Discussion
In this study, we found a significant decrease of VTA DA population
activity after long term, but not short term, abstinence from chronic
cocaine self-administration, a pattern paralleling the phenomenon of
incubation of craving (Grimm et al., 2001). We also found a significant
increase of BLA activity after long but not short-term, abstinence.
Finally, we show that the decrease in VTA DA activity depends on the
BLA-VP pathway.
We have previously shown that extended exposure to cocaine (25 days),
followed by abstinence (7 and 30 days) persistently decreases DA
activity in the VTA (Salin et al., 2021). Here, we extend these findings
using a shorter 10 day-self-administration protocol, showing a decrease
in DA population activity after 26-45 days but not 1-3 days of
abstinence. We found no changes of DA population activity during acute
withdrawal, suggesting no changes in tonic DA release in VTA target
structures (Floresco et al., 2003) at this time point. This is
consistent with some studies using microdialysis that showed no changes
in baseline DA during acute withdrawal (Calipari et al., 2014; Cameron
et al., 2016), but it is inconsistent with other work (Hurd et al.,
1989; Weiss et al., 1992) demonstrating reduced basal DA levels during
acute withdrawal. There are multiple differences between studies that
can account for these discrepancies, including the use of slice versusin vivo preparation, the type of self-administration paradigm
(length of the self-administration period for example), and the time of
microdialysis measurements relative to the last self-administration
sessions (12h, 24h or up to 72h after the last self-administration
session). In our study, DA neurons’ activity was recorded from 18h up to
72 h after the last session, suggesting that potential decreases in
population activity and subsequent decreased DA release might normalize
rapidly following discontinuation of cocaine self-administration.
Another explanation could be that tonic DA decreases independently of
population activity (via changes in presynaptic DA release or increased
reuptake). Although there are discrepancies in changes of DA level or DA
activity during acute withdrawal, a decrease in DA activity has been
consistently described in the literature following long-term withdrawal
(Salin et al., 2021;Shen et al., 2007). This decrease could participate
in the dysphoria described after protracted cocaine abstinence (Haake et
al., 2019).
Previous work has shown that activation of the BLA potently decreases
the number of DA neuron firing (Chang and Grace, 2014). Increased
activity in the BLA has been previously described after extended
withdrawal from chronic cocaine (Munshi et al., 2019), which is
consistent with the increased firing rate and percentage of spikes in
bursts after long-term abstinence in our study. However, Munshi et al
did not study BLA activity after acute withdrawal. Here, we show no
changes in the firing rate or the percentage of spikes firing in burst
after 1-3 days of withdrawal. These changes of firing rate in the BLA
across the abstinence period could be attributed to multiples factors,
such as changes in intrinsic excitability, in the expression of NMDA or
AMPA receptors, or changes in different inputs to pyramidal neurons.
Changes in membrane properties have been described in several
pathological conditions such as drug addiction (Kourrich et al., 2015).
Numerous studies have described increased excitability of BLA neurons
after chronic stress (Rosenkranz et al., 2010; Rau et al., 2015). Since
chronic stress and drug of abuse share common substrates (Belujon and
Grace, 2011; Munshi et al., 2019), the increased firing rate and
bursting activity of BLA neurons after long abstinence could be due to
increased neuronal excitability. Another factor that might contribute to
increased firing is changes in the expression of NMDA or AMPA receptors
in the BLA. Indeed, previous work has shown an increased in the
expression of GLUR1 and GLUR2 subunits as well as a decrease in the
expression of NR2B subunits in the BLA during cocaine abstinence (Lu et
al., 2005). Interneurons in the BLA tightly regulate excitability of
principal neurons (Ehrlich et al., 2009) by targeting their perisomatic
region (Bienvenu et al., 2012) and blocking activity in principal
neurons (Woodruff and Sah, 2007). Since activity of BLA principal
neurons is regulated by local interneurons, dysregulation of
interneurons’ activity in the BLA after chronic cocaine could increase
spontaneous activity of principal neurons. Although increased activity
of parvalbumin (PV) interneurons has been described in the central
amygdala after chronic opiates withdrawal (Wang et al., 2016), there is
a lack of information concerning PV interneurons in the BLA after
psychostimulant withdrawal. Moreover, BLA activity can be potently
modulated by the prefrontal cortex through inhibitory control via
reciprocal connections (Rosenkranz and Grace, 2002). Changes in activity
in the medial prefrontal cortex has been observed in abstinent cocaine
abusers (Bolla et al., 2004) which could lead to pathological activation
of the amygdala. Therefore, after protracted abstinence of chronic
cocaine hyperactivity of the BLA could underlie the reduced DA VTA
population activity observed in the present study.
Chronic stress has been shown to increase BLA activity (Munshi et al.,
2019) and decrease DA population activity in the VTA (Chang and Grace,
2014). Inactivation of the BLA in a model of chronic stress has also
been shown to increase DA population activity (Chang and Grace, 2014).
In our model, we tested the effect of BLA inactivation on DA population
activity after long-term abstinence. In agreement with the stress
literature (Chang and Grace, 2014), BLA inactivation increased
dopaminergic activity in cocaine rats after long-term withdrawal. These
results support the existence of common substrates between addiction and
stress-related emotional disorders (Belujon and Grace, 2011; Polter and
Kauer, 2014), and emphasize that normalization of BLA activity may be
involved in reducing abstinence-related emotional symptoms.
The BLA does not project directly to the VTA but has strong projections
to the VP (Maslowski-Cobuzzi and Napier, 1994)and the BLA-VP-VTA has
been shown to exert potent action on DA neuron activity states (Chang
and Grace, 2014). In our model, blocking glutamatergic afferents in the
VP after long-term abstinence restores DA activity suggesting that the
BLA-VP pathway is involved in the decreased DA activity after protracted
abstinence. Mahler et al. have shown that GABAergic neurons from the
rostral part of the VP (RVP) projecting to the VTA, but not the caudal
part, is activated during cue-induced reinstatement and that
inactivation of the RVP-VTA pathway reduces cocaine seeking behavior
after long-term abstinence (Mahler et al., 2014). In our study,
glutamatergic afferents were blocked in the RVP, suggesting that
increased activity of the BLA-RVP pathway leading to hypodopaminergic
activity in the VTA after long-term withdrawal might play a critical
role in cue-induced seeking. Other regions such as RMTg could be an
additional relay structure in restoring DA activity since it receives
direct projections from the BLA (Kaufling et al., 2009), and sends
direct GABAergic inhibitory afferents to the VTA (Lecca et al., 2012).
However, RMTg activation inhibits DA neurons firing rate (Lecca et al.,
2012), but firing rate of VTA neurons was not altered during withdrawal
in our study.
Conclusion
Our study sheds new lights on neuroadaptations occurring during
incubation of craving, a phenomenon believed to play a role in
persistent risks of relapse, which is crucial to improve novel
therapeutic strategies. In particular, we demonstrate that after
long-term withdrawal the decrease of DA activity in the VTA is
associated with hyperactivity of the BLA and involved a circuit
comprising the BLA and the VP. Dysfunctions of this circuit could
underlie dysphoria, which has been described during acute but also
long-term withdrawal from cocaine. Imaging studies focusing on the
interactions of regions in this specific network could be used as a
marker of vulnerability to dysphoria, craving and relapse.
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Figure legends