Authors:

Celeste Wu

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Elucidating the regenerative process in axolotls (Ambystoma mexicanum), a model organism for whole-limb regeneration, may have therapeutic potential for limb amputees. Although regeneration is now understood to be a body-wide process, the relationship between the brain and regeneration is unknown. This project conducts preliminary tests supporting a larger-scale experiment measuring neural electrical signals via an implanted probe. Implantation surgery and probe retrieval from the brain depend on the application and eventual removal of dental cement which is dissolved with acetone. However, acetone is hypothesized to shrink axolotl neurons, affecting neuron density which relates to the number of neurons recorded by each probe. Acetone also damages morphological and structural integrity. In this experiment, four axolotls underwent shortened brain surgery, and cement was applied to close the wound. The heads were harvested, placed in four timepoints for acetone immersion: 72 , 24, 12, and a control of 0 hours, and neuron size and density from each group was quantified with beta-III tubulin staining using ImageJ. The control established a neuron diameter of 22.151μm, with the 24 hour condition displaying slight, insignificant shrinking at 18.596μm. Immunofluorescence images from the 72 hour time point displayed widespread tissue degradation with a neuron size of 19.927μm From the control's neuron size, a neuron density of 30 neurons per electronic probe was calculated. This fundamental information will optimize future probe implantation surgeries and signal recordings, leading to a more streamlined process of implanting, recording, and analyzing electrical signals via probe. Unraveling the Mechanistic Circuits Driving Spontaneous Neuropathic Pain Elke Bentley, Xiangsunze Zeng, Clifford Woolf Harvard College | Quincy House | Neuroscience | 2028 Spontaneous neuropathic pain arises from damage or dysfunction in the nervous system and occurs without any external trigger. While opioids are commonly used to manage this condition, they act broadly across multiple cell types, including both those associated with pain and those associated with addiction. Our objective is to identify the specific dorsal root ganglion (DRG) cell type responsible for spontaneous neuropathic pain, enabling the development of targeted, non-addictive therapies. To model this condition, we employed a sciatic nerve transection (SNT) in mice to induce a neuroma, an aberrant fibrous mass formed by disorganized nerve regeneration known to drive chronic pain. Continuous 24-hour recordings using wireless EMG transmitters revealed flicking behavior as a reliable marker of spontaneous neuropathic pain in the chronic phase. In vivo GCaMP calcium imaging identified a causal relationship between small- diameter DRG neuron subtypes and neuropathic pain within the neuroma. Through in vivo optogenetic studies, we were able to reinforce this causal relationship by triggering these specific small- diameter DRG neuron subtypes to cause spontaneous neuropathic pain behavior (i.e. flicking). Single-nucleus RNA sequencing on DRG tissue and ex vivo immunohistochemistry on neuroma and DRG samples, revealed the specific sensory neurons and ion channels that cause the hyperexcitability within neurons that lead to high levels of pain. We are looking forward to a potential clinical study to create and test a treatment method targeting specifically this small-diameter DRG neuron subtype to combat spontaneous neuropathic pain in humans. Harvard Summer Undergraduate Research Village Program for Research in Science and Engineering

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Harvard / Human Developmental and Regenerative Biology / 2028

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Co-authors:

Celeste Wu