Metastasis, or the spread of cancer to a secondary site, is responsible for most cancer-related deaths. The tissue-specific environment that disseminated tumor cells experience influences whether they will proliferate and colonize the tissue, remain dormant, or die. Skeletal muscle (SkM) is one of the rarest sites for metastasis, despite making up nearly half of human body mass. What makes SkM so resistant to metastasis? We set out to test the hypothesis that the mechanical nature of SkM is responsible for the lack of metastases at this site. To do so, we used the mdx mouse model of Duchenne muscular dystrophy to probe if the destruction of SkM structure and function would make it a more hospitable host to disseminated tumor cells. Wild type and mdx mice were intramuscularly injected with EO771 murine mammary tumor cells and monitored for tumor outgrowth using bioluminescent imaging. Preliminary results suggest that tumor growth is increased in dystrophic (e.g. dysfunctional muscle) versus wild-type mice. A complementary and more reductionist approach to test whether mechanics influences tumor colonization of muscle is to employ a culture model that allows tumor cells seeded on top of a SkM layer to experience mechanical stretching akin to the contraction/relaxation movements of muscle. To accomplish this, we constructed a device that applies a cyclic stretch to a 3D organotypic SkM culture model on a flexible silicone plate. We predicted that stretching would reduce tumor cell survival, when compared to no stretching. While these experiments are in progress, we believe that these data may elucidate a relationship between mechanical activity and suppression of tumor outgrowth in SkM. This work will contribute to a more complete understanding of how SkM avoids tumor colonization and could inform future approaches that leverage tissue mechanics to treat or prevent metastasis.

Mitochondria are multi-functional organelles that regulate calcium signaling, an important signaling pathway that controls cellular processes ranging from transcriptional regulation to vesicular secretion. Calcium ions enter the mitochondria through the Mitochondrial Calcium Uniporter (MCU) complex, a highly selective calcium uniporter controlling calcium flux across the inner mitochondrial membrane. Previous data suggests uniporter inhibition alters lipid metabolism and induces a qualitative increase in cytosolic lipid droplets. Stemming from this initial observation, we created two research goals for this project: 1) to quantify and characterize lipids in response to MCU inhibition and 2) aims to explore the role of known or novel lipid synthesis pathways in MCU-dependent lipid accumulation. To understand the role of MCU in cellular physiology, we used CRISPR technology to knockout the MCU gene (MCU KO) in HeLa cell lines. We quantified differential lipid accumulation in single cell images through confocal fluorescence microscopy. In addition, potential mechanisms were investigated by Western Blot analysis and quantitative Polymerase Chain Reaction (qPCR). Here we verify that inhibition of mitochondrial calcium flux is responsible for lipid accumulation, a phenotype rescued by exogenous MCU expression. This accumulation is attributed to the upregulation of a transcription factor – Nuclear Factor of Activated T cells 4 (NFATc4). Gene knockdown of NFATc4 rescues the MCU phenotype, strongly suggesting NFATc4 as a downstream signaling factor of MCU inhibition. In follow-up experiments, we seek to identify MCU-regulated gene expression and understand their roles in regulation of metabolism. Although the function of increased cellular lipid content remains elusive, we theorize it may play a role in mitochondria-to-cytosol stress communication. Futher characterization of lipid-mediated stress pathways may identify novel targets for conveying increased cell stress tolerance.

As high levels of drug use account for thousands of drug related deaths every week, it’s important to investigate the neural mechanisms behind drug escalation as well as develop possible harm reduction strategies for drug taking. Therefore, the purpose of this experiment was to examine the effects of the Kappa Opioid receptor (KOR) on cocaine escalation in the mesolimbic system. Using a preclinical model to examine this hypothesis, CRISPR/SaCas9 was utilized in the ventral tegmental area (VTA) to selectively repress expression of KOR. After four weeks, the rats underwent cocaine self-administration during short access periods and then escalation was tested in long access periods. Lastly we utilized immunohistochemistry to confirm CRISPR/SaCas9 transduction in dopaminergic neurons. We found that decreased expression of the KOR decreased escalation during long access periods. Future research will examine the role of KOR from and in specific brain regions such that KOR expression will be decreased only in dopaminergic projections from the VTA into the NAc.

The locus coeruleus (LC) is a small nucleus of noradrenergic neurons in the pons, which, despite its size, has broad projections throughout the central nervous system (CNS). Functionally, the LC is believed to be involved in various critical functions, including the physiological response to stress, as well as mediating arousal. Previous investigations have demonstrated that optogenetic activation of the LC at a tonic frequency promotes wakefulness in rodents. While this observation causally implicates LC function in wakefulness, it is still not known how the LC is endogenously controlled to mediate arousal. One potential candidate in this control is the peptide nociceptin and its cognate receptor, the nociceptin opioid peptide receptor (NOPR), both of which are highly expressed around the LC. To investigate, we first conducted two pharmacological experiments using the NOPR agonist Ro64-6918 to assess its effects on locomotion and on the activity of LC noradrenergic neurons. To determine where the endogenous nociceptin signal to the LC originates, we performed an intracranial injection of a Cre-dependent retrograde virus (AAV2-DIO-eYFP) into the LC of a mouse expressing Cre recombinase in nociceptin-expressing neurons. We observed that Ro64-6198 (10 mg/kg) strongly reduced open field locomotor activity compared to vehicle treatment. Using in vivo 2-photon calcium imaging (GCaMP6s), we found that Ro64-6198 (5 mg/kg) profoundly reduced LC noradrenergic neuron activity. Wakefulness appeared reduced in both in vivo experiments. Finally, we identified nociceptin-expressing cells projecting to the LC in the peri-LC as well as a long-range projection from the bed nucleus of the stria terminalis (BNST). Together, these studies suggest that nociceptin acting on LC noradrenergic neurons reduces arousal, and that the endogenous sources of nociceptin may originate in the peri-LC and BNST. Future studies will investigate nociceptin-expressing neuron activity during sleep/wake transitions and whether this activity is sufficient to alter wakefulness.

The process of development in mammals is always accompanied with high amounts of brain plasticity. Similarly, the maturation of sex organs during puberty in mammals serves as the foundation for important cognitive development. Researching centers of high plasticity could lead to key discoveries for the molecular basis of behavior. The medial preoptic area (MPOA) is an example of a very active site in pubertal gene expression. Estrogen-receptor 1, or Esr1, is a gene that is essential for hormone binding. We aim to gain insights on the neural basis of behavior by analyzing Esr1’s level of control in the MPOA, and on sexual behavior. This was completed by identifying single-cell types, determining the full transcriptome of the MPOA, and then observing any changes in gene expression after selectively knocking out the Esr1 gene. We utilized scRNA sequencing to identify single-cell types. The mating behaviors of mice was also studied to pinpoint any phenotypic differences. We have found that the deletion of Esr1 leads to severe hinderances in maturation, as well as sexual behavior, due to reduced function of Cis-regulatory elements. This provides important clarity regarding the key effectors of brain development in the pubertal stages. We aim to apply this approach to uncover the transcriptional dynamics of other areas in the brain, eventually forming a full, detailed representation of the brain. This would be a massive step forward in understanding the neural foundation governing sexual behavior.

Leptin and ghrelin are two hormones essential to maintaining and regulating energy levels and food intake. These hormones have opposite effects on feeding behavior where leptin suppresses feeding and ghrelin potentiates it. While the roles of these two hormones have been widely researched, their relative effects on distinct neural populations are still largely undetermined. Previous electrophysiological and in vivo imaging experiments have shown that the activity of individual populations of glutamatergic hypothalamic projection neurons are differentially affected by feeding hormones. This data leads to the question of whether or not the projection populations have a bias for leptin and ghrelin receptors that could account for the difference in sensitivity. We hypothesize that Lepr and Ghsr will be expressed at different levels within the different projection populations. To study this, we injected two retrogradely trafficked viruses into the target locations, lateral habenula (Lhb) and ventral tegmental area (VTA), in Mus musculus and performed fluorescent in situ hybridization experiments in the lateral hypothalamus (LHA), dorsomedial hypothalamic nucleus (DMH), ventromedial hypothalamus (VMH), paraventricular nucleus (PVH), and arcuate nucleus (ARC) for viral based fluorophores as well as leptin and ghrelin receptors. The viral expression was imaged using fluorescence microscopy and quantified for within individual hypothalamic neurons. Analysis is currently underway to reveal any differences in fluorescence between the two projection populations and therefore, determine any disparities in hormone receptor expression. The objective of this research is to understand whether differences in expression of Lepr and Ghsr exist within LHb- and VTA-projecting glutamatergic hypothalamic neurons. This study could be indicative of how hormones regulate feeding behavior and their particular effects on the hypothalamus projection neurons. Further research concerning the downstream impacts of these opposing hormonal pathways could shed light on the neural networks that govern food and energy balance.

Tauopathies are a set of neurodegenerative diseases identifiable by the aggregation of insoluble intracellular deposits of the microtubule associated protein tau. Among others, this class of diseases includes frontotemporal dementia (FTD), Pick’s disease, and supranuclear palsy. Though the precise pathology of tauopathies is poorly understood, most forms are associated with hyperphosphorylated tau and impaired clearance of tau through the ubiquitin-proteasome and autophagosome pathways. We attempt to explore the degradative properties of one specific tau variant, a serine to threonine missense mutation (S356T), which is strongly associated with an early onset behavioral variant frontotemporal dementia (bvFTD). Posttranslational modification at the S356 residue in tau controls its proteasomal degradation. Therefore, we hypothesize that the S356T variant exhibits impaired tau clearance compared to wildtype tau. To test this hypothesis, we use the pharmacological inhibitor Cycloheximide that prevents protein synthesis in both human embryonic epithelial cells (HEK293) and neurons transfected with wildtype and S356T tau protein. Cycloheximide has been shown to inhibit translation by binding to the E-site of the 60S ribosomal subunit and preventing protein elongation. This drug allows us to measure protein degradation without being confounded with new protein synthesis. We expect the amount of tau, relative to total protein, to be elevated in the S356T transfected cells after application of cycloheximide. If our hypothesis is correct, we plan to explore possible mechanisms behind the aberrant degradative properties of the S356T tau variant. By exploring the mechanisms through which tau mutants contribute to neuropathology, we hope to provide molecular insight into tau proteostasis that if successful could offer a promising therapeutic target.

Anxiety, a heightened state of arousal without the presence of an immediate threat, can be incapacitating once it reaches a disease state. Located in the extended amygdala, the Bed Nucleus of the Stria Terminalis (BNST) has been implicated in sustained fear states and other anxiety-related conditions. BNST neurons have been shown to be diverse, co-releasing neuropeptides and other neurotransmitters, but the quantification and localization of these neuromodulators is not clear. In order to identify the co-expression of neuropeptides I employed in-situ hybridization in the mouse BNST, where RNA sequences specific to relevant peptides are recognized with a fluorescent probe. Then, I created pipelines that identify and group amplified peptide signals to cells, quantifying intensity and presence, and collect them by co-expression and subregions within the BNST. I hypothesize that co-expression of these peptides is regionally biased. Finally, identifying differences in cell-type distribution has implications for anxiety-behavior and may provide insight towards treatment of anxiety conditions.

The nucleus accumbens (NAc) is a midbrain region associated with addiction-related behaviors. The NAc consists of medium spiny neurons (MSNs) that project to the substantia nigra (SN) or the ventral pallidum (VP) forming the direct (Go) and indirect (No-Go) pathways, respectively. The Go and No-Go pathways are mostly dichotomous in their expression of distinct receptors and neuropeptides. Dopamine receptor (D1) and adenosine receptor (A2a) expression corresponds to the Go and No-Go pathways, respectively. My goal is to investigate whether collateralization exists (i.e., single neuron projecting to both the VP and SN). This will be assessed by injecting retrograde tracers into the output regions and quantifying expression in MSNs. I used D1-iCre and A2a-iCre transgenic rats that express codon-improved Cre recombinase (iCre) in neurons expressing D1 or A2a, respectively. When the iCre expressing neurons are infected with a canine adeno-associated virus (CAV) containing a double-floxed and inverted (DIO) copy of a fluorescent protein, this anatomical marker protein is inverted and expressed specifically in these cells. I bilaterally injected four D1-iCre and four A2a-iCre males with CAV-DIO-TdTomato into the VP and CAV-DIO-ZsGreen into the SN. D1 and A2a neurons projecting to the viral injection site will uptake the virus and retrogradely label D1- or A2a-expressing MSNs in the NAc. The expression of these fluorescent proteins within the NAc will be quantified to investigate the projections’ dichotomy and collateralization. The dichotomy could be validated if the tracers expressed in exclusive populations of the NAc. Based on mouse studies, I hypothesize D1-Cre rats may have minimal colocalization in NAc MSNs but A2a-Cre rats may only express TdTomato in the NAc. Addiction continues to affect millions of individuals. We aim to elucidate anatomical differences to gain a better understanding of these midbrain pathways, which could be critical for the future of clinical treatment.

Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa. The psychoactive and cannabimimetic behaviors associated with THC have been well described as being dependent on the partial agonist activity of THC at the endogenous cannabinoid 1 receptor (CB₁R). We are investigating the direct action of THC on the medial prefrontal cortex (mPFC, a brain region primarily responsible for executive function), and the effects of adolescent THC exposure on µ-opioid receptor (MOR) expression in adult periaqueductal grey (PAG, a brain region involved in opioid-mediated pain inhibition). To increase our understanding of the cannabimimetic behavioral effects of THC, and its direct pharmacological action in the brain, it is important to map the neuro-anatomical expression of target proteins. We examined expression patterns of CB₁R and MOR in the mPFC and the PAG, respectively. To do this, we utilized a form of in situ hybridization, RNAscope. We leveraged RNAscope by preparing tissue samples from brain regions of interest for treatment with mRNA-specific probes, allowing us to target CB₁R and MOR mRNA. After a series of washes and incubations, these fluorescent probes hybridize to our target mRNAs and allow us to visualize their expression under a confocal microscope. Analysis of mRNA expression informs us on the localization of the CB1R/MOR and known neuron types within our brain regions of interest. After imaging, we are able to utilize HALO software to analyze the levels of expression and co-localization of CB₁R/MORs with neuronal markers for glutamatergic and GABAergic neuron types. By creating and optimizing a workflow for extraction, preparation, hybridization, and analysis, we determined CB₁R mRNA is primarily co-localized with glutamatergic neurons in the mPFC. Moving forward, we are utilizing this RNAscope technique to investigate differential CB₁R expression GABA interneuron subpopulations in the mPFC. (Funded by DA051558)

 Mu-opioid receptors (MOR) are expressed on populations of neurons within the brain. Exogenous activation of these receptors by drugs of abuse, such as heroin and fentanyl, causes feelings of euphoria and can be highly addictive. During natural behavior, MORs in the brain are activated by the endogenous ligand enkephalin. Spiny projection neurons (SPN) in the nucleus accumbens (NAc) are known to express enkephalin and likely release it during neural activity. It is known that these enkephalin SPNs can be either excited or inhibited while animals consume natural rewards. However, it remains unclear whether these functionally distinct enkephalin SPN populations are anatomically intermixed or are anatomically separated within NAc. In this study, we used 2-photon calcium imaging through endoscopic lenses to examine the neural activity of enkephalin SPNs in NAc while mice consumed sucrose rewards. We characterized the reward-excitations or reward-inhibitions of individual enkephalin SPNs over multiple imaging sessions. Through precise post-mortem histological examination, we then verified the anatomical placements of our endoscopic lenses and associated the relative anatomical location of neuronal populations to their reward-related neural activity. We found that enkephalin SPNs in anterior NAc were consistently reward-inhibited while enkephalin SPNs in posterior NAc were reward-excited. This is the first demonstration of anterior-posterior axis differences in the reward-related modulation of enkephalin SPNs and is a key step to understanding how opioidergic neurons function in natural reward behavior.

Environmental stress and threat influence feeding behavior in animals, but how that interaction occurs is still largely unclear. Neurons in the bed nucleus of the stria terminalis (BNST), part of the extended amygdala, have dense projections to the parabrachial nucleus (PBN) in the brainstem. We have uncovered projections in these neural circuits that link the modulation of feeding and threat assessment in mice. This project aims to investigate and characterize these previously unrecognized neural circuits with the incorporation of a variety of optogenetic, surgical, and histological techniques. We used Cre-dependent anterograde and retrograde viral tracers in order to trace the anatomy of these neural circuits, and found functional projections from inhibitory (GABA) and excitatory (glutamate) populations in the BNST to neurons in the PBN. We also used translating ribosome affinity purification (TRAP) to isolate the mRNA of these projections. This proved useful in separating and identifying the molecular expression profile of different GABAergic and glutamatergic subpopulations. Furthermore, we used a variety of behavioral assays to determine the BNST-PBN circuits’ role in feeding and threat-response behavior. We used fiber photometry to track the activity of GABAergic (vGAT) and glutamatergic (vGLUT2) populations during these behaviors, and found that vGAT and vGLUT2 populations have differing roles in threat and feeding behaviors. vGAT neurons increase their activity during feeding and decrease in response to threat, while vGLUT2 neurons decrease their activity during feeding and increase in response to threat. We also used optogenetic activation of these neurons to determine their causal role in behavior. With activation, vGAT populations drive place preference, operant positive reinforcement, and increased feeding. Conversely, vGLUT2 populations drive place aversion, operant negative reinforcement, and reduced feeding. These findings characterize the distinct nature of BNST-PBN neural circuits and the mechanism behind the evaluation of threatful stimuli and the integration of feeding.

Although RNA is most commonly regarded as a passive carrier of genetic information, throughout biology RNA molecules exhibit an exceptionally broad scope of other, non-coding functions. Exciting recent work has demonstrated that RNA plays diverse and fundamental roles in helping to pattern subcellular architecture, including that of numerous subnuclear structures that are essential to cellular function. Many such architectural RNAs are also causally dysregulated in human diseases, suggesting that they may represent a novel resource for untapped therapeutic targets. Yet, the mechanisms by which these architectural RNAs function have remained elusive. An essential first step towards deciphering RNA’s cellular function is to elucidate the complex networks of proteins, RNAs, and genomic loci with which that RNA interacts. Yet, this kind of analysis is impossible using conventional approaches. To address this critical need, the Shechner lab has been developing Oligonucleotide-directed biotinylation (ODB), a straightforward and universal method for mapping RNA interaction networks without taking them out of their native cellular context. Our recent results demonstrate that ODB enables precise targeting of individual RNA interactions in situ, enabling high-resolution proteomic, transcriptomic, and genome-interaction analysis in situ. The two goals of my project are to: (1) develop generalized and extensible ODB strategies that can be applicable to a wide range of RNA targets of interest, and (2) expand ODB's range of in situ labeling chemistries, enabling higher-precision analysis. To achieve these goals, I am performing a series of ODB experiments on 9 selected long non-coding RNAs of interest and establishing a high-precision localization pattern across all the RNA targets within the cellular context. The establishment of this powerful and general method can enable unprecedented dissection into RNAs that have been notoriously difficult to analyze by traditional approaches, and may potentially reveal new avenues for novel therapeutic target-discovery.

Neuropeptide S (NPS) is a neuropeptide produced primarily in two regions of the hindbrain, the locus coeruleus (LC) and the Kolliker-Fuse nucleus. The LC-NPS population is particularly interesting because of the LC’s role in norepinephrine production and subsequent transmission throughout the brain. Previous work has found that when NPS is injected into the amygdala, it results in an anxiolytic phenotype, implicating NPS and its G-protein coupled receptor (NPSr1) in anxiety-related behaviors. Using fluorescent in situ hybridization, a method which allows visualization of single RNA molecules within cells via fluorescent probes, we found preliminarily, that the orbitofrontal cortex (OFC) has dense expression of NPSr1 RNA. This is significant as the OFC is involved in higher-order cognition including social, reward-learning, and anxiety-like behaviors. For example, OFC neurons respond to social interaction as well as food cues, and inactivation of the OFC results in increased anxiety-like behavior. The LC is also known to send projections to the OFC that have been largely unexplored. Therefore, to better understand and characterize the connection between the LC and OFC we utilized in vivo fiber photometry to assess endogenous OFC-NPSr1 activity during reward-learning, social interaction, and innate behaviors. Our studies aim to uncover the functional role of LC-NPS release in the OFC.

Reward is a driving force for animal and human behavior. Reinforcing behaviors with rewards leads to enhanced learning ability, which can either promote behaviors that increase survival, or lead to maladaptive behaviors. The nucleus accumbens (NAc) and ventral tegmental area (VTA) are brain regions established to be involved in reward processing and have significant neural connectivity. Recent studies have identified a long-range GABAergic neural circuit connecting these two regions, however previous studies focus primarily on dopaminergic neurons. These inhibitory GABAergic neurons synapse with cholinergic interneurons within the NAc shell (NAcSh). Further, the dorsal and ventral subdivisions within the NAcSh have been shown to have different neural connectivity. To investigate the role of this circuit, I performed fiber photometry recordings of neural activity in GABAergic terminals in the dorsal and ventral NAcSh during reward reinforced behavior in mice. The recordings show an increase in GABAergic neural during reward consumption in the ventral, but not the dorsal, NAcSh. I also recorded the activity of NAcSh cholinergic interneurons as well as acetylcholine activity in the dorsal and ventral NAc shell. These recordings show that cholinergic neural activity as well as acetylcholine activity are reduced during reward consumption in the ventral, but not dorsal, NAcSh, reflecting the inhibition by the GABA neurons during this time. I also used the inhibitory photo-activatable chloride pump JAWS to inhibit GABAergic projections during reward consumption, finding that animals made reduced reward seeking events and consumed fewer rewards when JAWS is activated. Collectively, these results indicate GABAergic projections from the VTA to specifically the ventral NAcSh function in reward reinforcement by inhibiting cholinergic activity during reward consumption. These results characterize a previously unknown neural circuit and help us better understand psychiatric disorders like depression and addiction that impact these circuits. 

 Cannabis sativa is one of the most widely used drugs in the world. In humans, Cannabis sativa is commonly used to alleviate anxiety and pain, among other things, in medical and recreational contexts. In mice, intraperitoneal (i.p.) injections of its primary psychoactive compound, Δ9-tetrahydrocannabinol (THC) produce a characteristic triad of behavioral responses consisting of hypolocomotion, hypothermia, and analgesia. However, injections of THC do not accurately represent how humans typically administer THC, which primarily consists of inhalation and oral consumption. To better model a typical route of administration used by humans, we developed a voluntary oral consumption paradigm in mice whereby THC is formulated in gelatin. Following habituation, mice were given ad libitum access to THC gelatin for 2 hours. We measured the triad behaviors immediately following consumption to determine whether voluntary oral consumption of THC-gelatin using this paradigm induces acute cannabimimetic behaviors. Due to the slow pharmacokinetic activity of orally consumed THC, we measured triad responses immediately, 1 hour, and 2 hours after consumption. To compare our relative THC-gelatin-induced cannabimimetic behaviors to published data, we replicated the triad experiment and demonstrated our ability to obtain dose-dependent triad responses by using i.p. injections. At high concentrations (4mg/15mL) of THC-gelatin, cannabimimetic behavioral responses matched those of mice treated with low-dose (3 mg/kg) of THC i.p. injections. From these initial studies we conclude that development of THC-gelatin formulation triggers characteristic cannabimimetic behavioral effects in mice. These results suggests that classical THC and cannabinoid-dependent behaviors in mice can feasibly be studied with a more translational model (Funded by DA051558). Optimizing an oral administration model of cannabinoids in mice will enable future research on the pharmacology of oral cannabinoid therapeutics.