The COVID-19 and Taste Lab: A Mini Course-Based Undergraduate Research Experience on Taste Differences and COVID-19 Susceptibility.

Traditional course-based undergraduate research experiences (CUREs) are common approaches to expose students to authentic laboratory practices. Traditional CUREs typically take up most of or an entire semester, require a laboratory section or may be a standalone lab course, and require significant financial and time commitments by the institution and instructors. As such, CUREs are harder to implement at institutions with fewer resources. Here, we developed a mini-CURE, which are typically shorter in duration, called the COVID-19 and Taste Lab (CT-LAB). The CT-LAB requires significantly fewer resources ($0.05/student) and time commitment (two class periods) than traditional CUREs. CT-LAB centers around the biological relationship between COVID-19 susceptibility and taste status (non-taster, taster, and supertaster) as well as potential implications for public policy behavior. Students participated in a class-wide study where they examined if taste status was related to COVID-19 susceptibility. They found that non-tasters had a higher likelihood of testing positive previously for COVID-19 compared to tasters and supertasters. To assess student outcomes of this CURE, students completed a pre- and post-test assessment including a content test, STEM identity survey, taste test, COVID-19 history test, and a modified CURE survey. Content test scores improved while STEM identity and attitudes about science were unchanged. A direct comparison to a repository of traditional CUREs shows that the CT-LAB produced comparable benefits to traditional CUREs primarily in skills that were particularly relevant for the CT-LAB. This work suggests that mini-CUREs, even as brief as two class periods, could be a way to improve student outcomes.

The Spine Lab: A Short-Duration, Fully-Remote Course-Based Undergraduate Research Experience.

Course-based undergraduate research experiences (CUREs) are increasingly common approaches to provide students with authentic laboratory experiences. Typically, CUREs are semester-long, in-person experiences that can be financially and time prohibitive for some institutions, faculty, and students. Here, we developed a short-duration, fully-online CURE, the Spine Lab, to provide an opportunity for students to conduct original research. In this CURE, we focused on synaptic spines in the mammalian brain; synapses are the unit structure that functions in rapid information processing. The students worked together in pairs and as a class to analyze cortical neuron spine density and structural morphology changes between a mouse line with learning impairments (forebrain-specific ß-catenin knockouts [ß-cat cKOs]) and control (Ctl) littermates. The students showed their results in an online poster presentation. Their findings show that spine density is significantly reduced, while spine structural maturation is unaltered in the ß-cat cKO. Defining pathophysiological changes caused by CTNNB1/ß-catenin loss-of-function provides important insights relevant to human disorders caused by disruptive mutations in this gene. To assess the benefits of this CURE, students completed a pre- and post-test assessment including a content quiz, STEM identity survey, and a standardized CURE survey. Participation in the Spine Lab correlated with improved content and STEM identity scores, and decreased negative attitudes about science. Moreover, direct comparison to the CURE database reveals that the Spine Lab produces comparable benefits to traditional CUREs. This work as a whole suggests that short-duration, fully-online CUREs can provide benefit to students and could be an inclusive tool to improve student outcomes.

Combined Infusion and Stimulation with Fast-Scan Cyclic Voltammetry (CIS-FSCV) to Assess Ventral Tegmental Area Receptor Regulation of Phasic Dopamine.

Phasic dopamine (DA) release from the ventral tegmental area (VTA) to the nucleus accumbens plays a pivotal role in reward processing and reinforcement learning. Understanding how the diverse neuronal inputs into the VTA control phasic DA release can provide a better picture of the circuitry that controls reward processing and reinforcement learning. Here, we describe a method that combines intra-VTA cannula infusions of pharmacological agonists and antagonists with stimulation-evoked phasic DA release (combined infusion and stimulation, or CIS) as measured by in vivo fast-scan cyclic voltammetry (FSCV). Using CIS-FSCV in anesthetized rats, a phasic DA response can be evoked by electrically stimulating the VTA with a bipolar electrode fitted with a cannula while recording in the nucleus accumbens core. Pharmacological agonists or antagonists can be infused directly at the stimulation site to investigate specific VTA receptors' roles in driving phasic DA release. A major benefit of CIS-FSCV is that VTA receptor function can be studied in vivo, building on in vitro studies.

The Biological Impact of Menthol on Tobacco Dependence.

In the 1920s, tobacco companies created a marketing campaign for what would one day be their most profitable series of products: mentholated tobacco cigarettes. Menthol provides the smoker with a pleasant mint flavor in addition to a cooling sensation of the mouth, throat, and lungs, giving relief from the painful irritation caused by tobacco smoke. Promising a healthier cigarette using pictures of doctors in white coats and even cartoon penguins, tobacco companies promoted these cigarettes to young, beginner smokers and those with respiratory health concerns. Today, smoking tobacco cigarettes causes one in five US Americans to die prematurely, crowning it as the leading cause of preventable death. In contrast to the dubious health claims by tobacco companies, mentholated cigarettes are in fact more addictive. Smokers of mentholated cigarettes have lower successful quit rates and in some cases are resistant to both behavioral and pharmacological treatment strategies. There is now considerable evidence, especially in the last 5 years, that suggest menthol might influence the addictive potential of nicotine-containing tobacco products via biological mechanisms. First, menthol alters the expression, stoichiometry, and function of nicotinic receptors. Second, menthol's chemosensory properties operate to mask aversive properties of using tobacco products. Third, menthol's chemosensory properties aid in serving as a conditioned cue that can both enhance nicotine intake and drive relapse. Fourth, menthol alters nicotine metabolism, increasing its bioavailability. This review discusses emerging evidence for these mechanisms, with an emphasis on preclinical findings that may shed light on why menthol smokers exhibit greater dependence. IMPLICATIONS: Mentholated cigarettes have been shown to have greater addictive potential than their nonmentholated counterparts. Evidence is pointing toward multiple mechanisms of action by which menthol may alter tobacco dependence. Understanding menthol's biological functions as it pertains to nicotine dependence will be helpful in crafting novel pharmacotherapies that might better serve menthol smokers. In addition, a better understanding of menthol's pharmacology as it relates to tobacco dependence will be valuable for informing policy decisions on the regulation of mentholated cigarettes.

Learning impairments and molecular changes in the brain caused by ß-catenin loss.

Intellectual disability (ID), defined as IQ<70, occurs in 2.5% of individuals. Elucidating the underlying molecular mechanisms is essential for developing therapeutic strategies. Several of the identified genes that link to ID in humans are predicted to cause malfunction of ß-catenin pathways, including mutations in CTNNB1 (ß-catenin) itself. To identify pathological changes caused by ß-catenin loss in the brain, we have generated a new ß-catenin conditional knockout mouse (ß-cat cKO) with targeted depletion of ß-catenin in forebrain neurons during the period of major synaptogenesis, a critical window for brain development and function. Compared with control littermates, ß-cat cKO mice display severe cognitive impairments. We tested for changes in two ß-catenin pathways essential for normal brain function, cadherin-based synaptic adhesion complexes and canonical Wnt (Wingless-related integration site) signal transduction. Relative to control littermates, ß-cat cKOs exhibit reduced levels of key synaptic adhesion and scaffold binding partners of ß-catenin, including N-cadherin, α-N-catenin, p120ctn and S-SCAM/Magi2. Unexpectedly, the expression levels of several canonical Wnt target genes were not altered in ß-cat cKOs. This lack of change led us to find that ß-catenin loss leads to upregulation of γ-catenin (plakoglobin), a partial functional homolog, whose neural-specific role is poorly defined. We show that γ-catenin interacts with several ß-catenin binding partners in neurons but is not able to fully substitute for ß-catenin loss, likely due to differences in the N-and C-termini between the catenins. Our findings identify severe learning impairments, upregulation of γ-catenin and reductions in synaptic adhesion and scaffold proteins as major consequences of ß-catenin loss.

Social Stimulus Causes Aberrant Activation of the Medial Prefrontal Cortex in a Mouse Model With Autism-Like Behaviors.

Autism spectrum disorder (ASD) is a highly prevalent and genetically heterogeneous brain disorder. Developing effective therapeutic interventions requires knowledge of the brain regions that malfunction and how they malfunction during ASD-relevant behaviors. Our study provides insights into brain regions activated by a novel social stimulus and how the activation pattern differs between mice that display autism-like disabilities and control littermates. Adenomatous polyposis coli (APC) conditional knockout (cKO) mice display reduced social interest, increased repetitive behaviors and dysfunction of the ß-catenin pathway, a convergent target of numerous ASD-linked human genes. Here, we exposed the mice to a novel social vs. non-social stimulus and measured neuronal activation by immunostaining for the protein c-Fos. We analyzed three brain regions known to play a role in social behavior. Compared with control littermates, APC cKOs display excessive activation, as evidenced by an increased number of excitatory pyramidal neurons stained for c-Fos in the medial prefrontal cortex (mPFC), selectively in the infralimbic sub-region. In contrast, two other social brain regions, the medial amygdala and piriform cortex show normal levels of neuron activation. Additionally, APC cKOs exhibit increased frequency of miniature excitatory postsynaptic currents (mEPSCs) in layer 5 pyramidal neurons of the infralimbic sub-region. Further, immunostaining is reduced for the inhibitory interneuron markers parvalbumin (PV) and somatostatin (SST) in the APC cKO mPFC. Our findings suggest aberrant excitatory-inhibitory balance and activation patterns. As ß-catenin is a core pathway in ASD, we identify the infralimbic sub-region of the mPFC as a critical brain region for autism-relevant social behavior.

Evaluating oral flavorant effects on nicotine self-administration behavior and phasic dopamine signaling.

Understanding how tobacco product flavor additives, such as flavorants in electronic cigarettes, influence smoking behavior and addiction is critical for informing public health policy decisions regarding tobacco product regulation. Here, we developed a combined intraoral (i.o.) and intravenous (i.v.) self-administration paradigm in rats to determine how flavorants influence self-administration behavior. By combining i.o. flavorant delivery with fast scan cyclic voltammetry (FSCV) or i.v. nicotine self-administration in adult, male rats, we examined whether flavors alter phasic dopamine (DA) signaling and nicotine self-administration. Oral administration of 10% sucrose or 0.32% saccharin, but not 0.005% menthol, increased phasic DA release in the nucleus accumbens (NAc). Oral sucrose or saccharin, when combined with i.v. nicotine delivery, also led to increased self-administration behavior. Specifically, combined i.o. sucrose and i.v. nicotine decreased responding compared to sucrose alone, and increased responding compared to nicotine alone. In contrast, i.o. flavorants did not alter motivational breakpoint in a progressive ratio task. Oral menthol, which did not alter i.v. nicotine administration, reversed oral nicotine aversion (50 and 100 mg/L) in a two-bottle choice test. Here, we demonstrate that i.o. appetitive flavorants that increase phasic DA signaling also increase self-administration behavior when combined with i.v. nicotine delivery. Additionally, oral menthol effects were specific to oral nicotine, and were not observed with i.v. nicotine-mediated reinforcement. Together, these preclinical findings have important implications regarding menthol and sweet flavorant additive effects on tobacco product use and can be used to inform policy decisions on tobacco product flavorant regulation.

Vitamin D3: A Role in Dopamine Circuit Regulation, Diet-Induced Obesity, and Drug Consumption.

The influence of micronutrients on dopamine systems is not well defined. Using mice, we show a potential role for reduced dietary vitamin D3 (cholecalciferol) in promoting diet-induced obesity (DIO), food intake, and drug consumption while on a high fat diet. To complement these deficiency studies, treatments with exogenous fully active vitamin D3 (calcitriol, 10 µg/kg, i.p.) were performed. Nondeficient mice that were made leptin resistant with a high fat diet displayed reduced food intake and body weight after an acute treatment with exogenous calcitriol. Dopamine neurons in the midbrain and their target neurons in the striatum were found to express vitamin D3 receptor protein. Acute calcitriol treatment led to transcriptional changes of dopamine-related genes in these regions in naive mice, enhanced amphetamine-induced dopamine release in both naive mice and rats, and increased locomotor activity after acute amphetamine treatment (2.5 mg/kg, i.p.). Alternatively, mice that were chronically fed either the reduced D3 high fat or chow diets displayed less activity after acute amphetamine treatment compared with their respective controls. Finally, high fat deficient mice that were trained to orally consume liquid amphetamine (90 mg/L) displayed increased consumption, while nondeficient mice treated with calcitriol showed reduced consumption. Our findings suggest that reduced dietary D3 may be a contributing environmental factor enhancing DIO as well as drug intake while eating a high fat diet. Moreover, these data demonstrate that dopamine circuits are modulated by D3 signaling, and may serve as direct or indirect targets for exogenous calcitriol.

Examination of Rapid Dopamine Dynamics with Fast Scan Cyclic Voltammetry During Intra-oral Tastant Administration in Awake Rats.

Rapid, phasic dopamine (DA) release in the mammalian brain plays a critical role in reward processing, reinforcement learning, and motivational control. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique with high spatial and temporal (sub-second) resolution that has been utilized to examine phasic DA release in several types of preparations. In vitro experiments in single-cells and brain slices and in vivo experiments in anesthetized rodents have been used to identify mechanisms that mediate dopamine release and uptake under normal conditions and in disease models. Over the last 20 years, in vivo FSCV experiments in awake, freely moving rodents have also provided insight of dopaminergic mechanisms in reward processing and reward learning. One major advantage of the awake, freely moving preparation is the ability to examine rapid DA fluctuations that are time-locked to specific behavioral events or to reward or cue presentation. However, one limitation of combined behavior and voltammetry experiments is the difficulty of dissociating DA effects that are specific to primary rewarding or aversive stimuli from co-occurring DA fluctuations that mediate reward-directed or other motor behaviors. Here, we describe a combined method using in vivo FSCV and intra-oral infusion in an awake rat to directly investigate DA responses to oral tastants. In these experiments, oral tastants are infused directly to the palate of the rat--bypassing reward-directed behavior and voluntary drinking behavior--allowing for direct examination of DA responses to tastant stimuli.

Muscarinic, but not nicotinic, acetylcholine receptor blockade in the ventral tegmental area attenuates cue-induced sucrose-seeking.

The mesolimbic dopamine (DA) system is known to play a role in cue-mediated reward-seeking for natural rewards and drugs of abuse. Specifically, cholinergic and glutamatergic receptors in the ventral tegmental area (VTA) have been shown to regulate cue-induced drug-seeking. However, the potential role of these VTA receptors in regulating cue-induced reward seeking for natural rewards is unknown. Here, we examined whether blockade of VTA acetylcholine receptors (AChRs) and N-methyl-d-aspartate receptors (NMDARs) would alter cue-induced sucrose seeking in male Sprague-Dawley rats. Subjects underwent 10 days of sucrose self-administration training (fixed ratio 1 schedule) followed by 7 days of forced abstinence. On withdrawal day 7, rats received bilateral VTA infusion of vehicle, the muscarinic AChR antagonist scopolamine (2.4 or 24 µg/side), the nicotinic AChR antagonist mecamylamine (3 or 30 µg/side), or the NMDAR antagonist AP-5 (0.1 or 1 µg/side) immediately prior to examination of cue-induced sucrose-seeking. Scopolamine infusion led to robust attenuation, but did not completely block, sucrose-seeking behavior. In contrast, VTA administration of mecamylamine or AP-5 did not alter cue-induced sucrose-seeking. Together, the data suggest that VTA muscarinic AChRs, but not nicotinic AChRs nor NMDARs, facilitate the ability of food-associated cues to drive seeking behavior for a food reward.

Differential role of ventral tegmental area acetylcholine and N-methyl-D-aspartate receptors in cocaine-seeking.

Exposure to drug-associated cues evokes drug-seeking behavior and is regarded as a major cause of relapse. Cues evoke burst firing of ventral tegmental area (VTA) dopamine (DA) neurons and phasic DA release in the nucleus accumbens (NAc). Cholinergic and glutamatergic input to the VTA is suggested to gate phasic DA activity. However, the role of VTA cholinergic and glutamatergic receptors in regulating phasic dopamine release and cue-induced drug-seeking in cocaine experienced subjects is not known. In male Sprague-Dawley rats, we found that VTA inactivation strongly inhibited, while VTA stimulation promoted, cocaine-seeking behavior during early withdrawal. Blockade of phasic activated D1 receptors in the NAc core also strongly inhibited cue-induced cocaine-seeking--suggesting an important role of phasic DA activity in the VTA to NAc core circuit. Next, we examined the role of VTA acetylcholine receptors (AChRs) and N-methyl-D-aspartate receptors (NMDARs) in regulating both NAc core phasic DA release and cue-induced cocaine-seeking. In cocaine naïve subjects, VTA infusion of the nicotinic acetylcholine receptor (AChR) antagonist mecamylamine, the muscarinic AChR antagonist scopolamine, or the NMDAR antagonist AP-5, led to robust attenuation of phasic DA release in the NAc core. During early cocaine withdrawal, VTA infusion of AP-5 had limited effects on NAc phasic DA release and cue-induced cocaine-seeking while VTA infusion of mecamylamine or scopolamine robustly inhibited both phasic DA release and cocaine-seeking. The results demonstrate that VTA AChRs, but not NMDARs, strongly regulate cue-induced cocaine-seeking and phasic DA release during early cocaine withdrawal.

Advances in studying phasic dopamine signaling in brain reward mechanisms.

The last sixty years of research has provided extraordinary advances of our knowledge of the reward system. Since its discovery as a neurotransmitter by Carlsson and colleagues (1), dopamine (DA) has emerged as an important mediator of reward processing. As a result, a number of electrochemical techniques have been developed to measure DA in the brain. Together, these techniques have begun to elucidate the complex roles of tonic and phasic DA signaling in reward processing and addiction. In this review, we will first provide a guide for the most commonly used electrochemical methods for DA detection and describe their utility in furthering our knowledge about DA's role in reward and addiction. Second, we will review the value of common in vitro and in vivo preparations and describe their ability to address different types of questions. Last, we will review recent data that has provided new mechanistic insight of in vivo phasic DA signaling and its role in reward processing and reward-mediated behavior.

Iron deficiency with or without anemia impairs prepulse inhibition of the startle reflex.

Iron deficiency (ID) during early life causes long-lasting detrimental cognitive sequelae, many of which are linked to alterations in hippocampus function, dopamine synthesis, and the modulation of dopaminergic circuitry by the hippocampus. These same features have been implicated in the origins of schizophrenia, a neuropsychiatric disorder with significant cognitive impairments. Deficits in sensorimotor gating represent a reliable endophenotype of schizophrenia that can be measured by prepulse inhibition (PPI) of the acoustic startle reflex. Using two rodent model systems, we investigated the influence of early-life ID on PPI in adulthood. To isolate the role of hippocampal iron in PPI, our mouse model utilized a timed (embryonic day 18.5), hippocampus-specific knockout of Slc11a2, a gene coding an important regulator of cellular iron uptake, the divalent metal transport type 1 protein (DMT-1). Our second model used a classic rat dietary-based global ID during gestation, a condition that closely mimics human gestational ID anemia (IDA). Both models exhibited impaired PPI in adulthood. Furthermore, our DMT-1 knockout model displayed reduced long-term potentiation (LTP) and elevated paired-pulse facilitation (PPF), electrophysiological results consistent with previous findings in the IDA rat model. These results, in combination with previous findings demonstrating impaired hippocampus functioning and altered dopaminergic and glutamatergic neurotransmission, suggest that iron availability within the hippocampus is critical for the neurodevelopmental processes underlying sensorimotor gating. Ultimately, evidence of reduced PPI in both of our models may offer insights into the roles of fetal ID and the hippocampus in the pathophysiology of schizophrenia.

Serine racemase deletion abolishes light-evoked NMDA receptor currents in retinal ganglion cells.

Glycine and/or D-serine are obligatory coagonists of the N-methyl-D-aspartate receptor (NMDAR). Serine racemase, the D-serine-synthesizing enzyme, is expressed by astrocytes and Müller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of D-serine to inner-retinal function. Retinal tissue levels of D-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of D-serine in inner-retinal function and development.

Canonical TGF-beta signaling is required for the balance of excitatory/inhibitory transmission within the hippocampus and prepulse inhibition of acoustic startle.

Smad4 is a unique nuclear transducer for all TGF-beta signaling pathways and regulates gene transcription during development and tissue homeostasis. To elucidate the postnatal role of TGF-beta signaling in the mammalian brain, we generated forebrain-specific Smad4 knock-out mice. Surprisingly, the mutants showed no alteration in long-term potentiation and water maze, suggesting that Smad4 is not required for spatial learning and memory. However, these mutant mice did show enhancement of paired-pulse facilitation in excitatory synaptic transmission and stronger paired-pulse depression of GABA(A) currents in the hippocampus. The alteration of hippocampal electrophysiology correlated with mouse hyperactivity in homecage and open field tests. Mutant mice also showed overgrooming as well as deficits of prepulse inhibition, a widely used endophenotype of schizophrenia. With a specific real-time PCR array focused on TGF-beta signaling pathway, we identified a novel regulation mechanism of the pathway in the hippocampal neurons, in which Smad4-mediated signaling suppresses the level of extracellular antagonism of TGF-beta ligands through transcriptional regulation of follistatin, a selective inhibitor to activin/TGF-beta signaling in the hippocampus. In summary, we suggest that the canonical TGF-beta signaling pathway is critical for use-dependent modulation of GABA(A) synaptic transmission and dendritic homeostasis; furthermore, a disruption in the balance of the excitatory and inhibitory hippocampal network can result in psychiatric-like behavior.