The endocannabinoid (EC) system regulates diverse physiological processes in humans, but its evolutionary origins remain incompletely understood. To investigate the early evolution of cannabinoid signaling, we characterized the EC system in the Pacific hagfish (Eptatretus stoutii), an early-diverging vertebrate. Transcriptomic analysis identified a single putative cannabinoid receptor (CBR), rather than the two receptors found in other vertebrates, along with key enzymes involved in the synthesis and degradation of the endogenous ligands anandamide (AEA) and 2-arachidonoylglycerol (2-AG). Sequence analysis revealed conservation of characteristic GPCR motifs and residues associated with ligand recognition. Phylogenetic analysis supported a close relationship between the hagfish receptor and vertebrate cannabinoid receptors, with some features suggesting a more CB1-like profile. Structural modeling with AlphaFold3, binding pocket prediction with P2Rank, and molecular docking with AutoDock Vina helped visualize a plausible ligand-binding pocket for both AEA and 2-AG. Functional testing using a BRET-based TRUPATH assay further demonstrated that the hagfish receptor is activated by both ligands and even RNB 61 (a CB2 selective agonist). Together, these findings provide transcriptomic, structural, and functional evidence for an ancestral endocannabinoid system in hagfish and offer insight into the early evolution of cannabinoid signaling in vertebrates.

Infantile amnesia describes how memories formed in early life are rapidly forgotten, while those formed in adulthood can last a lifetime. Infant memories are stored at the time of learning but become inaccessible later in development. The retrosplenial cortex (RSC) plays a critical role in contextual memory in adults, and the DeNardo lab recently identified silent infant engram cells in the RSC that are larger and less likely to be reactivated by natural cues. However, how RSC neuronal activity and upstream connectivity differ between infants and adults remains unknown. Last quarter, we used WGA retrograde tracing in TRAP2 mice to map inputs to RSC engram cells formed at postnatal day (P) 17 and P60 after contextual fear conditioning. While tracing reveals structural connectivity, fiber photometry captures how these circuits are active during behavior. This quarter, I will use dual-color calcium imaging in Vglut2-Cre mice to record excitatory and parvalbumin interneuron activity during learning and 14-day retrieval. Previously, it was found that parvalbumin cell number is consistent across development despite larger infant engrams, so I hypothesize that differences in parvalbumin activity may contribute to the larger engram. I will also record from TRAPed engram cells during retrieval in P17 and P60 mice, expecting infant engram cells to show less context specific activity consistent with noisy encoding. These experiments will clarify how RSC activity and connectivity mature to support lasting memory.

Subcortical white-matter stroke (WMS) causes extensive demyelination and axonal injury, which contribute to long-term neurological deficits. WMS elicits a rapid inflammatory response which can be initially protective, initiating injury clearance and regenerative processes, but pathological when prolonged. Inflammation resolution is partially dependent on the presence of myelin debris, where accumulation at the lesion site inhibits remyelination. Post-stroke outcomes depend on the capacity of phagocytic microglia to clear myelin debris quickly. Endothelial CXCL5 overexpression has been implicated in improved long-term tissue recovery by promoting myelin-degrading microglia; identifying therapeutic strategies that mimic this response is key in improving post-stroke outcomes. This project has begun to establish a protocol for in vitro quantification of myelin phagocytosis, which will be used to evaluate pharmacological compounds that improve microglial capacity for myelin debris clearance. Magnetic antibody-based isolation of lesion-derived debris produced a myelin enriched fraction validated by Western blot. These results were corroborated by immunofluorescence characterization. Debris was conjugated with a pHrodo amine dye as validated by plate reader fluorescence, enabling quantitative measurement of microglial phagocytic internalization via fluorescence-based assays in future screening experiments. Future findings will allow for development of myelin clearance accelerating therapeutics and improve recovery following WMS.

Schizophrenia is a heritable psychiatric disorder that can involve hallucinations, disorganized speech, cognitive deficits, and avolition. Adult schizophrenia patients show atypical functional connectivity (FC) of the brain’s default mode network (DMN), including the anterior cingulate cortex (ACC), a region implicated in cognitive control, as well as the processing of salient information (Menon et.al, 2011). However, it remains unclear how genetic liability for schizophrenia influences early brain development in ways that confer vulnerability to later mental illness. To address this gap, we leveraged infant neuroimaging data from the Developing Human Connectome Project to investigate how common genetic variation associated with schizophrenia impacts FC of the ACC in 261 neonates. Polygenic scores were calculated using summary statistics from a genome-wide association study of 76,755 schizophrenia cases and 243,649 controls (Trubetskoy et.al, 2022). Neonates with higher schizophrenia polygenic scores had weaker FC between the left ACC and the left supramarginal gyrus, left inferior parietal lobule, and left angular gyrus (cluster-corrected Z>2.3). Furthermore, neonates with higher schizophrenia polygenic scores also showed weaker FC between the right ACC and other areas of the DMN. These findings suggest that genetic liability for schizophrenia may impact FC of brain networks involved in language comprehension, emotion processing, and self-referential thought long before the typical adolescent emergence of clinical symptoms.

Higher levels of education have been associated with “cognitive reserve” represented in neuropsychological trajectories by apparent relative protection from symptomatic decline during the emergence of neurodegenerative processes occurring in brain tissue, as well as in differing profiles of regional brain metabolism as assessed in PET studies. Relationships between education levels and motor function associated with loss of integrity of presynaptic dopaminergic pathways in patients with Parkinson’s Disease (PD) remain to be similarly elucidated. Utilizing the Parkinson’s Progression Markers Initiative (PPMI), a large database of SPECT and PET images assessing dopaminergic systems in affected and healthy living human brains was acquired and analyzed from 1,464 participants, including 1,217 PD patients and 247 Healthy Control (HC) subjects. An index based on ratios of presynaptic dopamine transporter activity in putamen to white matter background within each subject was compared to subjects’ cognitive performance as measured by the Montreal Cognitive Assessment (MoCA) instrument, and their motor performance as measured by the Hoehn and Yahr Test. The present study indicates that educational status does not serve as a confound in the measurement of motor performance nor of presynaptic dopaminergic activity, in either PD patients or normal subjects.

Early life adversity (ELA) is a major risk factor for developing mood and anxiety disorders during childhood and later in life. Studies in human and animal models suggest exposure to ELA increases this risk by disrupting the development of emotion centers within the brain. One center being the nucleus accumbens (NAc) which plays a role in threat avoidance. ELA’s been shown to alter dendritic spine densities in regions of the brain that play a role in responses to stress and threats. However, the impact ELA has on NAc dendritic spines, post-synaptic sites of glutamatergic inputs, remains unknown. In our study, we use a limited bedding and nesting (LBN) mouse model of ELA, which disrupts normal maternal care and immunohistochemistry and genetic labeling techniques to examine how ELA impacts neurons within the NAc from early postnatal periods to adulthood. We hypothesize there will be noticeable differences in neuronal spine densities within the NAc in mice exposed to LBN. A three-way ANOVA test measuring the effects of sex, age, and rearing revealed a significant main effect of age and an interaction between rearing condition and sex. Consistent with this, LBN male mice had decreased medium-spiny neuron spine densities compared to age-matched SR males, while spine densities were increased in female LBN compared to age-matched controls SR female mice. Together, these findings represent a clear difference between the stress and sex for the rearing and standard conditions as seen through the spine densities of NAc neurons.

Short sleep harms neurodevelopment in adolescents, possibly by injuring the brain. Studies in healthy children show shorter sleep is linked with lower grey matter volume (GMV) in the hippocampus, a brain region key for memory and thinking. Lower GMV may signal reduced brain function. While this link is known in teenagers, it is unknown in healthy adults. This study asks whether sleep duration is linked to signs of brain injury in adults with consistent routines (defined here as employed). Specifically, in adults aged 22-35 years, which brain regions show changes in GMV based on sleep hours? We analyzed 596 healthy young adults from the Human Connectome Project. All participants were employed and had biomarker and MRI data. GMV was measured using voxel-based morphometry in SPM12 (a MATLAB neuroimaging package). We tested the link between GMV and hours of sleep with linear regression, adjusting for age, sex, body mass index (BMI), and HbA1c. Two regions showed GMV association with hours of sleep: An area next to the right cerebral white matter and in the right middle occipital gyrus (MOG), significant at P=0.05, corrected for multiple comparisons. We found a positive association between GMV and sleep duration in employed adults. Using employed participants anchored results in consistent sleep routines, making them applicable to everyday habits. These findings reinforce the importance of sleep at all ages. Further studies can explore how these regions affect cognition and whether they are linked to disease.

A key genetic contributor to Alzheimer’s disease (AD) is the risk factor apolipoprotein E4 (APOE4), which elevates disease risk compared with the neutral APOE3 variant. The two variants differ by a single nucleotide, making APOE4 a viable target for correction through CRISPR-based cytosine base editing. However, the delivery of CRISPR therapeutics to the central nervous system remains a significant challenge due to the blood–brain barrier (BBB). To deliver these editing components into the brain, where CRISPR therapies normally cannot reach, we use Synthetic Exosomes (SEs), which are microfluidically generated lipid vesicles designed to mimic the size and deformability of natural exosomes. SE performance will be evaluated based on BBB penetration and APOE4-to-APOE3 editing efficiency in AD mouse model. Because nanoparticle stability is essential for effective transport and BBB penetration, polyethylene glycol (PEG) is incorporated into SE formulations. PEG can extend circulation time, improve structural integrity, and limit aggregation. To optimize our SE formulation, we produced SEs with different PEG concentrations and evaluated how PEG concentration influences nanoparticle size distribution and post-lyophilization reconstitution, both critical for systemic stability and CNS delivery.

The cerebellum is a highly organized brain structure essential for motor coordination, learning, and cognitive function. Gene X is a neuron-specific RNA-binding protein that regulates alternative splicing. Dysregulation of Gene X has been implicated in neurological disorders, including motor dysfunction, epilepsy, and autism spectrum conditions. In this study, we used a Cre-LoxP knockout system to conditionally delete Gene X to investigate its role in cerebellar development. At postnatal day 21 (P21), sagittal brain sections from cKO and wild-type (WT) mice were stained with immunofluorescent markers to visualize cerebellar layers. ImageJ was used to quantify total cerebellar area, perimeter, and sublayers (granule, molecular, and Purkinje layers). While cKO mice showed trends in decreased cerebellar size and structure, Mann-Whitney U tests revealed no statistically significant differences at the current sample size. These findings show the potential role of Gene X in fine-tuning cerebellar development and neural circuit organization. Although statistical significance was not reached, the observed structural trends and existing literature suggest that Gene X may be an important regulator of cerebellar morphogenesis and function. This project is significant because it advances our understanding of how neuron-specific RNA splicing mechanisms contribute to brain development and neurological disease, possibly informing therapeutic strategies for disorders involving motor dysfunction and epilepsy.