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eGFP-tagging of the dopamine D2 receptor: re-examination of subcellular receptor localization in the substantia nigra and dorsolateral striatum of transgenic mice

Trinkle, Mason (2019) eGFP-tagging of the dopamine D2 receptor: re-examination of subcellular receptor localization in the substantia nigra and dorsolateral striatum of transgenic mice. Undergraduate Thesis, University of Pittsburgh. (Unpublished)

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The release of dopamine (DA) in the dorsal striatum modulates response to cortical input, facilitating fine motor skill planning and goal-directed behaviors. The neurons producing this DA signal originate in the midbrain substantia nigra pars compacta (SNc), where they autoregulate by releasing DA from their soma and dendrites onto D2-type autoreceptors (D2R) of neighboring cells. Exactly how DA transmits through the extracellular space, however, is not fully understood. Electrophysiological evidence suggests that DA release and receipt must be in close proximity to each other, possibly involving non-traditional synapses. This study sought to test for anatomical evidence supporting close proximity of D2Rs to dendrites that could transmit DA in the SNc.
Brain tissue from a transgenic mouse strain possessing a knock-in of enhanced green fluorescent protein (eGFP) on the D2R was labeled with immunogold antibodies directed against eGFP. This immunogold labeling was then analyzed by electron microscopy to determine the subcellular distribution of the D2R. Image analysis showed that in dendrites and axon terminals of the SNc, eGFP-D2R immunogold was found more often intracellularly than along the membrane. Intracellular immunogold was most often associated with the smooth endoplasmic reticulum, where it likely represents a transport form of D2R. Distal dendrites contained proportionally more immunogold on the plasma membrane suggesting specialized DA receipt sites. Immunogold for eGFP-D2R was also found at rare dendrodendritic synapses. Throughout all profiles in the SNc, eGFP-D2R immunogold particles were randomly spread along the membrane and rarely (7%) directly apposed to other dendrites. In a comparative study of the dorsolateral striatum in the same animals, both total and membrane-bound eGFP-D2R were significantly higher than in the SNc and more frequently apposed to dendrites. These results indicate that the findings in the SNc were not unduly affected by methodology. Furthermore, the outcomes are more consistent with volume transmission serving intercellular DA communication in the SNc than non-traditional synapses. A novel discovery was also made that D2Rs are present on the axon initial segments of SNc neurons. These may serve a potent autoregulatory function that has yet to be physiologically defined for DA cells.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Trinkle, Masonmat201@pitt.edumat2010000-0002-4556-5844
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSesack,
Committee MemberBender,
Committee MemberMeriney,
Committee MemberRubio,
Date: 16 April 2019
Date Type: Publication
Defense Date: 25 March 2019
Approval Date: 16 April 2019
Submission Date: 11 April 2019
Access Restriction: 3 year -- Restrict access to University of Pittsburgh for a period of 3 years.
Number of Pages: 80
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Neuroscience
David C. Frederick Honors College
Degree: BPhil - Bachelor of Philosophy
Thesis Type: Undergraduate Thesis
Refereed: Yes
Uncontrolled Keywords: Neuroscience, D2R, mouse, SNc, eGFP-D2R, DA, nigrostriatal
Date Deposited: 16 Apr 2019 19:34
Last Modified: 16 Apr 2022 05:15


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