Cocaine and Morphine Induced Silent Synapses

Study Notes: Mechanisms of Silent Synapses in Morphine and Cocaine Exposure

Introduction & Background

  • Main Question: How do cocaine and morphine, despite producing similar behavioral outcomes (locomotor responses), induce opposing synaptic modifications in the nucleus accumbens (NAc), and what are the cellular mechanisms underlying these changes?

    • Silent Glutamatergic Synapses:

    • Definition: Contain functional NMDARs (N-methyl-D-aspartate receptors) and are absent or unstable with AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors).

    • Previous Studies: Indicated generation of silent synapses via cocaine exposure in the NAc shell (NAcSh) by inserting new NMDARs into the synaptic cleft.

    • Current Study: Indicates morphine exposure also generates silent synapses in the NAcSh, but through the internalization of AMPARs from pre-existing synapses.

  • Key Peptides:

    • GluA2 peptide: Negative control for AMPAR internalization.

    • GluA23Y peptide: Inhibitor of endocytosis (i.e., the process of internalizing AMPARs).

Key Terms & Abbreviations

  • Nac: Nucleus accumbens.

  • NacSh: Nucleus accumbens shell.

  • MSN: Medium spiny neurons.

  • D1R: Dopamine D1 receptor (associated with learning and memory).

  • D2R: Dopamine D2 receptor (involved in motor function).

  • EPSC: Excitatory postsynaptic currents.

  • CV: Coefficient of variation, inversely proportional to the number of functional synapses and release probability.

  • Tat-GluA2: Treatment to block endocytosis of AMPARs.

  • GluN2B: Extrasynaptic NMDAR subunit.

  • tdTomato: Bacterial artificial chromosome transgenic mice used for visualizing MSN in basal ganglia pathways.

  • CPP: Conditioned place preference assessment.

  • Dil: Lipid staining (fluorescent when associated with membranes).

  • IP: Intraperitoneal injection (to administer saline, cocaine, or morphine).

  • SC: Subcutaneous injection (to administer morphine).

  • Silent Synapses: Commonly found in the developing brain, characterized by instability and maturation potential depending on usage frequency.

Experiment Design & Methods

  • Minimal Stimulation Assay:

    • Injection Protocol:

    • I.p. injection of cocaine HCl (15 mg/kg in saline), morphine sulfate pentahydrate (10 mg/kg in saline), or saline control, administered once daily for 5 days.

    • Electrophysiological recordings taken approximately 24 hours after the last injection.

  • Whole-cell Recordings from MSNs:

    • Conducted in the ventromedial NAcSh to observe EPSCs in cocaine-, morphine-, and saline-exposed mice.

  • Analysis of Silent Synapse Recordings:

    • Based on two theoretical assumptions:

    1. Presynaptic release sites are independent.

    2. Release probability across all synapses, including silent synapses, is identical.

    • Calculation of Silent Synapse Percentage:

    • Formula: 1 - rac{ ext{ln}(F{-70})}{ ext{ln}(F{+50})} where:

      • F_{-70} is the failure rate at -70 mV.

      • F_{+50} is the failure rate at +50 mV.

  • Statistical Analysis:

    • ANOVA with Bonferroni Post Test.

    • T-tests.

    • Animal Subjects: Male Sprague-Dawley rats (200-250 g; 50-80 days old), male B51 WT, or male Drd1a-tdTomato mice (~25 g; 50-80 days old).

Supplementary Observations

  • Supplementary Figure 2:

    • Sample data demonstrating successful vs. failed synaptic responses in the minimal stimulation assay using black and gray dots to denote EPSC amplitude.

Figures and Data Analysis

Figure 1: EPSC Analysis

  • 1a: Measurement of CV of isolated AMPAR/NMDAR-mediated excitatory currents at -70 mV and +50 mV, respectively.

  • 1b: Control EPSC of AMPAR (at -70 mV) and NMDAR (at +50 mV) mediated current.

  • 1c: Morphine-exposed EPSC shows decrease and scattering in AMPAR-mediated current, with changes in NMDAR current.

  • 1d: Quantification reveals decreased CV ratio of AMPAR/NMDAR currents in morphine-exposed, suggesting a reduction in AMPAR due to silent synapses.

  • EPSCs Recorded After 1 Day of Withdrawal:

    • Minimal stimulations elicited at both +50 mV (NMDAR) and -70 mV (AMPAR) to analyze responses at 1-day withdrawal post treatments.

  • Summary Findings:

    • Notable increased percentage of silent synapses in NAcSh MSNs in young rats (~50 days old).

  • Morphine Exposure Effects:

    • Gradual increase in silent synapse percentage during the 5-day exposure period, dramatically decreasing after a 7-day withdrawal period.

Figure 2: Kinetics and NMDAR Effects

  • 2a: NMDA and AMPA EPSC decay kinetics were measured after saline, cocaine, or morphine exposure.

  • 2b: Cocaine prolongs decay of NMDA current, while morphine does not affect decay kinetics.

  • 2d: Ro 256981 (NMDAR antagonist targeting GluN2B) shows inhibition of NMDAR and EPSCs more in cocaine-exposed rats.

  • Implications: Indicates GluN2B's role in the impact of cocaine use on NMDARs.

  • Effects on Silent Synapse Generation:

    • Co-administration of scrambled peptide showed no effect on cocaine or morphine-induced silent synapse generations.

  • GluA23Y Peptide Impact:

    • Specifically inhibits AMPAR endocytosis affecting morphine-induced silent synapses but not cocaine's effects.

Figures 3 & 4: Dendritic Changes and Spine Analysis

  • Dendritic Changes Post Drug Treatment:

    • Cocaine increases filopodia and long-thin spine density; morphine reshapes spines.

    • Effects are assessed through analyses of spine types: filopodia, long-thin, mushroom, stubby.

  • Key Observations:

    • Morphine's effect is on shape and not number, leading to stable total density.

    • Significant alteration in spine density and shape in cocaine-exposed vs. morphine-exposed states.

Limitations and Future Directions

  • Considerations include the influence of parvalbumin (PV) interneurons in the nucleus accumbens on drug conditioning.

  • Exploration of full addiction-related behaviors and how they correlate with silent synapse dynamics.

  • Questions regarding the criteria for animal subject selection and protocol choices.

  • Need to assess if findings hold for self-administered paradigms versus the administered protocols examined in the study.

Discussion & Unaddressed Questions

  • Three major questions remain:

    1. Is the generation of silent synapses also projection-specific based on the type of medium spiny neurons (MSNs)?

    2. When exposed to cocaine and morphine, do rules of silent synapse generation uphold?

    3. How do D1 and D2 MSN circuits coordinate in mediating drug-related behaviors after chronic exposure and withdrawal?

Follow-Up Studies

  • Continued exploration in related studies includes analysis of cocaine associative memory, distinct roles of D1R vs D2R in drug exposure, and the importance of PV interneurons in addiction behaviors.

  • Key references for follow-up include works by William J. Wright, Nicholas M. Graziane, and Zinsmaier et al. addressing cocaine-induced adaptations and potential cellular mechanisms overseeing addiction.