which
converts dopamine to norepinephrine (Kampangkaew et al., 2019).
Significantly, a genetic variation in the 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 Plcb1 gene in the
cue-induced reinstatement of cocaine seeking after extinction. Together
with previous findings in humans (Drgon et al., 2012; Cabana-Domínguez
et al., 2017) and mice (Eipper-Mains et al., 2013), 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 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 ofPlcb1 . However, the haploinsufficiency of 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 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 PLCB1.
To sum up, we studied, for the first time, the contribution of thePLCB1 gene to cocaine addictive properties using Plcb1+/-mice. Previous studies have revealed an up-regulation of 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 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 Plcb1+/- mutant mice. These
results highlight the importance of the 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.