Dorsal root ganglion (DRG) neurons, located in the dorsal root of spinal nerves, serve as critical relays for transmitting sensory information from the peripheral nervous system (PNS) to the central nervous system (CNS) (Basbaum et al., 2009). These neurons detect and convey diverse sensory stimuli, including touch, temperature, nociception, vibration, and proprioception, to the spinal cord for processing (Zimmermann et al., 2007). Current research on DRG neurons focuses on several key areas, such as pain mechanisms, neuronal plasticity, neuroinflammation, and neuromodulation (Bennett and Woods, 2014, Krames, 2014). A major emphasis is placed on understanding how DRG neurons contribute to chronic and neuropathic pain, particularly through the modulation of ion channels and signaling pathways (Waxman and Zamponi, 2014). Additionally, their structural and functional plasticity in response to injury or inflammation is a critical factor in persistent pain states (Costigan et al., 2009).

Given their role in sensory transmission, DRG neurons have emerged as promising targets for neuromodulatory therapies, such as dorsal root ganglion stimulation, for treating chronic pain (Deer et al., 2017). Advances in single-cell RNA sequencing (scRNA-seq) have further enabled the characterization of DRG neuron subtypes based on gene expression profiles (Usoskin et al., 2015). However, while rodent models have provided valuable insights, human DRG neurons exhibit distinct differences in gene expression and function (Ray et al., 2018). Thus, developing reliable in vitro human DRG neuron models is essential for translating preclinical findings into clinical applications and facilitating high-throughput drug screening (North et al., 2019).

The human DRG neuronal cell line HD10.6 represents a valuable in vitro model for studying nociceptive sensory neurons (Raymon et al., 1999). Derived from human embryonic DRG cells and immortalized via v-myc transfection, HD10.6 cells differentiate into neuron-like cells expressing key sensory neuron markers (Raymon et al., 1999). Differentiated HD10.6 neurons exhibit robust electrophysiological properties, including action potential firing and functional expression of nociceptive ion channels such as TRPV1 (Ray et al., 2018, Raymon et al., 1999). Transcriptomic analyses indicate that HD10.6 cells closely resemble human nociceptors, making them a suitable model for studying sensory signaling (North et al., 2019).

To further develop HD10.6 as a functional model for sensory activation imaging, we evaluated the genetically encoded calcium indicator (GECI) GCaMP6s as a molecular tool. GCaMP6s, a fusion protein comprising GFP, calmodulin (CaM), and the M13 peptide, exhibits high sensitivity to intracellular calcium fluctuations (Chen et al., 2013). Upon Ca²⁺ binding, conformational changes enhance GFP fluorescence, allowing real-time monitoring of neuronal activity (Chen et al., 2013). The "s" variant, with slower decay kinetics, is particularly suited for tracking sustained calcium dynamics (Chen et al., 2013).

In this study, we investigated the utility of GCaMP6s for quantitative imaging of sensory activation in HD10.6 neurons. We constructed and characterized adeno-associated virus serotype 9 (AAV9) vectors for efficient GCaMP6s delivery and validated their functionality in differentiated HD10.6 cells. This approach aims to establish a robust in vitro platform for studying human nociceptive signaling and screening potential analgesics.