snRNA-Seq for Very Small Punches of Brain Tissues

(Quanzhi Li Ph. D. Invent Biotechnologies Inc.)

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Wojick et al., [A nociceptive amygdala-striatal pathway modulating affective-motivational pain. Science Advances, (2025), 11(30)] identify a dedicated nociceptive amygdala-striatal circuit— BLA^noci to dorsomedial NAcSh—that selectively drives affective-motivational pain behavior. Their findings decouple the emotional and sensory components of pain and highlight a targetable pathway for non-opioid pain relief. The Single Nuclei Isolation Kit for Neuronal Tissues (BN-020, Invent Biotechnologies Inc.) was instrumental in achieving high-resolution transcriptomic insights from small, genetically defined neuronal populations, enabling confident conclusions about the molecular adaptations of BLA^noci neurons during chronic pain. This toolkit is a valuable resource for single-cell transcriptomics in neuroscience, especially when dealing with low-input or rare neuronal ensembles.

Key Findings:

• A stable, nociception-responsive neuronal ensemble (BLA^noci) was identified using TRAP2 mice across both acute and chronic neuropathic pain models.
• Inhibition of BLA^noci neurons selectively reduced affective-motivational pain behaviors without altering sensory thresholds.
• Single-nucleus RNA sequencing (snRNA-seq) revealed that chronic pain induces distinct transcriptional reprogramming in BLA^noci neurons, including synaptic plasticity and axonogenesis pathways.
• BLA^noci neurons project preferentially to the dorsomedial NAcSh, which emerged as a key relay center encoding pain valence signals.
• Chemogenetic inhibition of BLA^noci → NAcSh projections significantly reduced pain affect behaviors, establishing the necessity of this pathway in pain-related emotional states.

UMAP of all nuclei (n = 72,125) from uninjured mice (n = 9 total) captured by amygdala punches in 30 unique clusters (Fig. 2)

Role of Single Nucleus Isolation Kit:

The BN-020 kit played a crucial technical role in enabling single-nucleus RNA sequencing (snRNA-seq) from very limited amygdala tissue samples:

• Efficient isolation from small tissue volumes: The BLA^noci ensemble represented a small neuronal subset within the amygdala. The BN-020 kit allowed high-yield nuclear extraction from these rare cell populations, which is typically challenging in low-input settings.
• Preservation of nuclear RNA integrity: The protocol minimized RNA degradation, ensuring high-quality transcriptome profiling even from microdissected amygdala regions.
• Compatibility with TRAP2-labeled tissue: Enabled successful extraction of nuclei from tissue previously subjected to genetic labeling and recombination, maintaining fidelity of cell type-specific expression profiles.
• Enabled robust downstream snRNA-seq: Without BN-020, the team may have been limited in their ability to profile BLA^noci transcriptional changes due to the small cell numbers available for sequencing.