Cortical Contributions to Complex Learning

Robinson, Siobhan; DeRose, James

August 17, 2021

Cortical Contributions to Complex Learning

Sharp Imaging of Chemogenetically Tagged Neurons in Multilayer Rat Cortical Tissue

Intro

This article describes the use of sharp, high contrast imaging to facilitate assessment of viral-construct placement in rat cortical brain tissue. Complex learning is often investigated through use of a rodent model. To avoid the limitations associated with permanent rat brain lesions, chemogenetic-induced temporary inactivations can be conducted. This approach involves using a viral construct to deliver foreign DNA (a receptor) and a fluorescent marker to cells within specific brain regions. Next, temporary inactivation of the region of interest is achieved by administering a pharmacological agent that activates the receptor. Using this approach, learning, memory, motivation and other behaviors can be assessed both with and without brain activity in the region of interest. Critical to this research is verification of correct placement of the viral construct. The assessment of placement is easier with the sharp, haze-free images provided when using the THUNDER Imager Tissue and Instant Computational Clearing (ICC).

Introduction

One approach that neuroscientists use to investigate complex learning is to assess the effects of damage to specific brain regions in rodents. Chemogenetics [1-3] can be used to temporarily, rather than permanently inactive a region of interest. This two-step approach involves viral-mediated delivery of a piece of modified, foreign DNA (a receptor) and a fluorescent tag to cells within a specific brain region. Next, administration of a pharmacological agent activates the receptor and induces temporary inactivation of cells. Thus, investigators can combine chemogenetics with learning paradigms to assess behavior with and without neural activity in the region of interest. Assessment and verification of correct placement of the viral construct is an essential component of the research and requires the use of sophisticated imaging.

Challenges

An imaging solution that can quickly screen thick 3D cortical tissue specimens and achieve sharp, high-contrast imaging, where important details deep inside the specimen are clearly resolved, is important for cortical brain regions. These cortical regions are comprised of six distinct layers of neuron; having the ability to determine which layers contain labeled neurons provides important information about the neural circuitry underlying complex learning [2,3]. Conventional widefield microscopy offers users speed and detection sensitivity, but unfortunately there is often an out-of-focus blur or haze, due to signals from out-of-focus planes of thick specimens, reducing significantly image contrast [4].

Methods

Fluorescence images of rat cortical tissue with neurons expressing enhanced green fluorescent protein (eGFP) were acquired using a THUNDER Imager Tissue with a HC PL APO, 40x, 0.95 NA (numerical aperture) objective and DFC9000 GT camera. Instant Computational Clearing (ICC) was applied to remove the haze [4].

Results

The images below are of neurons in rat cortical tissue which express eGFP.

Fig. 1: A) Single plane image of eGFP-expressing neurons in cortical tissue of a rat brain; B) Same image as in 1A after Instant Computational Clearing; and C) Intensity line profile for 1A (red) and 1B (blue) where the blue line shows the removal of the out-of-focus light in the computationally cleared image (1B).

Conclusions

Conventional widefield fluorescence microscopy images appear hazy. However, use of the THUNDER technology Instant Computational Clearing (ICC) [4] significantly enhances the contrast. The THUNDER Imager allows for easier, more accurate assessment of the correct placement of a viral construct in rat cortical brain tissue compared to typical widefield imaging.