Molecular mechanisms underlying silent synapse generation and their role in drug-associated behaviors
by Paloma R. Huguet Rodríguez
Date of Examination:2025-06-23
Date of issue:2025-08-29
Advisor:Prof. Dr. Dr. Oliver Schlüter
Referee:Prof. Dr. Nils Brose
Referee:Prof. Dr. Silvio O. Rizzoli
Persistent Address:
http://dx.doi.org/10.53846/goediss-11440
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Abstract
English
Silent synapses are immature glutamatergic connections that express stable NMDARs, while AMPARs are absent. These synapses are abundant during early development and serve as plasticity substrates, offering synaptic opportunities to reorganize neural networks. In the adult brain, repetitive exposure to cocaine generates de novo silent synapses in the nucleus accumbens (NAc). Although network adaptations in the NAc might contribute to regulating drug-associated behaviors involving motivation and action, they do not fully account for all aspects of addiction. Here, using the electrophysiology procedure of minimal stimulation with synaptic failure analysis, I found that cocaine also increases silent synapses in the dorsal striatum and hippocampus, but not in other areas tested. The emergence of silent synapses in those areas suggests their involvement in processes such as habitual or compulsive drug use and associative learning. Astrocyte-neuronal crosstalk via thrombospondins (TSPs) and their neuronal receptor α2δ-1 is required for cocaine-induced silent synapse generation in the NAc. Using AAV-mediated knockdown, I found that α2δ-paralogs were differentially required throughout the brain for developmental and cocaine-induced silent synapse generation. Specifically, α2δ-1 was necessary in the striatum and α2δ-3 in the visual cortex, but both α2δ-1 and α2δ-3 were necessary in the hippocampus. This mechanistic diversity suggests why disrupting α2δ-1 function alone had previously limited effects on drug-associated behaviors. Together, these findings support the existence of distinct synapse subtypes defined by their molecular composition, which may underlie functional heterogeneity across neural networks. However, the α2δ-family lacks intracellular domains to transduce TSP-triggered signaling. To obtain a blueprint of the neuronal and astrocytic machinery involved in cocaine-induced silent synapse generation, I tested for proteins known to be involved in synaptogenesis and able to bind to TSPs, using knockdown strategies. The numerous identified proteins are involved in membrane morphology and cell adhesion regulation and likely participate in specific stages of the synaptogenic process. I propose that (1) α2δ-paralogs and integrins act as molecular bridges between pre- and postsynaptic compartments through TSP binding, (2) BAI, Fzd9, and Celsr neuronal receptors may transduce synaptogenic signals, such as TSP-signaling, and (3) Vangl proteins may serve as auxiliary subunits to modulate and fine-tune the process. Ultimately, these components likely converge on a shared intracellular signaling cascade. This thesis identified PSD-95β as a key mediator of the synaptogenic signaling cascade in the striatum with its N-terminal domains, specifically the L27 domain and interactions with the PDZ domains, mediating the signaling. Focusing on the astrocytic contribution, I utilized astrocyte-specific DREADD-hM3Dq activation and found that stimulating these cells alone was sufficient to generate silent synapses. Our follow-up findings suggest that this effect is dependent on mGluR5 and/or σ1, the latter being known to bind to cocaine. At a behavioral level, inhibition of σ1 prevented cocaine-induced locomotor sensitization, which is dependent on silent synapse generation and is a proxy for increased incentive salience with repeated drug exposure. These results support my hypothesis that astrocytic σ1 functions as a cocaine receptor, mediating its behavioral effects by generating silent synapses in neighboring neurons. To link CP-AMPARs, known to mediate cue-induced drug-craving and set the threshold for cocaine-memory retrieval, to silent synapse generation, I blocked α2δ-1 with gabapentin. As a result, CP-AMPAR expression was prevented, but not drug-associated memories. These findings crystallize a therapeutic potential to prevent drug craving by targeting silent synapse generation to prevent CP-AMPAR expression. Moreover, I used PSD-95 KO mice as a model of impaired silent synapse maturation. These mice showed impaired long-term memory retrieval but not retention, as previously thought. Notably, cue-induced rescue of this impairment correlated with the incorporation of CP-AMPARs, suggesting that these receptors might represent an active memory trace. Thus, CP-AMPARs may provide a novel approach to monitor and manipulate drug-associated memories or active memory traces in a generalized manner. Furthermore, non-contingent cocaine exposure erased drug-associated memories in PSD-95 KO and PSD-95 heterozygous mice but not in WT mice, indicating that the lack of synaptic strengthening through synaptic maturation facilitates the disruption of drug-associated memories. This finding is of great clinical relevance for the amelioration of drug-associated behaviors as associative memories typically trigger relapse and compulsive drug consumption. In summary, this thesis elucidated the molecular machinery underlying silent synapse generation, providing a blueprint of the involved signaling components and identifying novel molecular targets to manipulate the process. By leveraging this molecular knowledge, I reveal that silent synapse-based drug-associated behaviors, such as locomotor sensitization, can be prevented. Additionally, I analyzed the role of silent synapse maturation in drug-associated memories. This maturation proved essential for long-term memory retrieval and stability, providing novel insights into how stable memories are formed and can be erased. These findings propose a target with important clinical implications for drug addiction.
Keywords: Silent synapse; AMPAR; CP-AMPARs; PSD-95; synaptogenesis; synaptic plasticity; drug addiction; drug-associations; locomotor sensitization
