Cues in the environment that reliably predict rewards play a central role in guiding behavior. Learning about reward-paired cues is facilitated in part by dopaminergic neurons in the ventral tegmental area (VTAda neurons) that signal prediction error teaching signals to drive associations between cues and rewards. VTAGABA neurons may modulate prediction errors by providing VTA with an expectation for upcoming rewards, allowing VTA dopamine neurons to accurately calculate the difference between expected and received rewards. To test this, we have employed a sensory-specific Pavlovian instrumental transfer (PIT). PIT allows us to test how subjects first learn that two cues lead to two distinct rewards (white noise → pellet; click → sucrose). Separately, they then learn to make two distinct actions that produce either of the rewards (left lever → pellet; right lever → sucrose). Finally, during the critical PIT test, each cue is presented while subjects have the opportunity to perform the actions without reward feedback. If sensory-specific expectation for reward is elicited by the cue, subjects should make the action that is congruent with the expectation elicited by the cue (e.g. white noise → left lever). We tested whether VTAGABA neurons would be important for the generation of this sensory-specific expectation by optogenetically inhibiting VTAGABA neurons during cue presentation in the PIT test. We hypothesize that inhibition of VTAGABA neurons would impair performance in the PIT test as they harbor sensory-specific reward expectations, allowing them to modulate VTADA prediction errors and associative learning.

A thorough understanding of the neural systems implicated in maladaptive alcohol consumption is necessary to develop new treatment and prevention strategies for alcohol use disorder (AUD). The orbitofrontal cortex (OFC) plays a role in decision making, alcohol motivation, and dependence. Research on role of OFC in alcohol consumption is vital. Previous work has shown that OFC inhibition results in decreased consumption during cue-based reinstatement. The current study sought to investigate the role of OFC in alcohol consumption after a period of voluntary intermittent alcohol consumption. Subjects were adult Long-Evans male and female rats (n = 9) that either received null virus (n = 4) or hM4Di DREADD on a CaMKII promoter (n =5) bilaterally in ventrolateral OFC. Rats underwent a 2-week period of voluntary intermittent consumption of alcohol using a two-bottle choice procedure wherein the amount of consumption was recorded. Following the two week period, subjects were administered (i.p) vehicle (A) or CNO (B) before being placed into a testing chamber wherein lick bouts, latency to lick, and locomotor activity were recorded for 30 minutes on three separate days using an ABA design. We expected to observe escalation of alcohol consumption across a 2-week period, with females escalating and consuming more than males. In rats receiving null virus in OFC, we observed alcohol consumption escalate across days of testing. We observed an increase in lick frequency, decreases in lick latency, but no change in locomotor activity when in the testing chamber. Data collection in the DREADD group is ongoing.

The Wnt signaling pathway plays a key role in cell fate determination and organogenesis in embryonic development and maintenance of adult stem cells. In the canonical pathway, Wnt ligand binds to LRP 5/6 and Frizzled(Fz), resulting in LRP 5/6 disruption of the β-catenin destruction complex. Axin is then recruited by LRP5/6, and the PDZ domain of the Dishevelled (Dvl) protein binds to by the seventh transmembrane receptor of Fz, inhibiting the destruction complex.^4 When inactive, the destruction complex assists with phosphorylating β-catenin. Dysregulation of this pathway can correlate to improper stem cell growth, maturation, and deficiency, which can lead to limbal stem cell deficiency – characterized by ocular pain, and decreased visual acuity.^2 PDZ domain inhibitors can be used to study the effects of Wnt pathway inhibition.^1In literature, a range of concentrations have been used to inhibit the Wnt pathway; however, higher concentrations of inhibitors can possibly lead to unwanted off-target effects.^5 The ONE-Tox Luciferase and Cell Viability Assay(Promega) was used to quantify Wnt signaling inhibition of the PDZ domain inhibitors on stable transfected HEK-293 cells. Sulindac was tested at 8 and 12 µM of Wnt3a. At low concentrations, Wnt signaling pathway was activated (5 µM and 25 µM respectively). At higher concentrations, inhibition was seen. This further leads to the inquiry as to what exactly occurs at the lower concentrations of PDZ domain inhibitors, and which IC50 is best for favorable inhibition of the pathway.

Wingless/integrated (Wnt) signaling is a transduction pathway that determines cell migration, fate determination, and organogenesis in embryonic development. In adults, it's crucial for stem cell maintenance and renewal, proliferation, and differentiation. Degradation of this pathway leads to oncogenesis and disruption of stem cell self-renewal and maturation, leading to diseases caused by stem cell deficiency such as limbal stem cell deficiency1,4. This pathway is activated when the Wnt ligand binds to Frizzled and LRP5/6, recruiting Dishevelled which then inhibits the GSK-3β enzyme. Accumulation and translocation of β-catenin in the nucleus lead to complex transcription factors that activate the Wnt signaling pathway1. Previous literature shows Wnt signaling activation via GSK-3β inhibitors at nanomolar and micromolar concentrations, the latter being extremely potent. Specific activation of the Wnt pathway is crucial because high concentrations have possible off-target effects. Using stable transfected HEK 293 cells, Wnt activation via different concentrations of GSK3B inhibitors was studied. After treatment, Promega’s TOPFlash assay was used to determine cell viability (fluorescence) and Wnt signaling activation (luminescence). Different GSK3b inhibitors had different potency at similar concentrations. CHIR99021 had a dramatically higher activation starting at 10uM compared to lower concentrations(.5-1uM). L807mts had a much lower but steadier increase in activation. High activation, as seen in CHIR99021, can be the result of off-target effects that are not adequately understood. Understanding the extent that the Wnt pathway is activated via GSK B inhibitors is crucial in creating experimental models that can isolate and study the target of interest without off-target effects.