Spinal muscular atrophy, or SMA, is a term applied to any genetic disorder that affects the spinal motor neuron through gene mutations. Depending on the type of gene mutation, there is an altering of the survival muscular neuron protein (SMN protein) that can affect either the function, quality, or amount of that protein. Due to multiple locations within the genome that produce the SMA protein, different variations of the disease exist and are classified clinically based upon the pattern of nerve loss and the causative gene mutation, with variable onset depending on which gene location has been affected. SMA is unfortunately often fatal in early childhood. Nusinersen is a novel drug treatment that uses an anti-sense oligonucleotide injected directly into the CSF that alters the genome in order to increase the amount of functional SMA protein produced. Pain is unfortunately a common occurrence for those living with SMA, and advancements in treatment and survivability have created a larger patient population and an increased need to address their pain. The scope and experience of pain in the SMA patient can vary widely, manifesting in multiple organ systems simultaneously. Our team proposes utilizing metabolomics to study the cerebrospinal fluid (CSF) of SMA patients currently receiving intrathecal Nusinersen treatment in our clinics. The CSF is already drawn as part of this treatment and is discarded. By utilizing this normally discarded resource, we are provided with a unique opportunity to analyze a human vital fluid without presenting increased harm to the patient. The aim of this proposed study is to explore the metabolomic profile of the CSF in SMA patients, this study being an important first step in generating information and data critical to developing future hypothesis-driven research.

The opioid epidemic is a growing challenge facing the US, and adolescents are an under-researched population susceptible to opioid addiction and overdose. After surgery, many adolescents are prescribed pain medicines such as opioids to treat severe pain they may experience, but this exposes teens to opioids which have strong addictive properties. The aim of this study is to 1) understand teens’ perceptions and attitudes about prescription pain medicines that influence opioid use decisions and behaviors, and 2) develop a measure assessing perceptions and beliefs as risk factors for adolescent opioid misuse and abuse. We performed a literature review identifying existing measures that ask children, adolescents, and young adults about their perceptions of prescription pain medicines. Next we conducted semi-structured brief phone interviews about opioid decision making with adolescents aged 12-18 years, who either had recent surgery/ICU admission or were healthy, from 3 existing studies at Seattle Children’s Research Institute. After consent, interviews were audio recorded, transcribed, and coded to identify themes across the interviews. We conducted 15 interviews. Emerging nodes are “It’s important that I understand risks of opioids so I can balance this with helping my pain,” and “Having a support system, including family support, helps me use my opioids safely”. An example quote of the family support theme is “I just think it’s a lot easier to be able to monitor [my meds] when I had a strong support system” (15 year old participant). Once coding is complete, findings will be combined with expert input to develop a measure which will undergo pilot testing with adolescents. Understanding perceptions about prescription pain medicines will allow researchers to measure factors which place youth at higher risk for opioid addiction and to develop interventions for youth requiring opioid treatment, for example in the context of surgery.

Binge eating disorder is a debilitating disease which can arise from many kinds of traumas, pains, and stresses of life. Previous characterization of a binge-eating model developed by our lab shows that mice will consume greater quantities of high palatable diet (HPD) following exposure to specific types of psychological stressors including forced swim and foot shock compared to mice exposed to such psychological stressors. It was found that the claustrum of the brain had increased neural activity following bouts of binge-eating. One aspect of my research required me to quantify the density of neural activation in the claustrum from its most rostral to caudal area. We found that stressed mice displayed significantly higher levels of claustrum neural activation compared to controls. For the behavioral pattern we wanted to rule out influence of energy expenditure in the stress paradigm. Mice were given access to running wheels for an hour and then received access to HPD. Mice who displayed high levels of running had similar food intake to that of mice who did not display running activity. This suggests that psychological stress is an underlying component in this model for stress eating. As an ongoing project we are utilizing 1-photon imaging in the claustrum to monitor single cell activity across no stress vs. stress sessions and subsequent feeding behavior. We have thus far found increased neural activity in response to onset of a feeding bout in no stress conditions and we are investigating how stress modulates the effect of neurons tracked across time. This research potentially has great impact on the scientific community’s knowledge behind why psychological stressors contribute to binge-eating behaviors and could one day have astounding translational benefits for treating humans with binge-eating disorder.

Understanding the neural circuit mechanism underlying aversive memory formation is important for developing new treatments for patients with post-traumatic stress disorder (PTSD). The nociceptive circuit involved in such aversive memory formation remains incompletely characterized. Nociceptin opioid peptide receptors (NOPRs) in this circuit are expressed throughout the brain and have been implicated in nociceptive responses, anxiety, and aversive fear memory. To model aversive memory formation, we used a cued fear conditioning paradigm in mice. A mouse first learns to associate a tone and foot shock on day 1 and is assessed by its behavioral freezing response to tone only on day 2. We observed that using a high dose of a NOPR agonist on day 1 of the cued fear conditioning was associated with decreased arousal and prevented associative learning on day 2. Then, using thermal pain assays, we demonstrated a weakly anti-nociceptive effect after administration of the NOPR agonist, suggesting the disrupted fear learning is likely not due to loss of sensory transmission. Because disruption of fear learning was associated with decreased arousal, we screened for brain regions involved in the sedation from NOPR activation by selectively expressing NOPRs using a viral vector in different brain areas in a mouse lacking NOPRs. Interestingly, we found activating NOPRs in the parabrachial nucleus (PBN) in the brainstem was sufficient to produce sedation. Additionally, in situ RNA hybridization showed strong co-localization of oprl1 (NOPR gene) expressing cells and calca, a genetic marker encoding CGRP of a small group of cells in the ventrolateral PBN. Given previous literature has shown disrupted fear learning by inhibiting neural activity of those CGRP neurons, we hypothesize that NOPR-expressing PBN neurons may play an important role in aversive memory formation as well. Future investigations will explore the necessity and sufficiency of NOPR-expressing PBN neurons in aversive memory formation.

 Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa. In mice, intraperitoneal (i.p.) injections of THC, produce a characteristic triad of behavioral responses: 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. Thus, we have developed and optimized a paradigm of oral THC consumption in mice to better model a typical route of administration used by humans. Our model balances an acute consummatory window with a highly palatable, chocolate-flavored gelatin. This incentivizes mice to voluntarily consume enough THC to produce measurable cannabimimetic behaviors. Over a 3-day exposure paradigm we habituated mice to the gelatin where they had ad libitum access for 2 hours each day. We introduced THC into the gelatin and measured the triad of behaviors immediately following consumption to determine whether voluntary oral consumption induces the acute cannabimimetic behaviors. We found significant hypolocomotion, hypothermia, and analgesia at our highest concentration. Next, to determine whether these behaviors are caused by THC’s action at the primary endocannabinoid receptor, CB₁R, we treated mice with the inverse agonist SR1 prior to the behavioral tests. SR1 blocked the cannabimimetic behaviors induced by the consumption of THC-gelatin, suggesting the effects are CB₁R-dependent. To finalize this model, we have adapted our oral consumption paradigm to an acoustic startle behavioral model. Following our consumption paradigm, mice are subjected to tones of varying decibels and their startle response is measured. Moving forward we will continue acoustic startle testing to confirm preliminary data and expand the doses tested. Overall, these data verify that our model effectively induces cannabimimetic behaviors and can be used for future behavioral studies investigating a more translational route of administration compared to i.p.

The consumption of Cannabis has increased with legalization, rising 46% from 2019 to 2020 in the US. The primary psychoactive compound in Cannabis, ᐃ9-tetrahydrocannabinol (THC), modifies motivation and induces hypolocomotive effects that cause patients to stop using medical marijuana. Given the increasing frequency of Cannabis use and the unwanted side effects of THC, I sought to decipher the motivational and locomotive effects of THC on prefrontal cortex (PFC) activity during appetitive Pavlovian conditioning. I utilized biological sensors to measure neural activity (CamKIIa-GCaMP6f for calcium in projection neurons (N=3) and eCB2.0 for total endocannabinoid activity (N=6)) in WT mice aged 8-12 weeks. Neural activity (utilizing fiber photometry) and general behavior was recorded during appetitive Pavlovian conditioning. Here, a house light in the behavioral chamber (conditioned stimulus (CS)), initiated 6s before a sipper (sucrose) extended for 20 seconds (unconditioned stimulus (US)). After a random inter-trial interval of 60, 90, 120, or 150s, another event triggered a reward to consolidate an association between the house light (CS) and the reward (US). Mice experienced this conditioning for 25 minutes every day for 5 days. On day 6, I treated mice with either a moderate dose of THC (5 mg/kg) or vehicle to measure changes in neural and endocannabinoid activity during conditioning. I found that both endocannabinoid and calcium signaling were tightly locked to the CS and the US. Interestingly, trials where THC-treated mice did not interact with the sipper (THC-dependent demotivation), neural activity matched the pattern during training days. These data suggest time-locked neural activity linked to stimuli, separate from the locomotor output, to receive the reward. This study contributes to the understanding of THC’s effects on signaling during motivated versus locomotive behaviors to inform future THC-derived treatment paradigms.