Repeated seizures lead to progressive ventilatory dysfunction in SSKcnj16-/- rats.

Patients with uncontrolled epilepsy experience repeated seizures putting them at increased risk for sudden unexpected death in epilepsy (SUDEP). Data from human patients have led to the hypothesis that SUDEP results from severe cardiorespiratory suppression after a seizure, which may involve pathological deficiencies in the brainstem serotonin (5-HT) system. Rats with a genomic Kcnj16 mutation (SSKcnj16-/- rats) are susceptible to sound-induced generalized tonic-clonic seizures (GTCS) which, when repeated once daily for up to 10 days (10-day seizure protocol), increased mortality, particularly in male rats. Here, we test the hypothesis that repeated seizures across the 10-day protocol will cause a progressive ventilatory dysfunction due to time-dependent 5-HT deficiency. Initial severe seizures led to ictal and postictal apneas and transient decreases in breathing frequency, ventilatory drive, breath-to-breath variability, and brief hypoventilation. These seizure-induced effects on ventilation were exacerbated with increasing seizures and ventilatory chemoreflexes became further impaired after repeated seizures. Tissue analyses of key brainstem regions controlling breathing showed time-dependent 5-HT system suppression and increased immunoreactivity for IBA-1 (microglial marker) without changes in overall cell counts at 3, 7, and 10 days of seizures. Fluoxetine treatment in SSKcnj16-/- rats prevented repeated seizure-induced progressive respiratory suppression but failed to prevent seizure-related mortality. We conclude that repeated seizures cause a progressive compromise of ventilatory control in the immediate postictal period largely mediated by serotonin system suppression in brainstem regions of respiratory control. However, other unknown factors contribute to overall survival following repeated seizures in this model.NEW & NOTEWORTHY This study demonstrated that repeated seizures in a novel rat model (SSKcnj16-/- rats) caused a progressively greater ventilatory dysfunction in the immediate postictal period associated with brainstem serotonin (5-HT) suppression. Augmenting brain 5-HT with a selective serotonin reuptake inhibitor prevented the progressive ventilatory dysfunction induced by repeated seizures but failed to prevent seizure-related mortality, suggesting that repeated seizures may lead to cardiorespiratory suppression and failure through multiple mechanisms.

Adapting Case Studies for Synchronous and Asynchronous Online Courses.

Case studies are an effective active learning method that increases student engagement and are readily adaptable from in-person to online learning environments. In this perspective, Neuroscience Case Network fellows (NeuroCaseNet; NSF-RCN-UBE Grant #1624104) provide specific examples of how case studies were successfully adapted for synchronous and asynchronous online learning, including general strategies and best practices for adapting case studies into both online learning environments.

Effective Use of Student-Created Case Studies as Assessment in an Undergraduate Neuroscience Course.

Case studies and student-led learning activities are both effective active learning methods for increasing student engagement, promoting student learning, and improving student performance. Here, we describe combining these instructional methods to use student-created case studies as assessment for an online neurovirology module in a neuroanatomy and physiology course. First, students learned about neurovirology in a flipped classroom format using free, open-access virology resources. Then, students used iterative writing practices to write an interrupted case study incorporating a patient narrative and primary literature data on the neurovirulent virus of their choice, which was graded as a writing assessment. Finally, students exchanged case studies with their peers, and both taught and completed the case studies as low-stakes assessment. Student performance and evaluations support the efficacy of case studies as assessment, where iterative writing improved student performance, and students reported increased knowledge and confidence in the corresponding learning objectives. Overall, we believe that using student-created case studies as assessment is a valuable, student-led extension of effective case study pedagogy, and has wide applicability to a variety of undergraduate courses.

The Simpsons Neuron: A Case Study Exploring Neuronal Coding and the Scientific Method for Introductory and Advanced Neuroscience Courses.

A fictitious patient, Miguel, has been diagnosed with drug-resistant epilepsy and is awaiting neurosurgery. While in the hospital, Miguel agrees to participate in a research study in which depth electrodes are used to record neuronal activity in response to a range of stimuli. Interestingly, a neuron is identified that seems to respond selectively to video clips of the animated satirical TV show The Simpsons. Students are challenged to make observations, formulate and revise hypotheses, and interpret data, excerpted from an authentic dataset derived from actual patients in a 2008 Science paper. Students then consider implications for these data, evaluate their ability to generalize to non-human (rodent) models, and speculate about future directions for this research. Adaptations of this case have been implemented in introductory and advanced neuroscience courses. Students responded positively to the case, and reported gains in science competence and identity, particularly in the introductory courses. Suggestions for implementation and adaptation of this experience are offered. While this case has been implemented in undergraduate neuroscience courses, it might also be used in physiology, psychology, biology, research methods, or clinical courses.

Cardiorespiratory Response to Moderate Hypercapnia in Female College Students Expressing Behaviorally Inhibited Temperament.

Behaviorally inhibited (BI) temperament is marked by heightened behavioral sensitivity to environmental threats. The degree to which threat sensitivity is reflected in cardiorespiratory responses has been relatively unexplored. Female college students were exposed to modest hypercapnia (7.0% CO2) or ambient air (AA) while engaging in a computerized task with cued reinforcement features. All physiological variables except for blood pressure were processed in 4 min epochs corresponding to pre-exposure, exposure, and post-exposure. Primary respiratory measures were respiratory frequency (fb), tidal volume (VT), and minute ventilation (VE). Electrocardiograms (ECGs) were processed using ARTiiFACT software with resultant heart rate variability (HRV) measures in the frequency domain and time domain. Consistent with the literature, modest hypercapnia increased VT, Fb, and VE. No differences in respiratory parameters were detected between BI and non-behaviorally inhibited individuals (NI). For HRV in the time domain, RMSSD and NN50 values increased during CO2 inhalation which then returned to pre-exposure levels after CO2 cessation. Hypercapnia increased high frequency (HF) power which then recovered. BI exhibited reduced low frequency (LF) power during the pre-exposure period. For NI, LF power reduced over the subsequent phases ameliorating differences between BI and NI. Hypercapnia improved the task performance of BI. This is the largest study of female reactivity to hypercapnia and associated HRV to date. In general, hypercapnia increased time domain HRV and HF power, suggesting a strong vagal influence. Those expressing BI exhibited similar respiratory and HRV reactivity to NI despite inherently reduced LF power. Although 7% CO2 represents a mild challenge to the respiratory and cardiovascular systems, it is nonetheless sufficient to explore inherent difference in stress reactivity in those vulnerable to develop anxiety disorders.

Endogenous glutamatergic inputs to the Parabrachial Nucleus/Kölliker-Fuse Complex determine respiratory rate.

The Kölliker-Fuse Nucleus (KF) has been widely investigated for its contribution to "inspiratory off-switch" while more recent studies showed that activation of the Parabrachial Nucleus (PBN) shortened expiratory duration. This study used an adult, in vivo, decerebrate rabbit model to delineate the contribution of each site to inspiratory and expiratory duration through sequential block of glutamatergic excitation with the receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5). Glutamatergic disfacilitation caused large increases in inspiratory and expiratory duration and minor decrease in peak phrenic activity (PPA). Hypoxia only partially reversed respiratory rate depression but PPA was increased to >200 % of control. The contribution of PBN activity to inspiratory and expiratory duration was equal while block of the KF affected inspiratory duration more than expiratory. We conclude that in the in vivo preparation respiratory rate greatly depends on PBN/KF activity, which contributes to the "inspiratory on- "and "off-switch", but is of minor importance for the magnitude of phrenic motor output.

The contribution of endogenous glutamatergic input in the ventral respiratory column to respiratory rhythm.

Glutamate is the predominant excitatory neurotransmitter in the ventral respiratory column; however, the contribution of glutamatergic excitation in the individual subregions to respiratory rhythm generation has not been fully delineated. In an adult, in vivo, decerebrate rabbit model during conditions of mild hyperoxic hypercapnia we blocked glutamatergic excitation using the receptor antagonists 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5). Disfacilitation of the preBötzinger Complex caused a decrease in inspiratory and expiratory duration as well as peak phrenic amplitude and ultimately apnea. Disfacilitation of the Bötzinger Complex caused a decrease in inspiratory and expiratory duration; subsequent disfacilitation of the preBötzinger Complex resulted in complete loss of the respiratory pattern but maintained tonic inspiratory activity. We conclude that glutamatergic drive to the preBötzinger Complex is essential for respiratory rhythm generation. Glutamatergic drive to the Bötzinger Complex significantly affects inspiratory and expiratory phase duration. Bötzinger Complex neurons are responsible for maintaining the silent expiratory phase of the phrenic neurogram.

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.

Using Case Studies to Promote Student Engagement in Primary Literature Data Analysis and Evaluation.

Analyzing and evaluating primary literature data is a common learning objective in undergraduate neuroscience courses. However, students with more clinically focused career goals often dismiss the relevance of evaluating basic neuroscience literature. Here, we describe using case studies to promote student engagement in primary literature in a cellular and molecular neuroscience course. Two example literature-based case studies are provided: Untwisting Pretzel Syndrome, a neurodevelopment case exploring synapse formation in a pretzel syndrome patient, and The Trials of ALS, a neurodegeneration case exploring axon degeneration and repair in an amyotrophic lateral sclerosis patient. These cases were assigned after neurodevelopment and neurodegeneration lectures covering key concepts. Both cases begin by introducing the patient and hypothesizing symptoms and diagnoses, followed by scenes incorporating primary data to illustrate disease pathogenesis and treatments. Students complete questions embedded in these cases as homework, and class time is used to discuss their answers. Discussion emphasizes that there can be multiple "correct" answers, and the best answers are accurate and well-supported. Accordingly, students edit their answers in class, and these annotations are factored into a pass/fail grade on the case. Additional scenes and questions from the same case studies are used on the course's take-home exams, thereby allowing students to practice primary data analysis and evaluation before a graded assignment. Student evaluations support literature-based case studies as an effective learning tool, with students identifying cases as the most valuable aspect of the course, and reporting increased confidence in understanding cellular and molecular neuroscience.

A retrograde adeno-associated virus for collecting ribosome-bound mRNA from anatomically defined projection neurons.

The brain contains a large variety of projection neurons with different functional properties. The functional properties of projection neurons arise from their connectivity with other neurons and their molecular composition. We describe a novel tool for obtaining the gene expression profiles of projection neurons that are anatomically defined by the location of their soma and axon terminals. Our tool utilizes adeno-associated virus serotype 9 (AAV9), which we found to retrogradely transduce projection neurons after injection at the site of the axon terminals. We used AAV9 to express Enhanced Green Fluorescent Protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a), which enables the immunoprecipitation of EGFP-tagged ribosomes and associated mRNA with a method known as Translating Ribosome Affinity Purification (TRAP). To achieve high expression of the EGFP-L10a protein in projection neurons, we placed its expression under control of a 1.3 kb alpha-calcium/calmodulin-dependent protein kinase II (Camk2a) promoter. We injected the AAV9-Camk2a-TRAP virus in either the hippocampus or the bed nucleus of the stria terminalis (BNST) of the mouse brain. In both brain regions the 1.3 kb Camk2a promoter did not confer complete cell-type specificity around the site of injection, as EGFP-L10a expression was observed in Camk2a-expressing neurons as well as in neuronal and non-neuronal cells that did not express Camk2a. In contrast, cell-type specific expression was observed in Camk2a-positive projection neurons that were retrogradely transduced by AAV9-Camk2a-TRAP. Injection of AAV9-Camk2a-TRAP into the BNST enabled the use of TRAP to collect ribosome-bound mRNA from basal amygdala projection neurons that innervate the BNST. AAV9-Camk2a-TRAP provides a single-virus system that can be used for the molecular profiling of anatomically defined projection neurons in mice and other mammalian model organisms. In addition, AAV9-Camk2a-TRAP may enable the discovery of protein synthesis events that support information storage in projection neurons.