Resumen de Turning on and off the cerebellum in drug-induced memory
This comprehensive thesis encompasses an exploration of the cerebellum's role in drug-induced memories and its relationship with other critical regions of the addiction circuitry. Over the last decade, neuroscience research has illuminated the cerebellum's central involvement in addiction, particularly highlighting its pivotal role in the associative learning processes that link drug use to contextual cues (Miquel et al., 2009, 2016, 2020; Moulton et al., 2014).
Chapter 1 of this thesis builds upon prior research conducted by our team, which convincingly demonstrated that the posterior cerebellum's vermis (lobule VIII) has the capacity for undergoing significant changes in neural activity during the expression of drug-context memories (Carbo-Gas et al., 2014a; Carbo-Gas et al., 2014b; Carbo-Gas et al., 2017, Gil-Miravet et al., 2021; Guarque-Chabrera et al., 2022). While our earlier investigations into the cerebellar bases of drug-induced Pavlovian memory primarily involved mice or rats on unbiased procedures, this study seeks to replicate and expand upon these findings using a biased conditioned place preference procedure with rats. We assessed neural activity patterns using c-Fos expression within specific regions of the cerebellum, focusing intently on the granular layer within lobules VII and VIII of the vermis and paravermis, as well as Crus 1 and Crus 2 of the hemispheres. We compared rats expressing and not expressing preference towards cocaine-related cues, as well as a pseudo-conditioned group called the unpaired group as our experimental subjects. In accordance with our previous findings, results in Chapter 1 reveal consistent differences within the granular layer of lobule VIII between rats that exhibit a preference for cocaine-related contextual cues and those that do not. Remarkably, rats exhibiting a pronounced preference for cocaine-associated contextual cues such as odor and tactile stimuli showed increased neural activity in the granule cell layer of the dorsal posterior cerebellar vermis. Notably, we did not observe an effect of cocaine-induced conditioning on activity either in the paravermis or the hemispheres. Therefore, the findings from Chapter 1 unveil a potential medial-lateral gradient in the involvement of the cerebellar cortex in shaping Pavlovian memories associated with drug use.
The preference for the cocaine rewarded compartment correlated with the increased activity in the granule cell layer only in the posterior vermis but not with changes in the neural activity in neither paravermis nor hemispheres. Collectively, these findings align with results observed in human neuroimaging studies in which the presentation of drug-related cues triggers drug craving and stimulates cerebellar activity (Grant et al., 1996; Wang et al., 1999; Anderson et al., 2006; Schneider et al., 2001; Martin-Solch et al., 2001; Filbey et al., 2009; Olbrich et al., 2009; Lou et al., 2012; Xuan et al., 2019; Al-Khalil et al., 2020). and complement previous research conducted in our laboratory using different paradigms.
Chapter 2 expands on previous research by our laboratory and the preceding findings. By using lesioning and temporary deactivation techniques, our early studies suggested the involvement of the dorsal posterior vermis in the inhibitory regulation of preferences for stimuli associated with cocaine (Gil-Miravet et al., 2019). However, these interventions lacked cell-specificity. Thus, in the present chapter, we sought to investigate the dorsal cerebellum's role in the acquisition of conditioned preferences for stimuli associated with cocaine availability using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), a chemogenetic strategy. These experiments encompass both the activation and inhibition of the dorsal vermis in lobule VIII. The descriptive studies that precede this chapter unveiled the existence of two distinct neuronal populations within the cerebellar cortex - granule cells and Golgi interneurons - which may play an instrumental role in the formation of associative drug-context memories. (Carbo-Gas et al., 2014a; Carbo-Gas et al., 2014b; Carbo-Gas et al., 2017). Our observations validate the exclusive expression of the hSyn promoter by Golgi interneurons in the granule cell layer and interneurons within the molecular layer, including basket and stellate cells. The activation of these two inhibitory neuronal populations in the cerebellar cortex increased preference for stimuli associated with cocaine. However, their inhibition reduced conditioned preference for cocaine. These findings align with the results of Gil-Miravet (2019, 2021), which showed that conditioning training under a lesion of lobule VIII dramatically increased the fraction of animals that expressed cocaine-induced conditioned preference, involving the dorsal cerebellum in the inhibitory regulation of cocaine-induced Pavlovian learning. In the case of the excitatory DREADD, we hypothesize that the facilitation of cocaine-induced conditioning might be attributed to the excitation of interneurons in the dorsal vermis that in turn would reduce activity in Purkinje cells (PC) enhancing Deep Cerebellar Nulcei (DCN) activity. Conversely, the decrease in conditioned preference by the inhibitory DREADD is likely due to Purkinje cell disinhibition that would lead to a reduction in DCN activity. These findings provide valuable insights into how manipulating specific neural pathways in the cerebellum can impact the development of cocaine-induced preference.
To provide further context, Chapter 3 is part of an article already published in Frontiers in Systems Neuroscience that offers a theoretical model for understanding the cerebellum's role in cocaine-induced Pavlovian memory. The chapter also presents research using DREADDs, to further investigate and propose a causative working model for the role of the cerebellum in cocaine-induced conditioned memory. In these experiments, we inhibited the Interposed/Lateral DCN that connects Lobule VIII with the addiction pathway. Our findings suggest that inactivation of DCNs prevents the facilitating effect of a posterior vermis lesion on cocaine-induced preference conditioning. While these findings support our previous research (Gil-Miravet et al; 2019, 2021), they also introduce new questions and avenues for exploration.
Finally, in Chapter 4, we focus on the monosynaptic pathway connecting the Interposed DCN to the Ventral Tegmental areal (VTA). This pathway, as previously identified in studies by our research group and others (Carta et al., 2019; Gil-Miravet et al.,2021), also receives axonal input from Purkinje cells in the cerebellar vermis lobule VII (Gil-Miravet et al.,2021). This intricate network is believed to regulate activity and plasticity in the medial prefrontal cortex (mPC) and striatum, playing a pivotal role in cocaine-induced conditioned preference (Gil-Miravet et al., 2021). The primary objective of Chapter 4 is to test the hypothesis that the cerebellum regulates mPC cortex (IL) activity through the VTA using chemogenetic tools. To achieve this, we infused an inhibitory cre-dependent DREADD into the interposed nucleus and a cre-retrograde virus in the VTA. This allows us to exclusively express these receptors in the DCNs-VTA projections, and we subsequently examine the inhibition of neurons in the DCNs, VTA, and IL. Inhibition of the Interposed/Lateral DCNs leads to a decrease in neural activity within both the VTA and IL. These findings offer further support for prior research, which has indicated the existence of a monosynaptic excitatory projection from the DCN to the VTA (Gil-Miravet et al.,2021). This result highlights the intricate neural model involving the cerebellum, VTA, and IL.
In conclusion, this thesis advances our understanding of the cerebellum's multifaceted role in addiction, particularly in the context of drug-induced memories. The study elucidates the neural mechanisms, circuits, and pathways involved, shedding light on the cerebellum's influence on conditioned preferences and its potential as a therapeutic target for addiction intervention. These findings contribute to the ongoing efforts to combat addiction and address its enduring behavioral alterations.
