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Techniques | Approaches

TECHNIQUES: In the Bruchas laboratory we utilize a multidisciplinary approach to dissect the functional role of G-protein coupled receptors within intact circuits and systems.  In doing so, we use a variety of classical and cutting-edge technical approaches.  While we utilize these diverse series of methods, we strongly feel that techniques don't define science, that in fact a more mechanistic answer to a question is the goal.  In addition, we also develop novel engineering approaches and biological tools as necessary to better understand how GPCRs function in vivo to influence affective behavior.  This type of research requires a careful transition back-and-forth between in vitro studies, and in vivo studies.  Our most commonly used approaches are outlined below.

In vitro methods

Cell/Molecular

  • PCR, qRT-PCR, cloning, vector design, point mutations
  • primary cell culture [neuronal/glial culture]
  • transient and stable transfection of heterologous and primary cell types
  • confocal fluorescent microscopy, live cell imaging, receptor trafficking
  • optogenetic manipulation of cell signaling pathways
  • protein trafficking
  • radioligand binding
  • cAMP assays
  • Phospho-inositol hydrolysis (IP3 generation)
  • Calcium Imaging (Fluo-4, Fura-2)
  • primary culture

Biochemical

  • western blotting
  • immunoprecipitation
  • antibody isolation and purification
  • protein phosphorylation assays
  • structure-function analysis
  • Bioluminescence Resonance Energy Transfer (BRET)

In vivo methods

Behavioral

  • conditioned place preference/aversion
  • operant behaviors (i.e.; self-stimulation, sucrose, food).
  • social defeat stress
  • analgeisa/pain assays
  • social interaction/avoidance
  • optogenetics
  • morris water maze, novel object recognition
  • swim stress immobility
  • anxiety-like behaviors 
  • (elevated plus/zero maze, open field, light/dark box)

Physiology

  • In vivo optogenetics
  • In vivo single-unit, multichannel recordings
  • Novel optogenetic, and physiological hardware engineering and development
  • In vivo calcium imaging

Biochemical/Pharmacological

  • immunohistochemistry
  • local infusions
  • protein isolation
  • pump implantation

Surgical

  • rodent anesthesia
  • stereotaxic intracranial microinjection
  • intracranial cannulation, optogenetic implantation
  • GRIN lens implantation

Genetics / viral strategies

  • gene-trageting/LoxP-cre mouse genetics
  • viral construct generation and preparation
  • Adeno-associated (AAV) viral expression
  • Lenti-viral vector expression