Research reportMicroglia depletion in early life programs persistent changes in social, mood-related, and locomotor behavior in male and female rats
Introduction
Microglia are the innate immune cells of the central nervous system. Microglia colonize the rodent brain beginning on embryonic day 9.5 and reach peak numbers by the third postnatal week [15], [25], [40]. They release a variety of diffusible factors, such as chemokines and cytokines, and express a variety of receptors that allow them to respond to and modulate events under normal and abnormal circumstances in the brain. In the developing brain, microglia have been shown to prune synapses and regulate neurogenesis, apoptosis, and axonal innervation [7], [41], [45], [47], [51]. While many of these studies have investigated microglia-regulated processes in a cellular developmental context, few studies have examined whether microglia influence behavioral development in the absence of inflammation, stress or other pathology.
Previous studies have investigated the behavioral effects of depleting microglia from the adult rodent brain, and these studies have found very limited and transient effects of microglial depletion on social and anxiety behaviors [10], [55]. In development, models that are thought to activate microglia or alter their function, such as perinatal stress or inflammatory challenge, show changes in social, anxiety and despair-like behavior, as well as stress reactivity [6], [20], [31], [32], [37], [56], [63], [68]. These results suggest that microglia may be more important for developmental programming of behavior than the maintenance of behavior in adulthood. However, it is still unclear whether basal microglial function during development directly impacts the development of these behaviors and their expression later in life.
One strategy to determine the role of microglia on the development of later life behavior is to selectively deplete microglia through the use of liposomal clodronate. Clodronate is a cytotoxic drug that, when encapsulated in lipid droplets, is selectively taken up by phagocytic cells, where it subsequently induces apoptosis [60]. Liposomal clodronate specifically depletes macrophages while sparing other cell types, such as neurons, oligodendrocytes and astrocytes, when centrally injected into the central nervous system [12], [28], [59]. In the current studies we used central infusion of liposomal clodronate to deplete microglia from the developing brain and test whether microglia regulate the development of motivated behaviors in male and female rats. We found that microglia depletion in the early postnatal period led to decreased anxiety behavior and depressive-like behavior and increased locomotor activity in male and female rats, and reductions in stress-induced corticosterone release in females. The current studies underscore that microglia are necessary for the normal development of several motivated behaviors.
Section snippets
Animals
All procedures were conducted in accordance with The Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and approved by The Ohio State University Institutional Animal Care and Use Committee. One cohort of age matched juvenile and virgin adult male and female Sprague Dawley rats from four separate litters were used for all behavioral testing. Adult Sprague Dawley rats (Harlan) were mated in our facilities, or timed pregnant animals were ordered to
Iba1 immunofluorescence analysis
Fig. 2 shows representative pictures of microglia from each time point in the mPFC (left) and amygdala (right), and Fig. 3 summarizes microglial counts and densitometry across development following neonatal treatment with liposomal clodronate or vehicle.
In the mPFC, microglia counts per region of interest (ROI) indicated that microglia numbers were significantly reduced by 87.3% in clodronate treated rats compared to control treated rats at P2 (Fig. 3a; treatment: F1,8 = 85.4, p < 0.001; sex: F1,8 =
Discussion
In the current studies, we assessed the role of neonatal microglial depletion on behavioral development in male and female rats, and found that temporary neonatal loss of microglia perturbs the development of anxiety, social, and locomotor behaviors as well as the adult stress response. We found few sex differences in the effects of neonatal microglial depletion, suggesting that microglia during the neonatal period do not regulate baseline sex differences in the development of many of these
Acknowledgements
These studies were funded by NIHR21MH105826, NARSAD Young Investigator Award, and The Ohio State University Startup funds to KML and OSU Graduate Fellowship to LHN.
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