Methods

Optogenetics

Optogenetic methods are used to control the neuronal activity in a specific brain region. We combine the genetic and optical approaches to test the role of particular groups of neurons and their projections in feeding behavior and stress response in freely-moving rats. Genetic methods include viral expression in the targeted neurons the excitatory (channelrhodopsin, ChR2; transfected with AAV2-hSyn-ChR2-eYFP) or inhibitory (halorhodopsin, eNpHr; transfected with AAV7-hSyn-eNpHr3.0-mcherry-WPRE) light-sensitive opsins or control transfection with eYFP (using AAV2-hSyn-eYFP). A chronically implanted optic fiber is used to directly flash the light onto the expressed opsins. Blue light (465 nm) is used to excite ChR2-expressing neurons and orange light (620 nm) is used to inhibit eNpHr-expressing neurons.

Methods

In our optogenetic system we use two sources of light (LEDs) per behavioral chamber that is Med Associates automatic chamber allowing automatic detection of the behavioral feeding events (using infrared detectors at lickometers, feeder head entry detectors, etc.) and video recordings during optical stimulation of targeted neuronal groups in freely-moving rats.

Methods

A screenshot of software that we use to define the parameters of optical stimulation including the sources of light, the type and number of LEDs as well as the frequency, duration and intensity of light.

Methods

An example of expression of halorhodopsin (reporter gene is mcherry, fluorescent in red) in the paraventricular hypothalamic nucleus (PVN).

Methods

An example of expression of channelrhodopsin (reporter gene is YFP, fluorescent in green) in the nucleus accumbens shell (AcbSh).