Forged Without FIRE: Normal Brain Development in the Absence of Microglia

Abstract

Typical development of synaptic and neuronal properties can proceed without microglia in the cortex and thalamus

O'Keeffe M, Booker SA, Walsh D, Li M, Henley C, Simões de Oliveira L, Liu M, Wang X, Banqueri M, Ridley K, Dissanayake KN, Martinez-Gonzalez C, Craigie KJ, Vasoya D, Leah T, He X, Hume DA, Duguid I, Nolan MF, Qiu J, Wyllie DJA, Dando OR, Gonzalez-Sulser A, Gan J, Pridans C, Kind PC, Hardingham GE. Nat Neurosci. 2025 Feb;28(2):268-279. doi:10.1038/s41593-024-01833-x. Epub 2025 Jan 6. PMID: 39762658; PMCID: PMC11802452.

Brain-resident macrophages, microglia, have been proposed to have an active role in synaptic refinement and maturation, influencing plasticity and circuit-level connectivity. Here we show that several neurodevelopmental processes previously attributed to microglia can proceed without them. Using a genetically modified mouse that lacks microglia (Csf1r^{ΔFIRE/ΔFIRE}), we find that intrinsic properties, synapse number and synaptic maturation are largely normal in the hippocampal CA1 region and somatosensory cortex at stages where microglia have been implicated. Seizure susceptibility and hippocampal-prefrontal cortex coherence in awake behaving animals, processes that are disrupted in mice deficient in microglia-enriched genes, are also normal. Similarly, eye-specific segregation of inputs into the lateral geniculate nucleus proceeds normally in the absence of microglia. Single-cell and single-nucleus transcriptomic analyses of neurons and astrocytes did not uncover any substantial perturbation caused by microglial absence. Thus, the brain possesses remarkable adaptability to execute developmental synaptic refinement, maturation and connectivity in the absence of microglia.

Commentary

The loss of synapses is a form of neuronal plasticity within the nervous system. During development, in response to injury, and neurological disease, microglia can remove synaptic elements. The first described synaptic loss associated with microglia was reported by Blinzinger and Kreutzberg in 1968. This classic work demonstrated that facial nerve transection resulted in retracting synaptic boutons from the injured motor neurons. Synaptic bouton retraction coincided with microglial processes enwrapping the neuronal cell bodies. ¹ The spatial and temporal correlation led to the hypothesis that microglia actively participate in removing or “stripping” synapses from neurons, particularly under pathological conditions. Emerging evidence suggests that in the context of acquired epilepsy, microglia may contribute to aberrant synaptic stripping and, thereby, epileptogenic circuit remodeling.

The long-standing recapitulation-of-development hypothesis in acquired epilepsy proposes that neuronal death triggers the reactivation of developmental processes to replace synapses lost through neuronal death. ² The hypothesis can be exemplified by “mossy fiber sprouting,” which causes the formation of new recurrent excitatory circuits among dentate granule cells. A more general view would be that seizure-induced neuronal death in vulnerable brain regions causes multiple networks to form new excitatory circuits. The recapitulation-of-development hypothesis could also be restated wherein microglia-dependent synaptic stripping, rather than neuronal death, causes the formation of aberrant hyperactive circuits. The upshot to this modified hypothesis is that it incorporates an inflammatory cell component into the epileptogenic process and allows for the possibility that excitatory and/or inhibitory synapses can be eliminated by microglia, culminating in hyperactive neural networks and seizure generation. If this hypothesis is true, then it could explain why some brain injuries that cause neuronal death do not necessarily lead to epilepsy, as the critical mechanism would be removal of relevant synapses without sufficient replacement. In this context, microglia-mediated synaptic stripping could contribute to epileptogenic circuit remodeling in acquired epilepsies.

Microglia-mediated synaptic stripping during normal development has been inferred using mice with genetic knockout of complement proteins ³ and cell surface receptors, including Cx3cr1, Cr3, and Trem2.^4,5 Cx3cr1 global knockout mice show a transient increase in dendritic spines in the CA1 hippocampus at postnatal day 15 (P15) with abnormal electrophysiology. However, Cx3cr1-dependent differences in spines and physiology are rectified by P40. ⁴ In aggregate, these studies support a role for proteins and receptors highly, but not exclusively, expressed by microglia in sculpting circuits by eliminating spines in a complement-dependent manner. Despite the elegant approaches and imaging techniques used in these studies, it is worth noting that using a genetic knockout model of tagging, stripping, and engulfment is problematic because the gene products might have multiple functions. Moreover, tissue imaging has resolution limits and might result in false colocalization of microglial lysosomes or processes with synapses.

The role of microglia in affecting synaptic connectivity and, thereby, brain function has been challenged in studies utilizing an antagonist to colony-stimulating factor 1 receptor (CSF1R) to deplete microglia at P14. Microglia depletion did not affect visual signaling, neuronal tuning in the visual cortex, or ocular dominance plasticity, suggesting normal neuronal development and function in the absence of microglia and their spine-engulfing properties. ⁶ However, the CSF1R antagonist can target nonmicroglial cells, which confounds the interpretation.

Given the seemingly discordant findings regarding the role of microglia in spine stripping and neuronal plasticity, O’Keeffe et al. ⁷ took an alternative approach to address the role of microglia in brain development. The authors utilized Csf1r^{ΔFIRE/ΔFIRE} mice that have a homozygous deletion of the Fms-Intronic Regulatory Element (FIRE) in the promoter of the Csf1r gene. Csf1r^{ΔFIRE/ΔFIRE} mice lack macrophages in the developing embryo, resident macrophages in peripheral tissues including the skin, kidney, heart, and peritoneum, and brain-resident microglia, but the mice still have functional monocytes, brain-resident perivascular macrophages, and are healthy and fertile. ⁸ Operating on the premise that microglia-mediated synaptic stripping is essential for proper neuronal development and function, the authors examined the Csf1r^{ΔFIRE/ΔFIRE} mice and Csf1^r+/+ littermate controls for several intrinsic neuronal properties, including spine density and synaptic maturation and transmission in the hippocampal CA1 region and somatosensory cortex. For each parameter examined, the microglia-depleted Csf1r^{ΔFIRE/ΔFIRE} mice behave nearly identically to microglia-sufficient Csf1r^+/+ littermate controls. Seizure latency and duration induced by pentylene tetrazole were not different in Csf1r^{ΔFIRE/ΔFIRE} mice lacking microglia compared to microglia-sufficient controls. The authors conclude the brain can execute developmental synaptic refinement, maturation, and connectivity without microglia.

The authors must be commended for their systematic histological, electrophysiological, and behavioral examination of the Csf1r^{ΔFIRE/ΔFIRE} mice. Using both male and female mice increases experimental rigor and robustness, and the statistical tests are appropriate for each experimental condition. Sample size numbers were calculated to achieve satisfactory power based on the variance and effect size. Nevertheless, given that no statistically significant differences are observed in the absence of microglia, providing the post hoc statistical power would have been informative.

The genetic strain of the Csf1r^{ΔFIRE/ΔFIRE} mice was a mixed C57BL/6J and CBA background. This is notable because Csf1r^{ΔFIRE/ΔFIRE} mice on a congenic C57BL/6J background show perinatal lethality, and surviving mice develop hydrocephalus. ⁹ It is unclear if the early lethality and abnormal brain in the congenic Csf1r^{ΔFIRE/ΔFIRE} strain is due entirely or in part to the absence of microglia or another tissue-resident macrophage population. However, the observations in the congenic line indicate a more complex biology with genetic background as a profound phenotypic modifier and must be considered when interpreting the absence of overt phenotype on the mixed genetic background.

Despite exhaustive research concerning the role of microglia in organizing neuronal circuitry in epilepsy, several critical issues remain unresolved. First, it remains unclear if aberrant formation or removal of spines and synapses contributes to epileptogenesis or is a consequence of seizures. One of the experimental challenges in addressing this issue includes the complementary concerns of false positives (specificity) and false negatives (sensitivity). Thus, although it is quite challenging to quantify spine stripping and synaptic loss, it is even more difficult—if not impossible—to show that stripping or loss has not occurred. Second, the relevant cell types involved in spine and synaptic plasticity must be identified, and their roles must be defined. Intracellular cell signaling within neurons, such as the Ras-PI3k-mTOR and Ras-MAPK pathways, are involved in spine plasticity. ¹⁰ Microglia could mediate an important but nonessential role in the formation of functional neuronal circuits, as suggested by the work of O’Keeffe et al. ⁷ In a mouse model of multiple sclerosis, monocyte-derived macrophages initiate demyelination and are highly phagocytic. ¹¹ Thus, brain-invading monocytes could also play a role in the development of acquired epilepsies, but more work is required to demonstrate this. Finally, the neuronal structures being formed and eliminated require better characterization. Although spines serve as the primary locations for excitatory inputs, synapses can form at other sites. Thus, spine stripping might occur during development and disease without broad synapse elimination. Until these issues are addressed experimentally and the mechanisms and relevant cells driving spine and synapse elimination are identified, there remains a roadblock to developing therapies targeting synaptic stripping to limit or prevent acquired epilepsy.