Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition.

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via 'RiboTag,' morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

Polyimide Electrode-Based Electrical Stimulation Impedes Early Stage Muscle Graft Regeneration.

Given the increasing use of regenerative free muscle flaps for various reconstructive procedures and neuroprosthetic applications, there is great interest and value in their enhanced regeneration, revascularization, and reinnervation for improved functional recovery. Here, we implant polyimide-based mircroelectrodes on free flap grafts and perform electrical stimulation for 6 weeks in a murine model. Using electrophysiological and histological assessments, we compare outcomes of stimulated grafts with unstimulated control grafts. We find delayed reinnervation and abnormal electromyographic (EMG) signals, with significantly more polyphasia, lower compound muscle action potentials and higher fatigability in stimulated animals. These metrics are suggestive of myopathy in the free flap grafts stimulated with the electrode. Additionally, active inflammatory processes and partial necrosis are observed in grafts stimulated with the implanted electrode. The results suggest that under this treatment protocol, implanted epimysial electrodes and electrical stimulation to deinnervated, and devascularized flaps during the early recovery phase may be detrimental to regeneration. Future work should determine the optimal implantation and stimulation window for accelerating free muscle graft regeneration.

Transdermal optogenetic peripheral nerve stimulation.

OBJECTIVE: A fundamental limitation in both the scientific utility and clinical translation of peripheral nerve optogenetic technologies is the optical inaccessibility of the target nerve due to the significant scattering and absorption of light in biological tissues. To date, illuminating deep nerve targets has required implantable optical sources, including fiber-optic and LED-based systems, both of which have significant drawbacks. APPROACH: Here we report an alternative approach involving transdermal illumination. Utilizing an intramuscular injection of ultra-high concentration AAV6-hSyn-ChR2-EYFP in rats. MAIN RESULTS: We demonstrate transdermal stimulation of motor nerves at 4.4 mm and 1.9 mm depth with an incident laser power of 160 mW and 10 mW, respectively. Furthermore, we employ this technique to accurately control ankle position by modulating laser power or position on the skin surface. SIGNIFICANCE: These results have the potential to enable future scientific optogenetic studies of pathologies implicated in the peripheral nervous system for awake, freely-moving animals, as well as a basis for future clinical studies.