Experimental Approaches and Techniques

Our lab uses several methods to administer drugs to animals in order to examine the neurobiological and behavioral alterations due to drug and stress exposure. We use both involuntary and voluntary methods to modulate neural circuits and behavior.

Free and Voluntary intake: We use free and voluntary oral intake (top) to quantify consumption when no effort is required to obtain drug.

Operant Conditioning: Operant conditioning (middle) is a type of learning in which animals learn to associate an action with a consequence. Using operant conditioning, we can not only learn about animal's voluntary consumption, but we can also learn about their motivation for consumption. We use intravenous self-administration (middle-left) or oral self-administration (middle-right) with operant conditioning.

Drug inhalation: Our lab uses drug inhalation (bottom) to examine the effects of drug exposure on specific neural circuits which then inadvertently drive maladaptive behaviors and affective disorders.

We use these types of drug administration in combination with other techniques in the lab to dissect circuits involved in drug taking and seeking in order to separate how motivation drives circuitry changes. In addition we use operant conditioning and drug inhalation techniques to identify neural circuits affected after drug exposure.

Fast scan cyclic voltammetry (FSCV) is an analytical chemistry technique used to detect release and clearance of oxidative species from the synapse. It can be performed in an awake or anesthetized animals (in vivo) or in brain slices harvested from animals (ex vivo). Our lab uses this technique to assess dopamine release, dopamine transporter kinetics, and the function of other presynaptic membrane receptors that modulate dopamine transmission. FSCV uses a carbon-fiber microelectrode to oxidize and reduce dopamine. The change in current resulting from this redox reaction is a unique neurochemical signature of dopamine. FSCV has good spatial and temporal resolution. Furthermore, FSCV can be used in combination with other techniques to understand how a particular receptor modulates dopamine. We can then correlate the changes in receptor function or dopamine dynamics with the behavior obtained in our self-administration experiments.

Fiber photometry is an optics-based method used to monitor the neural activity of fluorophore-labelled discrete populations of neurons in freely moving animals. To measure this activity, genetically encoded fluorescent calcium indicators, such as GCaMP, are infused into brain regions of interest in a Cre-driven rat line. A chronically implanted fiber optic delivers excitation light to a targeted population of neurons tagged with these fluorescent calcium indicators and records the overall activity induced fluorescence. An epoch-based analysis allows correlation between this recorded neural activity and animal's behavior.

Optogenetics is a combination of light and genetic engineering. It allows us to control the activity of neurons using light-responsive ion channels, ultimately allowing us to excite or inhibit a selective population of neurons with light of a specific wavelength. A combination of viral vectors (or one viral vector) tagged with a fluorescent proteins are infused into brain region(s) of interest - often times in transgenic animals expressing promoter proteins in a specific population of neurons. This enables us to increase our specificity and we can modulate a single population of neurons. In our lab, we use optogenetics in conjunction with ex vivo voltammetry (bottom left) and behavioral (bottom right) methods to order to examine specific circuits in isolation.

Retrograde tracing helps us identify the source of projections to a selected region. Tracing can be accomplished using several methods.

Selective mono-synaptic tracing (top): Mono-synaptic projections and their localization can be identified using the retrograde labeling CAV2-Cre virus in conjunction with a Cre-dependent anterogradely transporting AAV virus. This dual virus approach increases specificity of projection identification and permits identification of cell-specific synapse formation from projecting neurons.

Non-selective mono-synaptic tracing (right): For simple mono-synaptic tract tracing and neuronal population identification, we use retrograde labeling beads with fluorophores.

Microscopy allows magnification and imaging of neuronal cells, including axons, synapses, and even synaptic vesicles. Fluorescence microscopes magnify images up to 60X the original and permits identification of distinct population of fluorophore-labelled neurons. Furthermore, it allows localization of specific neuronal projections and identification of membrane proteins that carry a fluorescent reporter protein. In addition to fluorescence microscopy, our lab uses confocal microscopy. Confocal microscopy allows for greater spatial resolution, thus increasing our ability to obtain three-dimentional reconstruction of projection tracts.

Our lab uses in vivo microdialysis — in awake and freely moving animals — in conjunction with high performance liquid chromatography (HPLC) to quantify norepinephrine, dopamine, and serotonin from various brain regions. A concentric and perforated probe is implanted through a guide cannula into the regions of interest. Artificial cerebro-spinal fluid is perfused through the probe, and brain samples are collected during the experiment. Dialysate samples are analyzed for neurotransmitters of interest using HPLC.