Existing techniques for monitoring neural activity in awake, behaving vertebrates are invasive and often require restraining the animal. Here we propose the use of bioluminescence to non- invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. The photoprotein GFP-apoAequorin (Ga) will be expressed in neurons of larval zebrafish and constituted in vivo with its substrate coelenterazine (CLZN) to form the Ca2+-sensitive bioluminescent sensor GFP-Aequorin (GA). Flashes of luminescence will then report spontaneous and evoked Ca2+ signals in the targeted neurons. These 'neuroluminescence'responses can be recorded with a large-area photon-counting detector while simultaneously monitoring behavior with an infrared-sensitive camera. Pilot studies have shown that transgenic, pan-neuronal GA fish produced large and fast neuroluminescent signals that could be recorded continuously for many days. The relationship of these light signals with the neurons underlying electrical activity will be explored by targeting patch-clamp recordings to individual neurons expressing the protein under pan-neuronal promoters. To explore the limits and the sensitivity of this technique, GA will specifically be targeted to the hypocretin-positive neurons of the hypothalamus, the serotonergic neurons of the raphe nuclei and the dopaminergic neurons of the ventral hypothalamus. To overcome a major limitation of existing bioluminescence monitoring strategies, which require a completely dark environment, we propose an extension of this method for fast temporal gating that is able to count single photons during normal lighting conditions. Thus, this assay will allow us to monitor, with high temporal resolution and stability, the activity of small subsets of neurons during unrestrained, visual behavior over a time period of many days. We believe that the fast, stable properties of GA's report of neural activity along with non- imaging detection strategies can provide a useful, easily implemented tool for monitoring the activity of genetically specified cell types during natural behavior;an attractive alternative to other more technically challenging imaging approaches currently being pursued.

Public Health Relevance

The ability to record from genetically identified neurons in freely behaving animals is highly desirable in modern neuroscience. We propose here a technology based on bioluminescence that exploits the translucence of the larval zebrafish combined with its availability for genetic manipulation. Bioluminescent and calcium sensitive proteins can be targeted to specific neurons of interest and will report neural activity with high temporal resolution and stability, during unrestrained, visual behavior over a time period of many days.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Research Project (R01)
Project #
5R01DA030304-02
Application #
8293110
Study Section
Special Emphasis Panel (ZRG1-CB-Z (56))
Program Officer
Wu, Da-Yu
Project Start
2011-07-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
2
Fiscal Year
2012
Total Cost
$324,200
Indirect Cost
$124,200
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Portugues, Ruben; Feierstein, Claudia E; Engert, Florian et al. (2014) Whole-brain activity maps reveal stereotyped, distributed networks for visuomotor behavior. Neuron 81:1328-43
Ahrens, Misha B; Huang, Kuo Hua; Narayan, Sujatha et al. (2013) Two-photon calcium imaging during fictive navigation in virtual environments. Front Neural Circuits 7:104