Dopamine D2 Receptor Activation on Striatal Inhibitory Collaterals

Abstract

The principal neurons of the striatum are GABAergic medium spiny neurons (MSNs), whose collateral synapses onto neighboring neurons play critical roles in striatal function. MSNs can be divided by dopamine receptor expression into D1 and D2 class MSNs, and despite overwhelming evidence for D2 receptors (D2R) in maintaining proper striatal function, it remains unclear how MSN collaterals are specifically altered by D2R activation. This is partly due to the low rate of inhibitory collateral detection between MSNs in conventional ex vivo slice recordings. Furthermore, most studies on MSN collaterals have been conducted either blind or in models in which only one MSN subtype can be distinguished. In this dissertation, I describe a dissociated culture system using striatal and cortical neurons harvested from genetically modified mice. These mice express tdTomato and EGFP downstream of the dopamine D1R and D2R promoters, respectively, allowing for simultaneous distinction between the two major subtypes of MSNs. These neurons develop spines, hyperpolarized resting membrane potentials and exhibit up-and-down states, while also maintaining expression of both fluorophores through time. Paired whole cell patch clamp recordings revealed an enhanced rate of inhibitory functional synapses than previously reported in slice recordings.

Using these cortico-striatal cultures, I report that chronic D2R stimulation, with the D2/3 receptor agonist quinpirole, regulates MSN collaterals in vitro by pre- and post-synaptic mechanisms. Quinpirole increased the rate and strength of collateral formation onto D2R-containing MSNs (D2 MSNs) as measured by paired whole-cell patch clamp recordings. Additionally, these neurons were more sensitive to low concentrations of GABA and exhibited an increase in gephyrin puncta density, suggesting increased postsynaptic GABAA receptors. Lastly, quinpirole treatment increased presynaptic GABA release sites as shown by increased frequency of sIPSCs and mIPSCs, correlating with increased VGAT puncta. Combined with the observation that there were no detectable differences in sensitivity to specific GABAA receptor modulators, I provide evidence that D2R activation powerfully transforms MSN collaterals via coordinated pre- and post-synaptic alterations. As the D2 MSNs are highly implicated in Parkinson's disease and other neurological disorders, these findings may contribute to understanding and treating the changes that occur in these pathological states.