Body painting, ultrasound, clinical examination, and peer-teaching: A student-centered approach to enhance musculoskeletal anatomy learning.

The presented course, established 2016 as a compulsory elective for 22nd-year bachelor medical students, aimed to enhance deep learning of upper and lower limb anatomy from a clinical perspective by a maximum of student-centered activities combining hands-on skills training with team-learning. Three cohorts (in total 60 students) participated in this study. Students rotated through body painting, ultrasound, and clinical investigation supervised by faculty or an experienced clinician. Teams of 3-4 students prepared presentations on clinical anatomy and pathological conditions, which by teacher- and peer assessments on average achieved >85% (mean 17.8/20 points ± 1.06). After each activity session, the students reported their learning experience through a reflective diary. Fifty students (83%) evaluated the course by a voluntary anonymous questionnaire combining Likert-type scale and free-text questions to assess, predominantly, perception of course activities and their perceived influence on learning anatomy. Journal reports and questionnaires revealed that the students highly valued the course, and 92% (29 females, 17 males) rated group work satisfying or well-perceived. The highest appreciation achieved ultrasound followed by clinical examination and body painting, which one third proposed to integrate into the regular dissection course. All students recommended the course to their younger peers. This course was feasible to integrate in the pre-existing curriculum. Limiting factors to offer this elective course to more students are availability of clinical teachers, technical equipment, and education rooms. Being student-directed tasks, body painting and reflective diary-writing would be feasible to implement without additional faculty, which we recommend to educators for student engagement activation.

Noninvasive Intravascular Microtransfusion in Colonial Tunicates.

Tunicates are a diverse group of worldwide marine filter-feeders that are vertebrates' closest invertebrate relatives. Colonial tunicates are the only know chordates that have been shown to undergo whole-body regeneration (WBR). Botrylloides in particular can regenerate one fully functional adult from a minute fragment of their vascular system in as little as 10 days. This regenerative process relies on the proliferation of circulating stem cells, likely supported by the activity of some of the 11 identified types of hemocytes. To study and challenge WBR, it is thus important to have the capacity to isolate, analyze, and manipulate hemolymph in regenerating colonies. Here we present a microtransfusion technique that permits the collection of pure hemocytes, the quantification of their purity, their labeling, and reinjection into colonial tunicates. To exemplify our approach, we present in addition a protocol to analyze the isolated hemocytes using flow cytometry. Our approach is minimally invasive, does not induce lethality, and therefore allows repeated transfusion into exactly the same colony with minimal disruption to the process being studied.

The Parvalbumin Hypothesis of Autism Spectrum Disorder.

The prevalence of autism spectrum disorder (ASD)-a type of neurodevelopmental disorder-is increasing and is around 2% in North America, Asia, and Europe. Besides the known genetic link, environmental, epigenetic, and metabolic factors have been implicated in ASD etiology. Although highly heterogeneous at the behavioral level, ASD comprises a set of core symptoms including impaired communication and social interaction skills as well as stereotyped and repetitive behaviors. This has led to the suggestion that a large part of the ASD phenotype is caused by changes in a few and common set of signaling pathways, the identification of which is a fundamental aim of autism research. Using advanced bioinformatics tools and the abundantly available genetic data, it is possible to classify the large number of ASD-associated genes according to cellular function and pathways. Cellular processes known to be impaired in ASD include gene regulation, synaptic transmission affecting the excitation/inhibition balance, neuronal Ca2+ signaling, development of short-/long-range connectivity (circuits and networks), and mitochondrial function. Such alterations often occur during early postnatal neurodevelopment. Among the neurons most affected in ASD as well as in schizophrenia are those expressing the Ca2+-binding protein parvalbumin (PV). These mainly inhibitory interneurons present in many different brain regions in humans and rodents are characterized by rapid, non-adaptive firing and have a high energy requirement. PV expression is often reduced at both messenger RNA (mRNA) and protein levels in human ASD brain samples and mouse ASD (and schizophrenia) models. Although the human PVALB gene is not a high-ranking susceptibility/risk gene for either disorder and is currently only listed in the SFARI Gene Archive, we propose and present supporting evidence for the Parvalbumin Hypothesis, which posits that decreased PV level is causally related to the etiology of ASD (and possibly schizophrenia).

The orbitofrontal cortex projects to the parvafox nucleus of the ventrolateral hypothalamus and to its targets in the ventromedial periaqueductal grey matter.

Although connections between the orbitofrontal cortex (OFC)-the seat of high cognitive functions-the lateral hypothalamus and the periaqueductal grey (PAG) have been recognized in the past, the precise targets of the descending fibres have not been identified. In the present study, viral tracer-transport experiments revealed neurons of the lateral (LO) and the ventrolateral (VLO) OFC (homologous to part of Area 13 in primates) to project to a circumscribed region in the ventrolateral hypothalamus, namely, the horizontally oriented, cylindrical parvalbumin- and Foxb1-expressing (parvafox) nucleus. The fine collaterals stem from coarse axons in the internal capsule and form excitatory synapses specifically with neurons of the parvafox nucleus, avoiding the rest of the hypothalamus. In its further caudal course, this contingent of LO/VLO-axons projects collaterals to the Su3- and the PV2 nuclei, which lie ventral to the aqueduct in the (PAG), where the terminals fields overlap those deriving from the parvafox nucleus itself. The targeting of the parvafox nucleus by the LO/VLO-projections, and the overlapping of their terminal fields within the PAG, suggest that the two cerebral sites interact closely. An involvement of this LO/VLO-driven circuit in the somatic manifestation of behavioural events is conceivable.

The Foxb1-expressing neurons of the ventrolateral hypothalamic parvafox nucleus project to defensive circuits.

The parvafox nucleus is an elongated structure that is lodged within the ventrolateral hypothalamus and lies along the optic tract. It comprises axially located parvalbumin (Parv)-positive neurons and a peripheral cuff of Foxb1-expressing ones. In the present study, injections of Cre-dependent adenoviral constructs were targeted to the ventrolateral hypothalamus of Foxb1/Cre mice to label specifically and map the efferent connections of the Foxb1-expressing subpopulation of neurons of the parvafox nucleus. These neurons project more widely than do the Parv-positive ones and implicate a part of the axons known to emanate from the lateral hypothalamus. High labeling densities were found in the dorsolateral and the upper lateral portion of the periaqueductal gray (PAG), the Su3 and PV2 nuclei of the ventrolateral PAG, the cuneiform nucleus, the mesencephalic reticular formation, and the superior colliculus. Intermediate densities of terminals were encountered in the septum, bed nucleus of the stria terminalis, substantia innominata, various thalamic and hypothalamic nuclei, pedunculopontine nucleus, Barrington's nucleus, retrofacial nucleus, and retroambigual nucleus. Scattered terminals were observed in the olfactory bulbs, the prefrontal cortex and the lamina X of the cervical spinal cord. Because the terminals were demonstrated to express the glutamate transporter VGlut2, the projections are presumed to be excitatory. A common denominator of the main target sites of the Foxb1-positive axons of the parvafox nucleus appears to be an involvement in the defensive reactions to life-threatening situations. The hypothalamic parvafox nucleus may contribute to the autonomic manifestations that accompany the expression of emotions. J. Comp. Neurol. 524:2955-2981, 2016. © 2016 Wiley Periodicals, Inc.

Birthdate of parvalbumin-neurons in the Parvafox-nucleus of the lateral hypothalamus.

The Parvafox-nucleus in the lateral hypothalamus is characterized by the presence of two distinct neural populations, the Parvalbumin (Parv) and the Foxb1-expressing ones. Foxb1-neurons are born at day 10 in the subventricular zone of the mouse mammillary region. It would be interesting to know if the subpopulation of Parv- neurons develop independently at different times and then meet the Foxb1- expressing neurons in the lateral hypothalamus, their final settling place. The aim of this study was to define the period of birth of the Parv-positive neurons using an in-vivo Bromodeoxyuridine-based method in rats. Parv-neurons are generated from embryonic day 10 to day 13, with a peak at day 12. Thus, it appears that the birthdates of the two subpopulations in these two species is similar, perhaps suggesting that they are born from the same neuroepithelial region.

Coaxiality of Foxb1- and parvalbumin-expressing neurons in the lateral hypothalamic PV1-nucleus.

In the ventrolateral hypothalamus, the PV1-nucleus is defined by its population of parvalbumin-expressing neurons. During embryogenesis, the ventrolateral hypothalamus is colonized also by Foxb1-expressing neurons. In adult Foxb1-EGFP mice, many immunofluorescent neurons were found within the region that is occupied by the PV1-nucleus. They formed a cloud around the axial cord of the parvalbumin-immunopositive cells, which they greatly outnumber (3:1). Only a small proportion of the neurons in the PV1-nucleus co-expressed both parvalbumin and Foxb1. In the light of these findings, a redesignation of this lateral hypothalamic structure as the PV1-Foxb1 nucleus would more accurately reflect its specific biochemical properties.