Projects

Neurocircuitry and cell-type specificity involved in stress and addiction

The nucleus accumbens (NAc) plays a critical role in controlling motivation to obtain reward, and ~95% of neurons are medium spiny neurons (MSNs) chemically coded into two subtypes that selectively express D1 or D2 dopamine receptors. These two populations appear to subserve distinct behavioral functions, with D1-MSN activation generally promoting and D2-MSN activation generally inhibiting behaviors. Here we aim to understand how stress induces enduring adaptations within MSN synapses and whether this encodes information about negative or positive stimuli (e.g. seeking responses precipitated by a stress-conditioned cue). Furthermore, at the molecular level, we observed that activated matrix metalloproteases signal through β3-integrins to sustain drug-seeking elicited by drug-associated cues and to potentiate excitatory synapses in NAcore. We also aim to study whether the coding in D1 or D2-MSN is mediated by β3 integrin.

Role of the pentapartite synapse in stress and addiction

We have been exploring the molecular and cellular underpinnings of stress disorder and made important discoveries in the field, such as that a single stress event induces long-lasting cortico-striatal synaptic potentiation at the pentapartite synapse in nucleus accumbens core (NAcore), which resemble drug induced adaptations. Pentapartite synapse is defined as pre and postsynaptic neurons, astrocytes, microglia and extracellular matrix. Furthermore, we found that by pairing the stressful event with a novel odor (stress cue) in order to elicit a conditioned stress response, we were able to recapitulate some of the behavioral and physiological aspects of PTSD. Here our goal is to first understand how the components of pentapartite synapse communicate and how it occurs temporally. Second, whether the exposure to a stress cue, that is sufficient to reinstate drug-seeking in drug-trained animals, induces neuroplasticity at the pentapartite synapse.

Novel Therapeutic Treatments for PSTD and SUD

We show that a single traumatic event induces enduring cortico-striatal synaptic plasticity and increases the vulnerability to drug use and relapse. Preliminary data show that an increase in our studied immediate early gene in NAc is necessary for drug-reward association, and required for cocaine-reward behaviour. In addition, the studied gene has been involved in synaptic plasticity in NAc induced by drugs and stress. In this project, we proposed to use a short synthetic antisense oligonucleotide to specifically target this specific gene that could be a good target for PTSD and SUD therapies.

Techniques

Calcium Imaging Recordings

Calcium imaging recording uses the genetically encoded calcium indicator, GCaMP, to record the neural activity of genetically defined subpopulations of neurons through a miniscope in freely moving rodents. We use this technique to do cell-type specific recordings in nucleus accumbens core (NAcore) while animals are performing different behaviors task, for example as defensive burying or self-administration. This technique allows us to understand cell-type and temporally specific signaling that encodes information about the underlying neurobiology of stress and addiction.

Confocal Microscopy

Confocal microscopy or confocal laser scanning microscopy is an optical imaging technique for increasing optical resolution and contrast of a micrograph/object. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. Objects like neurons, astrocytes or microglia can be label using viral constructs, or like in the picture using lipophilic colorant DiI (Fig A, B1 & C1). After capturing multiple z-tack, images are exported and analyzed using morphological analysis software, Bitplane Autoquant (Fig B2 & C2). Both techniques are extensively used in the scientific research.

Operant Conditioning

Operant conditioning is a type of learning that involves associating voluntary behaviour with a consequence. We use operant responding for a number of experiments associated with drug voluntary intake, for example cocaine, heroin, cannabinoids or nicotine, or natural rewards intake, for example food or sucrose. We have operant self-administration chambers for mice and rats. This voluntary process is a critical aspect of human drug addiction, and using operant tasks we can dissect the neural circuits involved in discrete components of behaviors associated with motivation. 

Morphological and Protein Analysis

After capturing multiple two-dimensional images at different depths, images are exported and analyzed using morphological analysis software, BitPlane Autoquant. Pictures on the right show AAV1-ef1-dflox-mCherry transfection in NAcore and method for quantifying dendritic protein immunoreactivity in labeled neurons. Micrograph sequence of the method used to quantify p-cofilin (green) expression in NAcore dendritic segments labeled with viral construct (red). 1. Micrograph of segment in the merged confocal imaging showing p-cofilin puncta and Cre-dependent labeling of D1-MSN dendrites.

1. Isolation of the dendritic segment using IMARIS software.
2. Masked dendrite and surrounding p-cofilin signal.
3. Isolated dendritic segment (red) and p-cofilin puncta within the dendrite (white).
4. Quantified colocalization between dendrite and p-cofilin puncta. Scale bar=5µm.