The dorsomedial striatum (DMS) is a brain region that functions in mediating goal-directed action but the cellular, molecular, and circuit-level substrates controlling this remain unknown. Interestingly, the kappa opioid receptor (KOR) and its ligand dynorphin (dyn) are present in about half the cells in the DMS. KOR is a G protein-coupled receptor known to have dysphoric effects through dyn. Given the effects of KOR in the DMS are relatively unknown, we investigated the role of the dyn/KOR system on goal-directed action. We hypothesized that dyn-KOR signaling in the DMS constrained goal-directed actions and motivation. To investigate, we used transgenic mouse strains engineered to express loxp sites on either side of either pre-dynorphin or KOR (pDyn or KOR cKO) genes. We performed viral intracranial microinjections in the DMS of either an anterograde AAV expressing Cre recombinase (AAV-Cre) to remove pDyn in DMS cells or an AAVretro-Cre to delete KOR in neurons projecting to the DMS. We assessed goal-directed action in these mice via instrumental conditioning at varying contingencies, as well as using a wildtype control strain. Furthermore, we utilized progressive ratio testing where the amount of nose pokes required for a reward, a sucrose pellet, increased exponentially, as a measure of motivation. A greater number of nose pokes made indicated higher levels of motivation. Next, reversal training was done where the nose poke port that is active between the two was switched to determine whether the difference in nose pokes made was due to a learning issue or a true difference in goal-directed activity. Our studies aimed to address the involvement of dyn-KOR modulation of goal-directed behavior in the DMS.

People suffering from stressful events are likely to experience negative affective states. Previous studies have shown that stress induces the release of neuropeptides including dynorphin, which acts presynaptically on kappa opioid receptors (KOR) and may act to inhibit neurotransmitter release in the limbic brain areas. In this project, we investigated the interaction between KOR and dopaminergic systems in the ventral shell of the Nucleus Accumbens (NAc) to determine how dopamine release is altered during in vivo dynorphin activation of KORs. To monitor and manipulate dopamine and KOR dynamics, we injected genetically encoded fluorescent indicators of dopamine (dLight) and KOR (kLight) respectively and implanted optical fibers in the NAc ventral shell in transgenic mice brains. In collaboration with Lin Tian at UC Davis, we also characterized the in vivo fidelity and accuracy of a newly developed kLight—version 1.2a. To light-stimulate dynorphin neurons in vivo, we injected a red-shifted channelrhodopsin, Chrimson that acts as a light-gated ion channel to depolarize dynorphin neurons. We recorded dopamine/KOR activities during behavioral experiments including Pavlovian conditioning using sucrose pellets. We hypothesized that upon dynorphin neuron activation, there will be a decrease in the amount of dopamine release in the NAc, an increase in KOR light activity and fewer motivated behaviors observed in mice to obtain sucrose pellets. Here we will present our findings measuring dopamine and kappa opioid function in the NAc. These results could have implications for neuropsychiatric diseases including depression and addiction.

Traumatic cervical spinal cord injury (SCI) results in a wide range of outcomes from partial paralysis to complete tetraplegia depending on the location of injury along the length of the cervical spinal cord. Importantly, there is a high density of motor neurons in the cervical region which are involved in important motor outputs such as breathing and hand function. The present study aims to minimize the secondary damage to the spinal cord after the primary insult, by addressing two substantial contributors to neuron death: first, surgical decompression is conducted to reduce local tissue swelling after injury, and second, administration of the metabolite oxaloacetate (OAA) to minimize excitotoxicity by stimulating glutamate transport away from injured neurons. We hypothesized that animals treated with decompression, OAA, or both, would have increased neuronal survival, general tissue sparing, and improved behavioral outcomes than those without treatment, and that combined treatment would be more effective than each individual treatment. We tested the treatments using a clinically relevant rat model for bilateral, moderately severe cervical spinal cord injury. Following treatments, we determined effectiveness by assessing animals’ forelimb function and quantifying motor neuron and white matter sparing in the injured tissue. Results consistent with the hypothesis would have meaningful impacts for future cervical SCI patients, as even a limited increase in tissue sparing in the cervical region have profound functional outcomes for patients’ independence and opportunities. Future studies will work to visualize parameters for segmental tissue at risk after acute injury in order to specify each patient’s treatment and maximize their opportunities for recovery.

Millions of people have vision diseases that are not yet treatable, leading to blindness. Mouse models exist for some inherited retinal diseases, and thus have helped develop vision loss therapies. However, other common retinal diseases like glaucoma lack accurate mouse models. Human pluripotent stem cells (hPSCs) are a promising technology that provide a new way to model human retinal diseases. hPSCs can be induced to become layered, 3D mini-retinas called retinal organoids. Retinal organoids mirror early neurogenesis of the human retina, and thus can be used for modeling developmental disorders. However, we and other research groups have shown that as retinal organoids mature, they lose many features of the normal human retina. In particular, the neurons that are damaged during glaucoma, called retinal ganglion cells (RGCs), are not well preserved in organoids. As the organoid matures, the RGC layer becomes disorganized and RGCs migrate through the retina. RGCs are the projection neurons of the human retina, so their axons extend through the optic nerve and carry visual information to the brain. Unlike in human development, retinal organoids are grown in isolation from their brain targets. We wondered whether we could preserve RGC organization by providing organoid RGCs with synaptic targets. We investigated this hypothesis by combining the retinal organoids with their natural targets in the brain, the lateral geniculate nucleus and the superior colliculus. It is not yet known how to make these brain regions from hPSCs, so we used newborn mouse brain and combined the retinal organoids with these brain regions into structures called “assembloids.” We are now testing whether these assembloids preserve the RGC survival and laminar organization that retinal organoids lack. Promoting RGC survival and organization will allow organoids to become better in vitro models for glaucoma and improve the outcome for patients with vision loss.

Preterm birth (PTB), delivery prior to 37 weeks of gestation, is the leading cause of neonatal death. According to the Center of Disease Control and Prevention, the survival rate in the US was more than 96% in 2013. Survivors of PTB are able to participate in normal activities such as sports despite having a higher rate of long-term morbidity. The higher rate of morbidity is thought to be caused by factors contributing to PTB. These include a combination of systemic neuroinflammation, perinatal hypoxia-ischemia, and/or exposure of the fetus to acute and chronic infection. However, it is not well known how early exposure to inflammation affects longer term cognitive function, particularly in an individual who might be at risk of ‘second hit’ stimulus. Therefore, this project aims to study how exposure to early inflammation induced by endotoxin lipopolysaccharide (LPS) affects the ability of brain cells to respond to a second hit stimulus. In this study, neonatal rats with LPS-induced neuroinflammation were used as the primary source of injury. Brains from LPS-exposed rats were isolated and exposed to oxygen-glucose deprivation (OGD) to model hypoxia as a second hit stimulus. This study showed that rats exposed perinatally to LPS followed by exposure to OGD had an increased number of microglia in the hippocampus as well as a hyper-ramified microglial phenotype in comparison to control animals without an exposure to OGD. Additionally, this study showed that OGD exposed slices had an increased number of localized nucleic propidium iodide stained cells indicating a higher cell death compared to the slices without exposure to OGD. These results support that exposure of slices from LPS rat model to OGD as a second hit stimulus can be used as a model to probe the mechanism underlying neuroinflammation associated with PTB and the susceptibility to a second hit stimulus.

Neonatal brain injury, caused by hypoxia-ischemia, is a major cause of neurological disability. Therapeutic hypothermia (TH) is the current standard of care for a subgroup of babies born with brain injury, but 50% of babies treated with TH still have poor outcomes. Initiated by hypoxia-ischemia, inflammatory processes contribute both to cell damage and tissue remodeling. During development, changes in the brain extracellular matrix (ECM) structure and composition have been shown to affect cell migration, proliferation, synaptic plasticity, and tissue architecture. However, the relationship between neuroinflammation and ECM reorganization remains largely unexplored in the neonatal brain. There is a need to characterize the brain microenvironment following neonatal brain injury to better understand disease progression. Nanoparticles, which have proven capable of probing biological systems, can be an optimal platform for evaluating structural changes in brain ECM as a function of normal or pathological processes. We aim to characterize ECM structural changes in multiple brain regions in an oxygen glucose deprivation (OGD) brain slice model in rats that has neuropathological hallmarks of hypoxic-ischemic (HI) brain injury. For this, 300 μm-thick organotypic whole hemisphere brain slices were prepared from postnatal day 28 rats. Slices were incubated for 2 hours in an oxygen-deprived chamber with media lacking glucose and pathological changes are observed after 24 hours. We perform multiple particle tracking (MPT) to quantify nanoparticle diffusion in ECM, immunohistochemistry to visualize the ECM and inflammatory processes such as mitochondrial morphology, and RT-qPCR to obtain ECM mRNA expression data. This combinatorial approach will capture changes in ECM structure at different time and length profiles. We have found mitochondrial fission and upregulation of inflammatory cytokines, demonstrating our ability to induce neuroinflammation in slices. By combining nanoscale diffusion data with micron-scale structural data, our findings will provide new insights into pathology-dependent ECM structural changes in neonatal brain injury.

Alzheimer’s Disease (AD) is a progressive, debilitating, neurodegenerative disorder where patients lose their ability to think and carry out tasks. This disease is characterized by aggregation of the β-amyloid (Aβ) peptide. Cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) are derivatives of marijuana which have been shown to possess neuroprotective properties. Experimental work in this field, is limited in its scope when probing mechanisms driving the phenomenon of Aβ peptide aggregation. Molecular dynamics (MD) simulations have been used to understand the intra-peptide interactions and potential impact of cannabinoids. In order to understand the effects of cosolvent structure on the mechanism of amyloid aggregation, we used classical molecular dynamics simulations of Aβ derived switch-peptides in the presence of model cannabinoids (i.e. CBD and THC). Aβ peptides transform from functional peptides into beta-sheets and therefore impact function within the brain. We tracked beta-sheet formation as a function of time to understand if cannabinoids sterically inhibit interactions between and within peptides. Preliminary results indicate that CBD and THC demonstrate a trapping effect on aggregated peptides. The impact of synthetic cannabinoids are much less understood, prompting additional interest in investigating the interactions among these molecules.

Treatment of neurological disease has made little progress due to the inability of many therapeutics to access the brain environment. However, delivery vehicles like nanoparticles can allow therapeutics to overcome brain-specific biological barriers including the blood-brain barrier (BBB), the dense extracellular space (ECS), and cellular targeting. The ability of nanoparticles to overcome these barriers is influenced by surface properties which can be modified through the formulation process. One understudied parameter is the choice of surfactant, molecules which stabilize nanoparticle formation and likely form an interface between the nanoparticle and brain environment. First, we investigated the potential toxicity of several commonly used surfactants on brain cells and slices. We added surfactant solutions to mouse microglial cells (BV2) or cultured brain slices and assessed cell viability two days later with colorimetric assays. Our results showed that while surfactants cholic acid (CHA) and polysorbate 80 (P80) caused toxicity at high doses, they were nontoxic at the low doses involved with nanoparticle formulation. Other surfactants, including Pluronic® F127 (F127) and poly(vinyl alcohol) (PVA), were nontoxic throughout the tested dose range. Interestingly, although the F127 compound is nontoxic on its own, nanoparticles formulated with F127 reduced cell viability. This result was not observed with any other nanoparticle-surfactant combination. Confocal microscopy indicated higher intracellular accumulation of the nanoparticles formulated with F127 compared to all other formulations, suggesting that toxicity is mediated by nanoparticle internalization and surfactant choice. Finally, we used a live cell imaging technique to capture videos of the nanoparticle internalization process. Building off these results, ongoing experiments will evaluate several nanoparticle-surfactant formulations on their ability to accumulate within brain tissue after in vivo administration. Findings from this work will guide nanoparticle design for future clinical translation.