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.
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.
References
- D.I. Fournier, H.Y. Cheng, S. Robinson, T.P. Todd, Cortical contributions to higher-order conditioning: a review of retrosplenial cortex function, Front. Behav. Neurosci. (2021) vol. 15, art. 682426, DOI: 10.3389/fnbeh.2021.682426,
- E.M. Taylor-Yeremeeva, S.C. Wisser, T.L. Chakoma, S.J. Aldrich, A.E. Denney, E.K. Donahue, J.S. Adelman, P.C.J. Ihle, S. Robinson, Appetitive and aversive sensory preconditioning in rats is impaired by disruption of the postrhinal cortex, Neurobiol. Learn & Mem. (2021) 107461 DOI: 10.1016/j.nlm.2021.107461.
- S. Robinson, T.P. Todd, A.R. Pasternak, B.W. Luikart, P.D. Skelton, D.J. Urban, D.J. Bucci, Chemogenetic silencing of neurons in retrosplenial cortex disrupts sensory preconditioning, J. Neurosci. (2014) vol. 34, iss. 33, pp. 10982-10988, DOI: 10.1523/JNEUROSCI.1349-14.2014.
- J. Schumacher, L. Bertrand, THUNDER Technology Note: THUNDER Imagers: How do they really work? Science Lab (2019) Leica Microsystems.
Related Articles
-
Central Nervous System (CNS) Development and Activity in Organisms
This article shows how studying central nervous system (CNS) development in Drosophila-melanogaster…
May 12, 2023Read article -
Neural Basis of Breathing, Vocalization, and Diseases of the Larynx
This article shows how research on diseases of the larynx can be improved with a THUNDER Imager and…
May 02, 2023Read article -
Going Beyond Deconvolution
Widefield fluorescence microscopy is often used to visualize structures in life science specimens…
Mar 22, 2023Read article