The early life programming of neuroimmune function

Microglia are the resident immune cells of the central nervous system (CNS). In adulthood, the role of microglia is to survey the neuronal environment and respond to foreign pathogens and injury. Prenatally, they are involved in the development of the brain. During this early postnatal period, microglia development can be impacted by environmental factors such as diet. Many studies have focused on microglia in terms of disease, so the role that microglia play during the early postnatal period and how microglia impact long-term development remains poorly understood. This thesis investigated the specific role of microglia in postnatal development. In this thesis, we hypothesise that disruption of microglia during the early life developmental period may permanently disrupt the long-term development of the brain. To test our hypothesis, we have used a rat microglia and macrophage ablation model to ablate microglia during the neonatal period at postnatal day (P)7 and P14. We then assessed microglia, astrocytes and immature and mature neurons, acutely (after 48 hrs at P9 and P16 respectively), during the juvenile period at P21 and in adulthood. In adulthood we have also measured if microglia affects memory and motor learning. Previously, we have effectively utilised a Cx3Cr1-Dtr Wistar rat model to acutely ablate microglia from the adult CNS. Here (in Chapter 2), we have established the use of this model during the neonatal period to investigate the acute consequences of microglia ablation on the early postnatal CNS. So, we injected neonatal rats with either saline or diphtheria toxin (DT) at either P7 or P14. After 48 hrs, we assessed how the acute ablation of microglia affected microglial, astrocyte, neuronal and inflammatory cytokine gene expression.  In this study, we were able to establish the ablation model during the early postnatal period, and found that despite reductions in microglia, astrocytes and neurons in the brain remain intact and normal. Next (in Chapter 2) we used this model to assess the effect of microglial ablation on adult microglia and astrocyte phagocytic function. For this study we used flow cytometric analysis and ex-vivo slice culture of repopulated microglia (where microglia numbers have returned to basal levels at 7 days after ablation) to assess the phagocytic ability of microglia and astrocytes when present with 2 µm coated microspheres. Here, we were able to demonstrate that microglia ablation increases the phagocytic capacity of microglia and astrocytes, the former in a brain-region-specific manner. These data provide early evidence that our model adequately functions in the neonatal period and establishes it for use in our future studies, as the characterisation allow us to investigate the long-term effects of microglia on the brain. Following the establishment of our acute microglia ablation model in the neonatal period, we then assessed this model during the adolescent period (at P21) (in Chapter 3) and in adulthood (at approximately P63) (in Chapter 4). Again, animals were injected at either P7 or 14 with either saline or DT and left to develop to their respective ages (P21 or P63). At both timepoints, we assessed microglia, astrocyte and neuron number and gene expression, along with any potential changes to both pro- and anti-inflammatory cytokines in the hippocampus. In the adults, we also assessed if early life microglia ablation had any impact on motor and spatial learning and anxiety. In our young animals (at P21), hippocampal microglia numbers remained low in P7 ablated animals and microglia complexity remained reduced after P14 ablation. As adults, this effect on the key immune cells lingered, which led to a small but significant increase in CA1 mature neuron numbers and a significant increase in astrocyte density in the subgranular/ granular region in the dentate gyrus in adults that had their microglia ablated at P14. Spatial learning and anxiety like behaviour remained unchanged, however performance in the rotarod test of motor learning suffered in microglia ablated animals. Here, our data reveal that transient depletion of microglia during the neonatal period may impact the brain briefly, but long-term consequences may still manifest even though the brain remains relatively unchanged. Our final study (Chapter 5) investigated if juvenile administration of minocycline will be able to ameliorate naturally occurring microglia disruption (obesity) later in life. In this study, we examined microglia, circulating cytokines and pro-and anti-inflammatory cytokine gene expression in the hippocampus and hypothalamus following neonatal overfeeding juvenile administration of minocycline in adulthood. We also investigated if hippocampal-dependent spatial memory is affected. Our data show that microgliosis from neonatal overfeeding is exacerbated in response to the bacterial mimetic lipopolysaccharide (LPS). Minocycline was able to attenuate this inflammatory profile in the hypothalamus but not in the hippocampus. However, despite abrogating the microglia pro-inflammatory profile, it did not reverse weight gain or fat mass. These data indicate that the persistent obesity seen with neonatal overfeeding may be independent of brain inflammation, but that attenuating microglial activity after neonatal overfeeding may still be beneficial for improving responses to neuroimmune challenge. Our initial hypothesis was to determine if disrupting microglia during the early life developmental period may permanently impair the long-term development of the brain. As microglia are crucial to the early life development of the brain, we aimed to investigate the fundamental role of microglia at this time. In the hippocampus we found that disrupting early life microglia may not entirely disrupt the development of the brain long-term. Our findings from this thesis suggest that despite early life insults to brain and potential disruption to microglia, hippocampal development remain intact long term. These findings provide additional insight on how early life microglia can acutely impact dorsal hippocampal development, its effects on spatial memory and how this may manifest in the long term. The dorsal hippocampus is well known to be primarily involved in cognitive function, while the ventral hippocampus is involved in the processing of stress and anxiety (Fanselow & Dong, 2010). It has also been shown that microglia regulate neurogenesis within the dorsal hippocampus (De Lucia et al., 2016). However, other brain regions may not be as resilient as the hippocampus and remain to be tested. We note that other brain regions, including other divisions of the hippocampus may exhibit other effects, however, the results within this thesis suggests that the dorsal hippocampus may clinically play a key role in the limitation of negative effects from pharmacological treatments which are able to cross the blood brain barrier, and thus preserving hippocampal function.