<em>Ex Vivo</em> Optogenetic Dissection of Fear Circuits in Brain Slices

Abstract

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

A peptidergic amygdala microcircuit modulates sexually dimorphic contextual fear

“…5C and Fig.S5). The electrophysiological and morphological properties of mICC neurons in our studies matched previously described properties of mICC neurons [15][16][17].…”

“…We wanted to determine if altering the activity of PACAPergic neurons that innervate the mICCs changes fear behavior. A previous study showed that the ex vivo optogenetic method can be used to analyze and study the role of intercalated cells in regulating fear behaviors, so we utilized optogenetics to answer our question [16]. For these experiments, we used the Adcyap1-2A-Cre mice and carried out the same intersectional virus labeling strategy described previously.…”

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

“…9. Make a skin incision of approximately 1 cm on top of the head using scissors 20 ( Figure 1B,E) and a hinged "double replica table" (see Figure 3B), which when opened produces a tensile fracture through the frozen specimen (Figure 2). This allows to retain and replicate both sides of the fractured specimen.…”

Mistletoe-Based Drugs Work in Synergy with Radio-Chemotherapy in the Treatment of GliomaIn VitroandIn Vivoin Glioblastoma Bearing Mice

“…Murine hippocampal organotypic slice cultures were prepared as previously described [50]. After one week in culture the slices were treated for 24 or 48 h with ISCADOR Qu, Aviscumine, and native ML-1 or were left untreated.…”