Year: 2024
Type: article
Abstract: Microglia were discovered in the central nervous system (CNS) by Pío del Río Hortega over a century ago as tiny cells with branched architecture distinguishable from astrocytes and neurons [1]. Since then, microglia have been characterized as resident immune cells of the CNS that play key roles in brain homeostasis. Until now, interactions between microglia and neurons while under, and recovering from, anesthesia have been obscure. A recent study by Koichiro Haruwaka, Long-Jun Wu, et al. shows a role for microglia-neuron interactions that has important implications for clinical settings where, during emergence from anesthesia, some patients have undesirable reactions called delirium that include extreme drowsiness or hyperactivity. In this work [2], Haruwaka et al. imaged conscious animal brain and tissue slices treated with, and recovering from, the anesthetic isoflurane to study microglia behavior using two-photon in vivo imaging. Together with correlative high-resolution 3D electron microscopy, they revealed dynamic tissue architecture that helps explain how microglia may function to shield the action of inhibitory neurons resulting in neuronal hyperactivity during post-anesthesia delirium. Haruwaka et al. implanted a transparent window in the crania of mice so that excitatory neurons in layer II/III of the somatosensory cortex could be visualized with two-photon imaging, and then with confocal and super-resolution light microscopy in fixed slices. Mouse brains were transfected with virus cocktails that marked neuronal cells of interest, PV (recombinant parvalbumin-expressing neurons) boutons, to monitor Ca++ signaling. To identify specific cells, they also used a variety of antibodies to label neuronal elements as well as microglia. Whereas most contacts (synapses) between neurons are excitatory, a few are inhibitory (GABAergic), and the GABAergic synapses were among those that were labeled. When the mice were awake, excitatory neurons exhibited spontaneous Ca++ signaling, which was suppressed by anesthesia. During emergence from anesthesia, almost half of the neurons resumed activity above baseline levels, about a quarter reduced their activity relative to the awake baseline, and another quarter showed no substantial change. These and other results demonstrate a transient period of neuronal hyperactivity during emergence from anesthesia, in some cases. Light microscopy methods showed that during anesthesia the territory occupied by the microglia and their processes increased gradually, and contact between microglial processes and GABAergic synapses became more extensive with more contacts but did not expand further during emergence from anesthesia. The observation of microglial processes positioning themselves between neurons and inhibitory synapses suggests that the microglia may be shielding the neurons, potentially as a response to counteract sedation. To examine these contacts further at higher resolution, tissue was first examined by two-photon microscopy, then selected and prepared for ultrastructural analysis. Specifically, the same cell interactions observed using two-photon microscopy were examined using a correlative serial block face scanning EM (SBFSEM) technique where thin layers of plastic-embedded tissue were removed from the block face to expose deeper levels, and the block face was imaged by backscatter SEM (Figure 1). This was repeated many times, and the resulting images were digitally segmented to highlight specific features of individual cells and their processes and reconstructed to provide 3D volumes of tissue at high resolution. Correlative two-photon and serial block face SEM imaging of the same brain region illustrating how microglia are positioned to shield the action of inhibitory neurons. A, Two-photon in vivo imaging of microglia-PV bouton interactions adjacent to a neuronal soma. Arrowhead (right) indicates the site of interaction. B, The boxed area in (A) was followed by SBF SEM and 3D reconstruction. C, Enlarged 3D serial reconstruction of a microglial process, PV bouton with vesicles, and neuronal soma. D, Dashed line in (C) shows the region of interest for the representative electron microscopy image slice showing that a microglial process shields a PV bouton. Examination of the 3D volume reconstructions clearly revealed that microglial processes extended into GABAergic synapses. Thus, the processes were in a position where they could shield the postsynaptic receptors from being stimulated. Prevention of inhibitory stimulation would result in an overall increase in excitation. Haruwaka et al. suggested that shielding inhibitory receptors could explain how, in some cases, a hyperactive state (delirium) could occur during emergence from anesthesia. A better understanding of this previously unappreciated role of microglia perhaps causing delirium could lead to innovative methods to address post-anesthesia complications.
Source: Microscopy Today
Authors: Stephen W Carmichael, Jeffrey L Salisbury
Institution: Mayo Clinic
Language: English
Subfield: Neurology
Field: Neuroscience
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W4400875644