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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