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). Previously we found that microglia populate the VTA at significantly lower density and exhibit sparse branching 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 morphologies. In the past year, we have expanded these analyses to show that there is no clear correlation between BG microglial density and neuronal density, as assessed by immunostaining for NeuN. However, similar analysis of overall cell density through DAPI staining showed that there is a consistent microglia-to-neuron ratio in the VTA, SNc, and NAc, suggesting that overall cell density may be one factor that regulates microglial cell numbers in these brain regions. In addition, we performed 3D whole-cell morphological reconstructions that demonstrate that NAc microglia have much greater process branching complexity and total process length compared to other BG microglia, which could suggest a heightened interaction with nearby synapses or other surrounding structures. We are currently performing analysis of glutamatergic synapse density within the basal ganglia through immunostaining for VGlut1, 2, and 3 to determine if microglial process branching complexity is related to the number of excitatory synapses within the surrounding tissue. To determine if these regional differences in microglial structure and distribution are accompanied by distinctions in functional status, we analyzed the abundance and localization of lysosomes within BG microglia through immunostaining for CD68. This analysis indicated that SNr microglia have elevated lysosome content compared to other BG microglia, suggesting that they differ from their counterparts in their phagocytotic or metabolic status. Differences in microglial functional status have been associated with shifts in their membrane properties. Previously, we used electrophysiological recording from microglial cells to show that microglia in the VTA and the adjacent SNr differed significantly in their membrane capacitance, resting potential, and the expression of voltage-gated potassium channels. We have expanded this analysis to include microglia in the SNc and have used pharmacology to more accurately demonstrate the identity of the potassium channels expressed by most SNr microglia. To complete our analysis of regional differences in microglial functional status, we have developed a workflow that allows whole transcriptome RNA sequencing of microglial cells isolated from distinct BG regions as well as the cortex (Ctx). Known microglial genes were expressed at high levels in all samples and genes expressed by neurons, astrocytes, and oligodendrocyte lineage cells were absent, confirming that pure populations of microglia had been isolated from each brain region. RT-PCR analysis of purinergic receptor expression from an independent cohort of animals showed the same pattern of expression as that observed by RNAseq, supporting the quality and reliability of the RNAseq dataset. While a core set of genes were expressed by microglia in all the analyzed brain regions, 34% of genes were expressed by microglia in only one brain region. In particular, VTA microglia exhibited the greatest number of uniquely expressed genes as well as the greatest number of genes that were expressed at significantly higher or lower levels than those observed in microglia from other brain regions. We are currently working to use independent techniques to validate some of the specific genes that appear to be uniquely expressed by microglia in specific BG nuclei. Together, these findings indicate that there are functional differences among microglia in different regions of the BG and raise questions about whether BG microglia will exhibit variable responses to pathological changes in BG circuit activity. Future experiments will use this RNA sequencing approach to determine how BG microglia and astrocytes are altered following both acute and chronic exposure to cocaine. To further define whether these cells influence the membrane properties and synaptic transmission of neurons within the BG, we are using transgenic strategies to ablate microglia within the CNS (CX3CR1-CreER;rfs-DTA mice). Recently, we have carried out important control experiments to demonstrate that this approach does in fact kill microglial cells. We used immunostaining for caspase3 in the early stages of ablation to show that this marker of programmed cell death can be detected within BG microglia. In addition, if microglia are allowed to repopulate the CNS following ablation and mice are treated with BrdU during this repopulation, numerous BrdU+ microglia are observed, indicating that the cells had been eliminated and were undergoing cell division to reinstate their numbers, as opposed to having temporarily downregulated key microglial cell markers. Experiments aimed at determining whether microglial elimination influences the membrane properties of BG neurons or drug-induced plasticity and behaviors are ongoing. Aging is a major risk factor for numerous neurodegenerative diseases. The changes that microglia undergo during normal aging and whether they influence the degenerative susceptibility of specific neuronal populations has not been well explored. To determine whether the regional differences in microglia phenotype that we observed in the young adult are preserved throughout life, we examined BG microglia in 18 month and 22-24 month old mice. Significant increases in microglial density were observed in the NAc, VTA, SNc, and SNr by 22 months of age. However, the magnitude of this increase was much greater in the VTA and significant increases in microglial density were already apparent in the VTA at 18 months of age. Regional differences in microglial process branching complexity are maintained at 18 months of age, although overall tissue coverage by microglial cell processes and somas is increased in all analyzed regions. Future analysis will address whether this increase in tissue coverage is due to increased soma size, increased soma number, increased process thickness, or increased process branching. Intracellular inclusions of undegradable proteins and lipids, known as lipofuscin, are observed in cells throughout the BG by 18 months of age. Preliminary analysis indicates that the majority of this material is accumulated in either neurons or microglia, and not other CNS cells. Future analysis will quantify whether there are regional differences in the extent of neuronal or microglial lipofuscin accumulation and whether this is related to the degree of increased microglial density.
|De Biase, Lindsay M; Schuebel, Kornel E; Fusfeld, Zachary H et al. (2017) Local Cues Establish and Maintain Region-Specific Phenotypes of Basal Ganglia Microglia. Neuron 95:341-356.e6