AAV5-CAG-GCaMP6s

The trigeminal sensory innervation of the cranial meninges is thought to have nociceptive functions and mediate headaches. However, the activity of meningeal afferents under natural conditions in awake animals remains unexplored. Here, researchers used two-dimensional and three-dimensional two-photon calcium imaging to track the activity of meningeal afferent fibers in awake mice. Surprisingly, a large number of afferents were activated under non-noxious conditions such as locomotion. They estimated locomotion-associated meningeal deformations and found afferents with different dynamics and adapted to different degrees of meningeal expansion, compression, shear, and Z-axis motion. Moreover, these mechanosensitive afferents often adapted to different directions of meningeal expansion or compression. Thus, in addition to their role in headache-related pain, meningeal sensory neurons track the dynamic mechanical state of the meninges under natural conditions.

Here, researchers developed a calcium imaging method to simultaneously monitor the activity of dozens of meningeal afferent peripheral nerve terminals within the intact cranial cavity of awake, behaving mice through chronically implanted cranial windows. Microinjection of AAV5-CAG-GCaMP6 into the TG resulted in labeling of TG neuronal somata and afferent fibers in the ipsilateral dural meninges (Figures 1A-1C). GCaMP6 colocalized with calcitonin gene-related peptide (CGRP) in a subset of fibers (Figure 1C), confirming expression of peptidergic and non-peptidergic meningeal primary afferent neurons.

Figure 1. Anatomy of meningeal innervation. (A) Schematic of the injection strategy for AAV-mediated expression of GCaMP6s in trigeminal ganglion neurons without damaging the ipsilateral meninges. (B) Histological sections demonstrate GCaMP6s expression in a subset of neurons in the trigeminal ganglion. (C) Using immunohistochemistry, researchers found CGRP expression in 31.7-62.2% of dural GCaMP6s-labeled fibers, indicating that AAV transduces both peptidergic and non-peptidergic meningeal afferents. (Blaeser A S, et al., 2022)