Immediate Early Gene Expression in Striatal D1 and D2 Spiny Projection Neuron during Striatum-Dependent Reinforcement Learning
Session Number
Project ID: BHVSO 13
Advisor(s)
Dr. Jones G. Parker; Northwestern, Feinberg School of Medicine
Madison Martin; Northwestern, Feinberg School of Medicine
Niki Moya; Northwestern, Feinberg School of Medicine
Discipline
Behavioral and Social Sciences
Start Date
22-4-2020 8:50 AM
End Date
22-4-2020 9:05 AM
Abstract
Phasic fluctuations in dopamine are thought to drive divergent changes in D1 and D2 spiny projection neuron (SPN) activity by regulating intracellular signaling and gene expression cascades that modify their excitatory synaptic strength. These changes may be crucial for brain processes such as reinforcement learning. In our research, we quantified the expression of the immediate early gene Fos, a marker of neuronal activity, in D1- and D2-SPNs in mice trained on a head-fixed, striatum-dependent fear conditioning task. We quantified Fos expression in two groups of mice: mice that selectively express the red-fluorescent protein tdTomato in D2-SPNs (A2A-tdTomato mice) and mice expressing a Fos-GFP fusion protein in both SPN types (Fos-GFP mice). Subsequent to training, we harvested brains from mice that either learned or failed to learn to run in place on a wheel while head fixed in response to an auditory tone that predicted a mild tail shock. Immediately after training in A2A-tdTomato mice or 30 min after training in Fos-GFP mice, we euthanized the mice and and transcardially perfused them with a phosphate buffered saline solution followed by the same solution containing 4% paraformaldehyde. After brain fixation, we acquired 100-µm thick brain sections using a vibratome. We then immunostained these brain slices with a rabbit anti-Fos primary antibody and amplified this immunostaining using either a donkey anti-rabbit secondary antibody conjugated to a green fluorophore in A2A-tdTomato mice or conjugated to a red fluorophore in Fos-GFP mice. We then used a two-photon microscope to acquire fluorescent images of striatal tissue. We used ImageJ, Matlab, and Excel to quantify the number and fluorescence intensity of Fos positive D1- and D2-SPNs in A2a-tdTomato mice and the overlap in native Fos immunostaining with Fos-GFP transgene expression in Fos-GFP mice. This information lays the groundwork for future investigations to pinpoint the mechanisms by which striatal neural activity is altered to drive reinforcement learning, which could develop therapies that more precisely target specific domains of dysfunction in diseases associated with the striatum.
Immediate Early Gene Expression in Striatal D1 and D2 Spiny Projection Neuron during Striatum-Dependent Reinforcement Learning
Phasic fluctuations in dopamine are thought to drive divergent changes in D1 and D2 spiny projection neuron (SPN) activity by regulating intracellular signaling and gene expression cascades that modify their excitatory synaptic strength. These changes may be crucial for brain processes such as reinforcement learning. In our research, we quantified the expression of the immediate early gene Fos, a marker of neuronal activity, in D1- and D2-SPNs in mice trained on a head-fixed, striatum-dependent fear conditioning task. We quantified Fos expression in two groups of mice: mice that selectively express the red-fluorescent protein tdTomato in D2-SPNs (A2A-tdTomato mice) and mice expressing a Fos-GFP fusion protein in both SPN types (Fos-GFP mice). Subsequent to training, we harvested brains from mice that either learned or failed to learn to run in place on a wheel while head fixed in response to an auditory tone that predicted a mild tail shock. Immediately after training in A2A-tdTomato mice or 30 min after training in Fos-GFP mice, we euthanized the mice and and transcardially perfused them with a phosphate buffered saline solution followed by the same solution containing 4% paraformaldehyde. After brain fixation, we acquired 100-µm thick brain sections using a vibratome. We then immunostained these brain slices with a rabbit anti-Fos primary antibody and amplified this immunostaining using either a donkey anti-rabbit secondary antibody conjugated to a green fluorophore in A2A-tdTomato mice or conjugated to a red fluorophore in Fos-GFP mice. We then used a two-photon microscope to acquire fluorescent images of striatal tissue. We used ImageJ, Matlab, and Excel to quantify the number and fluorescence intensity of Fos positive D1- and D2-SPNs in A2a-tdTomato mice and the overlap in native Fos immunostaining with Fos-GFP transgene expression in Fos-GFP mice. This information lays the groundwork for future investigations to pinpoint the mechanisms by which striatal neural activity is altered to drive reinforcement learning, which could develop therapies that more precisely target specific domains of dysfunction in diseases associated with the striatum.