In order to define the basic properties of microglia within the basal ganglia (BG) of the adult CNS, we used CX3CR1-EGFP transgenic mice to visualize microglia within the ventral tegmental area (VTA), nucleus accumbens (NAc), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr). In these transgenic mice, EGFP is expressed specifically within microglia and allows both the density and fine morphology of these cells to be quantified. Furthermore, EGFP expression allows microglia in acute brain slices to be targeted for electrophysiological recording and analysis. These analyses revealed that microglial cell properties are not uniform across distinct BG regions. Microglia within the VTA are found at significantly lower density and exhibit very sparse branching and simplified morphology compared to other BG regions. In contrast, microglia within the SNr are present at a dramatically high density and both SNr and NAc microglia display highly-ramified and complex morphology. Electrophysiological analysis revealed similar heterogeneity of microglia within these brain regions. Microglia in the VTA and the adjacent SNr differed significantly in their membrane capacitance, resting potential, and the expression of presumed delayed rectifier potassium channels. Together, these findings indicate that microglia throughout the brain cannot be considered equivalent. In addition, this heterogeneity raises important questions about whether microglia in distinct BG regions have different capacities to interact with surrounding neurons and whether their response to CNS insults will differ. Ongoing experiments include acute and chronic administration of drugs of abuse as well as lipopolysaccharide, an inflammatory agent, to probe for differential reactive responses within these cells. We have also used CX3CR1-EGFP mice to visualize microglia within the BG of the developing CNS, with a particular focus on the VTA and NAc. Quantification of microglial cell density during early postnatal periods (P6-8, P10-12, P14-16, P20-22) revealed that significant differences were present between the VTA and NAc, indicating that the regional heterogeneity observed in the adult is established shortly after microglia begin to colonize the CNS. As has been observed in other brain regions, density of microglia in the VTA and NAc peaked during the second and third postnatal weeks and then begin to taper to adult levels. However, microglial density peaked earlier in the NAc, suggesting that maturation of microglia within these two brain regions does not follow an identical time course. Dramatic changes in cell morphology across development were also evident, as microglia in both the NAc and the VTA transitioned from very simple, thick, bipolar branches to the thin, highly- ramified cell processes present in the adult. Future directions for this developmental analysis include characterization of additional brain regions, such as the medial prefrontal cortex, and experiments aimed at determining whether microglia in the early postnatal BG are engaged in phagocytosis of synapses. If microglia participate in circuit refinement within these brain regions, this raises important questions about the developmental consequences of perturbations of microglial cell function, such as childhood infections that penetrate the CNS. The data summarized above expand our understanding of microglial cells within the developing and adult BG. To begin to define whether these cells contribute to circuit refinement during development and whether they influence membrane properties and synaptic transmission of neurons within the BG of the adult, we are using transgenic strategies to specifically ablate microglia within the CNS. CX3CR1-CreER mice, which express inducible cre recombinase within microglia have been crossed to both rosa-flox-stop-diphtheria toxin (rfs-DTA) mice and rosa-flox-stop-diphtheria toxin receptor (rfs-DTR) mice. In double transgenic mice, injection of 4-hydroxytamoxifen activates Cre within microglia and those cells begin expressing DTA or DTR. In CX3CR1-CreER;rfs-DTA mice, this direct expression of diphtheria toxin within microglia causes the cells to undergo programmed cell death. In CX3CR1-CreER;rfs-DTR mice, subsequent injections with diphtheria toxin will only affect those cells expressing DTR, again pushing microglia into programmed cell death. We are currently characterizing the appropriate dose and administration regimen of 4-hydroxytamoxifen and diphtheria toxin to achieve near-complete ablation of CNS microglia and are defining the time window during which the cells remain absent from the CNS. In the future, these mice will allow us to determine whether microglia influence the basic properties of neurons in VTA and NAc and whether there are changes in the ability to induce synaptic plasticity and changes in drug-related behaviors in the absence of microglia. Furthermore, ablation of microglia during early postnatal periods will allow us to perturb microglial phagocytosis of synapses and to probe whether this results in lasting deficits in circuit function, such as a heightened susceptibility to the development of drug-seeking behaviors.

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National Institute on Drug Abuse
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