Promoting Scientific Exchange and Student Training Through Scientific Meetings; Insights from a Joint Virtual Undergraduate Neuroscience Conference During the COVID-19 Pandemic.

Participation in scientific conferences is a fundamental part of neuroscience and student training. Many conference opportunities have been cancelled, limited, or changed in response to the COVID-19 pandemic. This paper is a conference report from a joint virtual 2021 meeting of two regional undergraduate neuroscience conferences, the Midwest/Great Lakes Undergraduate Research Symposium in Neuroscience (mGluRs) and the Midwest Regional Neuroscience Conference (MidBrains). We discuss our conference planning logistics, benefits and challenges of the virtual conference format, student feedback on the virtual meeting, additional benefits of a joint meeting, and "take home" messages and considerations for future conferences. We hope insights from our experience can benefit future conference organizers in planning scientific conferences, both for in-person and virtual settings.

Learning Experimental Design through Targeted Student-Centric Journal Club with Screencasting.

Knowledge and application of experimental design principles are essential components of scientific methodology, and experience with these skills is fundamental for participating in scientific research. However, undergraduates often enter the research laboratory with little training in designing and interpreting their own experiments. In the context of a research university laboratory, we designed a journal club training exercise to address this need. Students were instructed on methods for interpreting scientific literature using a screencast, a digital recording of a slide presentation narrated by an instructor. Students subsequently examined a series of research publications with a focus on the experimental designs and data interpretation in a two-session group discussion journal club format. We have found this approach to be an efficient and productive method for engaging students in learning about principles of experimental design and further preparing them for success in laboratory research.

Characterizing the Undergraduate Neuroscience Major in the U.S.: An Examination of Course Requirements and Institution-Program Associations.

Neuroscience is a rapidly expanding field, and many colleges and universities throughout the country are implementing new neuroscience degree programs. Despite the field's growth and popularity, little data exists on the structural character of current undergraduate neuroscience programs. We collected and examined comprehensive data on existing undergraduate neuroscience programs, including academic major requirements and institution characteristics such as size, financial resources, and research opportunities. Thirty-one variables covering information about course requirements, department characteristics, financial resources, and institution characteristics were collected from 118 colleges and universities in the United States that offer a major titled "neuroscience" or "neural sciences." Data was collected from publicly available sources (online databases, institutions' neuroscience program websites) and then analyzed to define the average curriculum and identify associations between institution and program characteristics. Our results suggest that the average undergraduate neuroscience major requires 3 chemistry, 3 biology, 3 laboratory, 2-3 neuroscience, 1 physics, 1 math, and 2 psychology courses, suggesting that most neuroscience programs emphasize the natural sciences over the social sciences. Additionally, while 98% of institutions in our database offer research opportunities, only 31% required majors to perform research. Of note, 70% of institutions offering a neuroscience major do not have a neuroscience department, suggesting that most institutions offer neuroscience as an interdisciplinary major spanning several departments. Finally, smaller liberal arts colleges account for the majority of institutions offering a neuroscience major. Overall, these findings may be useful for informing groups interested in undergraduate neuroscience training, including institutions looking to improve or establish programs, students wanting to major in neuroscience and employers hiring neuroscience graduates.

Basal microRNA expression patterns in reward circuitry of selectively bred high-responder and low-responder rats vary by brain region and genotype.

Mental health disorders involving altered reward, emotionality, and anxiety are thought to result from the interaction of individual predisposition (genetic factors) and personal experience (environmental factors), although the mechanisms that contribute to an individual's vulnerability to these disorders remain poorly understood. We used an animal model of individual variation [inbred high-responder/low-responder (bHR-bLR) rodents] known to vary in reward, anxiety, and emotional processing to examine neuroanatomical expression patterns of microRNAs (miRNAs). Laser capture microdissection was used to dissect the prelimbic cortex and the nucleus accumbens core and shell prior to analysis of basal miRNA expression in bHR and bLR male rats. These studies identified 187 miRNAs differentially expressed by genotype in at least one brain region, 10 of which were validated by qPCR. Four of these 10 qPCR-validated miRNAs demonstrated differential expression across multiple brain regions, and all miRNAs with validated differential expression between genotypes had lower expression in bHR animals compared with bLR animals. microRNA (miR)-484 and miR-128a expression differences between the prelimbic cortex of bHR and bLR animals were validated by semiquantitative in situ hybridization. miRNA expression analysis independent of genotype identified 101 miRNAs differentially expressed by brain region, seven of which validated by qPCR. Dnmt3a mRNA, a validated target of miR-29b, varied in a direction opposite that of miR-29b's differential expression between bHR and bLR animals. These data provide evidence that basal central nervous system miRNA expression varies in the bHR-bLR model, implicating microRNAs as potential epigenetic regulators of key neural circuits and individual differences associated with mental health disorders.

Glucocorticoid treatment of astrocytes results in temporally dynamic transcriptome regulation and astrocyte-enriched mRNA changes in vitro.

While general effects of glucocorticoids are well established, the specific cellular mechanisms by which these hormones exert tissue-dependent effects continue to be elaborated. Diseases that demonstrate altered glucocorticoid signaling have been associated with alterations in astrocytes, yet relatively little is known about the effects of glucocorticoids upon this cell type. We have analyzed mRNA expression patterns following glucocorticoid treatment of mouse primary astrocyte cultures. Microarray analysis of cortical astrocyte cultures treated with dexamethasone over an eight-point, 24 h time course identified 854 unique genes with ≥twofold change in mRNA expression at one or more time points. Clustering analysis associated subsets of these mRNA expression changes with gene ontology categories known to be impacted by glucocorticoids. Numerous mRNAs regulated by dexamethasone were also regulated by the natural ligand corticosterone; all of the mRNAs regulated ≥twofold by corticosterone were substantially attenuated by cotreatment with the glucocorticoid receptor antagonist RU486. Of the mRNAs demonstrating ≥twofold expression change in response to both glucocorticoids, 33 mRNAs were previously associated with glucocorticoid regulation, and 36 mRNAs were novel glucocorticoid targets. All genes tested by qPCR for glucocorticoid regulation in cortical astrocyte cultures were also regulated by glucocorticoids in hippocampal astrocyte cultures (18/18). Interestingly, a portion of glucocorticoid-regulated genes were astrocyte enriched; the percentage of astrocyte-enriched genes per total number of regulated genes was highest for the early time points and steadily decreased over the time course. These findings suggest that astrocytes in vitro may initially deploy cell type-specific patterns of mRNA regulatory responses to glucocorticoids and subsequently activate additional cell type-independent responses.

Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription.

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.

Validation of Protein Knockout in Mutant Zebrafish Lines Using In Vitro Translation Assays.

Advances in genome-editing technology have made creation of zebrafish mutant lines accessible to the community. Experimental validation of protein knockout is a critical step in verifying null mutants, but this can be a difficult task. Absence of protein can be confirmed by Western blotting; however, this approach requires target-specific antibodies that are generally not available for zebrafish proteins. We address this issue using in vitro translation assays, a fast and standard procedure that can be easily implemented.

The microRNA network is altered in anterior cingulate cortex of patients with unipolar and bipolar depression.

MicroRNAs (miRNAs) are small, non-coding RNAs acting as post-transcriptional regulators of gene expression. Though implicated in multiple CNS disorders, miRNAs have not been examined in any psychiatric disease state in anterior cingulate cortex (AnCg), a brain region centrally involved in regulating mood. We performed qPCR analyses of 29 miRNAs previously implicated in psychiatric illness (major depressive disorder (MDD), bipolar disorder (BP) and/or schizophrenia (SZ)) in AnCg of patients with MDD and BP versus controls. miR-132, miR-133a and miR-212 were initially identified as differentially expressed in BP, miR-184 in MDD and miR-34a in both MDD and BP (although none survived multiple correction testing and must be considered preliminary). In silico target prediction algorithms identified putative targets of differentially expressed miRNAs. Nuclear Co-Activator 1 (NCOA1), Nuclear Co-Repressor 2 (NCOR2) and Phosphodiesterase 4B (PDE4B) were selected based upon predicted targeting by miR-34a (with NCOR2 and PDE4B both targeted by miR-184) and published relevance to psychiatric illness. Luciferase assays identified PDE4B as a target of miR-34a and miR-184, while NCOA1 and NCOR2 were targeted by miR-34a and 184, respectively. qPCR analyses were performed to determine whether changes in miRNA levels correlated with mRNA levels of validated targets. NCOA1 showed an inverse correlation with miR-34a in BP, while NCOR2 demonstrated a positive correlation. In sum, this is the first study to demonstrate miRNA changes in AnCg in psychiatric illness and validate miR-34a as differentially expressed in CNS in MDD. These findings support a mechanistic role for miRNAs in the regulation of stress-responsive genes disrupted in psychiatric illness.

Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo.

Previous studies have primarily interpreted gene expression regulation by glucocorticoids in the brain in terms of impact on neurons; however, less is known about the corresponding impact of glucocorticoids on glia and specifically astrocytes in vivo. Recent microarray experiments have identified glucocorticoid-sensitive mRNAs in primary astrocyte cell culture, including a number of mRNAs that have reported astrocyte-enriched expression patterns relative to other brain cell types. Here, we have tested whether elevations of glucocorticoids regulate a subset of these mRNAs in vivo following acute and chronic corticosterone exposure in adult mice. Acute corticosterone exposure was achieved by a single injection of 10 mg/kg corticosterone, and tissue samples were harvested 2 h post-injection. Chronic corticosterone exposure was achieved by administering 10 mg/mL corticosterone via drinking water for 2 weeks. Gene expression was then assessed in two brain regions associated with glucocorticoid action (prefrontal cortex and hippocampus) by qPCR and by in situ hybridization. The majority of measured mRNAs regulated by glucocorticoids in astrocytes in vitro were similarly regulated by acute and/or chronic glucocorticoid exposure in vivo. In addition, the expression levels for mRNAs regulated in at least one corticosterone exposure condition (acute/chronic) demonstrated moderate positive correlation between the two conditions by brain region. In situ hybridization analyses suggest that select mRNAs are regulated by chronic corticosterone exposure specifically in astroctyes based on (1) similar general expression patterns between corticosterone-treated and vehicle-treated animals and (2) similar expression patterns to the pan-astrocyte marker Aldh1l1. Our findings demonstrate that glucocorticoids regulate astrocyte-enriched mRNAs in vivo and suggest that glucocorticoids regulate gene expression in the brain in a cell type-dependent fashion.