A Neuronal Activity-Boosting Microglial Function in Post-Anesthetic Emergence: How Microglial-Neuronal Crosstalk May Alter States of Consciousness

Glial cells have often been referred to as the support cells of the brain. While they do have numerous supportive functions, there is emerging research showing they play an active role in shaping the brain and behaviour. Studying the cellular and molecular crosstalk between brain cell types is immensely valuable as this research topic continues to demonstrate that many brain functions are a result of a system of cells working together, rather than any cell type independently. To this point, Haruwaka and colleagues (2024) have furthered the field’s understanding of how microglia, the brain’s immune cells, monitor and regulate neuronal activity through highly specific extension and protrusion of their cellular processes [1]. In the present paper, the authors use a number of genetic tools in combination with confocal, two-photon, and electron microscopic approaches to uncover this unique microglial function that is responsible for boosting neuronal synchronization during the emergence from anesthesia. The current findings reshape our understanding of how microglia can precisely and transiently alter synaptic function in the brain, likely apply to other altered states of consciousness, such as sleep, and have implications for understanding the limitations of various methodological approaches to studying microglia.

Using in vivo two-photon imaging of calcium flux in the mouse somatosensory cortex, Haruwaka and colleagues demonstrate that during the post-anesthetic or ‘waking-up’ period (as early as 15 minutes after anesthetic cessation) neuronal cells engage in synchronized hyperactivity—a staggering 50% of neurons visualized in this region were found to increase their spontaneous activity compared to baseline. Following this hyperactivity, the mice displayed an increase in both pain sensitivity and locomotion. Interestingly, after depleting 98% of microglia (assessed via density of IBA1+ cells) from the brain using a pharmacological intervention, this neuronal hyperactivity during the emergence period did not occur. Furthermore, after depleting microglia, the same mice lacked the mechanical hypersensitivity that they previously displayed—leading the authors to further investigate the involvement of these immune cells in this process. Similar observations proposing a novel microglial role in facilitating anesthesia were also obtained recently using pharmacological and genetic manipulation [2, 3].

Next, Haruwaka and colleagues investigated the number of microglial processes making contact with neurons during wakefulness, isoflurane anesthesia, and emergence from anesthesia. They demonstrated that the amount of neuronal hyperactivity was positively associated with microglial contacts with neuronal cell bodies. Previously, microglia have been shown to alter their morphology to respond to neuronal activity, a function that is particularly driven through purinergic receptors (e.g., P2Y12) on microglial processes towards purines such as adenosine triphosphate [4]. In the current investigation, the authors isolated a phenotype of microglial process morphology, named “bulbous endings”, whose presence was also positively associated with neuronal hyperactivity. To further investigate this morphological phenotype, brain sections were co-stained against a vesicular gamma-aminobutyric acid (GABA) transporter (VGAT) that is found on GABAergic vesicles, which demonstrated a profound positive association between VGAT and microglial bulbous endings. The proportion of microglial bulbous endings overlapping with GABAergic terminals went from 1.4% in awake mice to 8.7% in anesthesia. The authors extensively characterized these interactions between microglia and GABAergic terminals using three-dimensional confocal and electron microscopy—visualizing microglial processes contacting, enwrapping, and engulfing these inhibitory boutons during anesthesia and emergence from anesthesia. The authors argue that this seemingly small increase in inhibitory shielding by microglia can have an outsized effect on neuronal activity and be necessary for neuronal hyperactivity during anesthesia emergence. The authors supported this hypothesis by demonstrating that chemogenetic inhibition of parvalbumin-expressing inhibitory interneurons in microglia-depleted mice was not sufficient for mimicking the functions of microglia during emergence from anesthesia, indicating that this specific shielding is necessary for the observed effects on neuronal hyperactivity.

To delve deeper into the mechanism behind this novel microglial morphological specialization, the authors selectively removed β-adrenergic receptors on microglia via Cx3cr1^CreER/+ and Adrb2^fl/fl mice. The loss of this receptor resulted in altered microglial process dynamics, associated with reduced process motility and surveyed territory. The authors confirmed that these microglial β-adrenergic receptors are necessary for synchronous neuronal activity and downstream sensitization to pain by monitoring electrical activity during the emergence from anesthesia. Additionally, knockout of the microglial β-adrenergic receptors prevented the previously demonstrated functional change in microglial bulbous endings and their colabelling with VGAT contacts on neurons prior to, during, and in emergence from anesthesia. Altogether, Hurawaka and colleagues demonstrate that norepinephrine activity on microglia is necessary for microglia-driven disinhibition, via a transient physical process blockade of excitatory neurotransmission that drives an increase in excitatory activity within the somatosensory cortex and results in downstream alterations in behaviour and tactile sensitivity.

The results of the current paper stand out for a few notable reasons. Firstly, the transience of this microglial function further demonstrates the fast-acting morphological alterations of these brain-resident immune cells and their ability to respond to and regulate neuronal activity in real time. Next, the specificity of this microglial morphological specialization to inhibitory synapses is equally fascinating as it demonstrates the potential for excitatory or inhibitory specificity in microglial morphological alteration and involvement in neuronal regulation. This opens further investigation about a wide variety of conditions, both in health and disease, where microglia may temporarily shape the excitation-to-inhibition balance in an adaptive or maladaptive manner. Lastly, the authors emphasized that these microglial bulbous endings do not lead to the eventual loss of the inhibitory synapses through mechanisms such as phagocytosis or trogocytosis, a function of microglia previously described in the literature [5].

The current findings further emphasize that norepinephrine is—perhaps surprisingly—a potent modulator of microglial properties including their interactions with neurons. Previously, norepinephrine was notably shown to reduce microglial surveillance and slow response to injury in awake mice [6, 7]. Related to this, in the discussion of their findings, Haruwaka and colleagues suggest that one significant implication of their results is that microglia may play a critical role in mediating transitions between brain states responsible for sleep; and recent work has provided novel evidence for this very function. Ma and colleagues (2024) demonstrated that microglial P2Y12-Gi coupled intracellular calcium regulates norepinephrine signalling responsible for maintaining sleep and modulating wake-to-sleep and sleep-to-wake transitions [8]. While there are inherent mechanistic differences between sleep and anesthesia, together these findings suggest that microglia may be central to natural and drug-induced state changes in consciousness by transiently altering excitatory and inhibitory signalling—primarily driven through their response to norepinephrine. Alternative states of brain activity, particularly sleep, which appear to be driven by glial cells including microglia are also necessary for maintaining brain health via essential microglial functions such as surveillance and phagocytosis [9] and neuron-glial lipid metabolism [10]. Thus, it appears microglia may exert a precise role in regulating the neuronal hyper- and hypo-activity under different states of consciousness, and conditions that interfere with these microglial-neuronal crosstalk likely have detrimental implications for brain health. We hope that the striking findings of the current paper inspire further research into the functional implications of microglia-neuronal crosstalk with the aim of understanding and identifying targets for microglial pharmacology in the treatment of brain disorders.

Lastly, it is worth noting that these findings additionally have implications for the interpretation of previous findings using many microscopy approaches that investigate microglia under anesthesia (e.g., two-photon in vivo microscopy) or post-mortem analyses when animals are exposed to anesthesia prior to brain fixation by transcardiac perfusion or immersion (e.g., immunofluorescence and electron microscopy). Haruwaka and colleagues have further displayed that microglial morphology can be quickly and substantially altered during anesthesia; thus, this caveat must be considered when conducting in vivo and post-mortem investigations of microglial morphology and interactions with neurons (Fig. 1).

Fig. 1.

Microglia alter neuronal activity in different states of consciousness. Microglial surveillance and interactions with neuronal elements are reduced during awake/conscious awareness. However, there is mounting evidence that microglia play an important role in the transitions to and maintenance of altered states of consciousness through specific and transient alterations in morphology. This effect seems to be associated with the action of norepinephrine on microglia.