Microglial cells, the primary immune cells of the brain and central nervous system, are known to undergo striking morphological changes in response to sensory or neural stimuli. However, the mechanisms and specific signals which cause these changes remain unknown.
In this study published in the journal Science Advances, researchers used visual flickering sensory stimulation to reveal the underlying mechanisms governing microglial morphology and cytokine expression in mice.
They found that stimulating neural activity using different flickering frequencies produced significantly different microglial and cytokine responses and identified the critical causal role of nuclear factor κB. Their results elucidate how brain rhythms affect brain function.
Brain rhythms and immune function
Brain rhythms are distinct patterns of neuronal activity associated with specific behaviors, arousal levels, and sleep states. These rhythms change in response to environmental stimuli and specific task requirements (e.g., increased attentiveness). The two primary brain rhythms are the gamma rhythm (~30 to 100 Hz), associated with attention and sensory processing, and the beta rhythm (~12.5 to 30 Hz), associated with suppressing motor behaviors and active concentration.
The influence of brain rhythms and neural entrainment (the process by which the neural activity phase syncs with sensory rhythms) on neuronal electrical function is well documented. However, the influence of these processes on other brain functions, including immune response, remains understudied.
Microglial cells are a specialized population of macrophages found in the brain and central nervous system (CNS). Like other macrophages, they are responsible for the detection, phagocytosis (engulfment), and destruction of foreign potential pathogens and old and damaged somatic cells.
Microglia are the primary immune cells of the brain and CNS. They have been found to secrete extracellular signals and are the predominant source of cytokines, proteins responsible for immune cell growth and activity.
Previous work has shown that microglia dynamically alter their morphology and processes in response to sensory and neural activity changes. They exist in three main states – elongated phenotypes that scan their surrounding environments, shortened phenotypes with enlarged soma when pathogens are detected, and ameboid phenotypes lacking processes for pathogen removal.
Microglial and cytokine response mechanisms to specific neural activity patterns have hitherto been shown to up- or downregulate these cells and protein, respectively. Previous studies by the research group that conducted this study have demonstrated that specific flickering sensory stimulation (flicker at 40 Hz) induces microglial recruitment for pathogen removal in mice with Alzheimer’s disease.
Other work, however, has shown that microglia respond differently in diseased and healthy individuals (the pathological context), so these stimulations’ effects on healthy animals remain uncharacterized.
Cytokines are secreted by microglia, and to a significantly reduced extent, by neurons. Protein expression is governed by phospho-protein signaling pathways which are present in both cell types. The nuclear factor κB (NF-κB) is one of the main signaling pathways, and flicker at 40 Hz has been shown to increase activation in this pathway in Alzheimer’s disease murine models.
This makes the NF-κB a prime candidate for investigating the effects of sensory stimulation and brain rhythms on microglial responses in healthy animals.
What are flicker experiments, how are they used, and what do they tell us?
‘Flicker’ refers to a noninvasive form of visual sensory stimulation in which animals are exposed to light pulses at specific frequencies. In the present study, researchers used 20 (beta rhythm frequency) and 40 Hz (gamma rhythm frequency) flickers on murine models to evaluate the role of brain rhythms in altering microglial structure and function and cytokine secretion via NF-κB.
Researchers first verified that flicker elicited the corresponding activity patterns in the primary visual cortex using wide-field voltage imaging, a technique that detects voltage fluctuations within neurons. Their results revealed strong neural responses in the primary visual cortex at both 20 and 40 Hz, consistent with beta and gamma brain rhythms, respectively.
Exposure of mice to an hour of flicker elicited significant changes in microglial morphology – 20 Hz exposure induced the elongated scanning phenotype, while 40 Hz exposure caused the ameboid engulfing phenotype. However, these changes did not correlate with changes in Allograft inflammatory factor 1 (Aif1) transcription. Iba-1, the protein encoded by Aif1, is generally unregulated during the activation of microglial cells.
To elucidate the molecular mechanisms and immune-related gene pathways governing microglial morphological responses to beta and gamma rhythms, researchers employed RNA sequencing analyses. They found that 20 Hz and 40 Hz flickers elicit differential transcription factor expression, particularly those that control cytokine expression.
These results were consistent with the authors' work on Alzheimer’s disease mice, albeit more subdued. This implies that healthy and diseased brains respond differently to frequency-specific cytokine modulation.
RNA sequencing results further revealed that flicker stimulation induces cytokine expression by both microglial and neuron cells. Mice whose microglia were depleted before flicker exposure were used to identify which cytokines were expressed by microglia versus neurons – cytokines expressed by microglia would show decreased expression. In contrast, those expressed by neurons would have their expression levels intact compared to normal mice.
Single-nuclear RNA-sequencing (snRNA-seq) was used to identify and characterize genes and their differential expression in microglia and neurons.
Although 20 and 40 Hz are modestly different stimuli, differential gene expression analysis revealed that 40 Hz flicker had pronounced effects on L2/3, L6, and L5 neuronal populations, with fewer differentially expressed genes (DEGs) in oligodendrocytes, astrocytes, and microglia/PVMs…
Prichard et al. (2023)
Flicker at 40 Hz was found to induce the upregulation of genes responsible for neurotransmission and synaptic plasticity. Genes associated with transcription pathways and cell signaling, especially those related to NF-κB and/or mitogen-activated protein kinase (MAPK), were also upregulated suggesting that 40 Hz (gamma rhythm) is responsible for alterations in multiple biological functions across cell types.
To evaluate the role of MAPK and NF-κB signaling pathways in cytokine expression, researchers treated mice with inhibitors of these pathways prior to flicker exposure. They found that expression was significantly downregulated in treated mice compared to those without pathway inhibition.
In sum, our data show that different frequencies of flicker elicit different brain rhythms with divergent microglial and cytokine responses. Furthermore, our results show that much of the 40 Hz flicker–induced expression of cytokines arises from microglia, although not all, and that changes in microglia and cytokine expression depend on pNF-κB.
Prichard et al. (2023)
The study used flicker experiments on healthy mice to elucidate the mechanisms underlying microglial morphology change and cytokine expression, the two most important mediators of brain and CNS immune response.
Their results revealed that 20 (beta) and 40 Hz (gamma rhythm frequency) flickers elucidate significant and distinct morphological alterations in microglia and altered cytokine expression by both neurons and microglial cells.
Results confirmed the hypothesis that the NF-κB signaling pathway plays a crucial role in cytokine expression. The researchers suggest their work will help uncover the mechanisms of brain neuron-microglia communication when responding to flicker and flicker-induced brain rhythms.
In conclusion, these results reveal the mechanistic model under which neural/brain rhythm activates neuronal NF-κB phospho-signaling, resulting in cytokine release from these cells. This, in turn, alters microglial functional morphology.
Furthermore, because we find that flicker effects generalize beyond the context of Alzheimer’s pathology, this sensory-induced and brain rhythm–induced regulation of microglia and cytokine responses could be harnessed to mitigate pathological neuroimmune activity present in multiple diseases.
Prichard et al. (2023)