Classroom-Based Research Experiences to Support Underserved STEM Student Success: From Introductory Inquiry to Optogenetics in the Embryonic Chicken.

In order to help overcome barriers to success for undergraduate STEM students from disadvantaged backgrounds, we developed two classroom-based research experiences (REs), Connecting Life (CL) and the Summer Research Institute (SRI). These REs were implemented over a two-year period (2014-2015) for regional community college students as part of the Southern Illinois Bridges to the Baccalaureate (SI Bridges) program. CL and SRI, broadly centered in biomedical sciences research, are designed to be offered in tandem. CL utilizes a guided inquiry approach with microscopy work-stations in experimental cell biology to experientially introduce research while building skills and confidence. CL serves as the gateway experience for the SRI, an intensive summer RE in which scholars engage in authentic research using modern technologies including optogenetics. We piloted the REs in year 1 (9 scholars) and made refinements in year 2 (10 scholars). Participants ("Bridges scholars") were enrolled full-time at one of two regional, rural community colleges, and came on-site to Southern Illinois University at Carbondale (SIUC) for the paid REs. Here we report the development, design and implementation of CL and the SRI, and report improved STEM research-related attitudes and aptitudes as a result of these experiences. Our findings suggest that guided inquiry with increasingly technical authentic research projects in a classroom-based and supportive learning community-style setting is a positive model for the transformation of underserved community college students into confident, motivated scientists with research-ready skills, and is likely translatable to other research novices.

Sensory regulation of spontaneous limb movements in the midstage embryonic chick.

It is becoming increasingly apparent that somatosensation plays an important role in regulating prenatal movement and developmental plasticity. Numerous studies performed on embryonic chicks and perinatal rats are beginning to implicate proprioception to be particularly important in modulating motility very soon after afferent connections are made in the spinal cord. In this report, we demonstrate new approaches in the chick embryo to explore the role of sensation in modulating embryonic movement. Force recordings from the legs of chick embryos on E9 and E11, during spontaneous motility, demonstrate changes in sensory regulation consistent with the concept that sensory regulation is functioning one day after sensory synapse formation and that the complexity of this regulation increases by E11. Additionally, we present new video data showing activation of embryonic motility on E5 and E9 in embryos expressing channelrhodopsin in the spinal cord as a novel way to approach the issues of sensorimotor development.

Pyridoxine treatment alters embryonic motility in chicks: Implications for the role of proprioception.

Somatosensory feedback is important for the modulation of normal locomotion in adult animals, but we do not have a good understanding of when somatosensory information is first used to modulate motility during embryogenesis or how somatosensation is first used to regulate motor output. We used pyridoxine administration (vitamin B6 ), which is known to mostly kill proprioceptive neurons in adult mammals and embryonic chicks, to explore the role of proprioceptive feedback during early embryonic motility in the chick. Injection of pyridoxine on embryonic day 7 (E7) and E8 reduced the amplitude of leg movements recorded on E9 and the number of large, healthy neurons in the ventral-lateral portion of the DRGs. We conclude that proprioception is initially used during embryogenesis to modulate the strength of motor output, but that it is not incorporated into other aspects of pattern generation until later in development as poly-synaptic pathways develop.

A system for the determination of planar force vectors from spontaneously active chicken embryos.

Generally, a combination of kinematic, electromyographic (EMG), and force measurements are used to understand how an organism generates and controls movement. The chicken embryo has been a very useful model system for understanding the early stages of embryonic motility in vertebrates. Unfortunately, the size and delicate nature of embryos makes studies of motility during embryogenesis very challenging. Both kinematic and EMG recordings have been achieved in embryonic chickens, but two-dimensional force vector recordings have not. Here, we describe a dual-axis system for measuring force generated by the leg of embryonic chickens. The system employs two strain gauges to measure planar forces oriented with the plane of motion of the leg. This system responds to forces according to the principles of Pythagorean geometry, which allows a simple computational program to determine the force vector (magnitude and direction) generated during spontaneous motor activity. The system is able to determine force vectors for forces >0.5 mN accurately and allows for simultaneous kinematic and EMG recordings. This sensitivity is sufficient for force vector measurements encompassing most embryonic leg movements in midstage chicken embryos allowing for a more complete understanding of embryonic motility. Variations on this system are discussed to enable nonideal or alternative sensor arrangements and to allow for translation of this approach to other delicate model systems.

Optogenetic regulation of leg movement in midstage chick embryos through peripheral nerve stimulation.

Numerous disorders that affect proper development, including the structure and function of the nervous system, are associated with altered embryonic movement. Ongoing challenges are to understand in detail how embryonic movement is generated and to understand better the connection between proper movement and normal nervous system function. Controlled manipulation of embryonic limb movement and neuronal activity to assess short- and long-term outcomes can be difficult. Optogenetics is a powerful new approach to modulate neuronal activity in vivo. In this study, we have used an optogenetics approach to activate peripheral motor axons and thus alter leg motility in the embryonic chick. We used electroporation of a transposon-based expression system to produce ChIEF, a channelrhodopsin-2 variant, in the lumbosacral spinal cord of chick embryos. The transposon-based system allows for stable incorporation of transgenes into the genomic DNA of recipient cells. ChIEF protein is detectable within 24 h of electroporation, largely membrane-localized, and found throughout embryonic development in both central and peripheral processes. The optical clarity of thin embryonic tissue allows detailed innervation patterns of ChIEF-containing motor axons to be visualized in the living embryo in ovo, and pulses of blue light delivered to the thigh can elicit stereotyped flexures of the leg when the embryo is at rest. Continuous illumination can disrupt full extension of the leg during spontaneous movements. Therefore, our results establish an optogenetics approach to alter normal peripheral axon function and to probe the role of movement and neuronal activity in sensorimotor development throughout embryogenesis.

Impaired Glucose Tolerance and Reduced Plasma Insulin Precede Decreased AKT Phosphorylation and GLUT3 Translocation in the Hippocampus of Old 3xTg-AD Mice.

Several studies have demonstrated that mouse models of Alzheimer's disease (AD) can exhibit impaired peripheral glucose tolerance. Further, in the APP/PS1 mouse model, this is observed prior to the appearance of AD-related neuropathology (e.g., amyloid-ß plaques; Aß) or cognitive impairment. In the current study, we examined whether impaired glucose tolerance also preceded AD-like changes in the triple transgenic model of AD (3xTg-AD). Glucose tolerance testing (GTT), insulin ELISAs, and insulin tolerance testing (ITT) were performed at ages prior to (1-3 months and 6-8 months old) and post-pathology (16-18 months old). Additionally, we examined for altered insulin signaling in the hippocampus. Western blots were used to evaluate the two-primary insulin signaling pathways: PI3K/AKT and MAPK/ERK. Since the PI3K/AKT pathway affects several downstream targets associated with metabolism (e.g., GSK3, glucose transporters), western blots were used to examine possible alterations in the expression, translocation, or activation of these targets. We found that 3xTg-AD mice display impaired glucose tolerance as early as 1 month of age, concomitant with a decrease in plasma insulin levels well prior to the detection of plaques (∼14 months old), aggregates of hyperphosphorylated tau (∼18 months old), and cognitive decline (≥18 months old). These alterations in peripheral metabolism were seen at all time points examined. In comparison, PI3K/AKT, but not MAPK/ERK, signaling was altered in the hippocampus only in 18-20-month-old 3xTg-AD mice, a time point at which there was a reduction in GLUT3 translocation to the plasma membrane. Taken together, our results provide further evidence that disruptions in energy metabolism may represent a foundational step in the development of AD.

Toward a self-deploying shape memory polymer neuronal electrode.

The widespread application of neuronal probes for chronic recording of brain activity and functional stimulation has been slow to develop partially due to long-term biocompatibility problems with existing metallic and ceramic probes and the tissue damage caused during probe insertion. Stiff probes are easily inserted into soft brain tissue but cause astrocytic scars that become insulating sheaths between electrodes and neurons. In this communication, we explore the feasibility of a new approach to the composition and implantation of chronic electrode arrays. We demonstrate that softer polymer-based probes can be inserted into the olfactory bulb of a mouse and that slow insertion of the probes reduces astrocytic scarring. We further present the development of a micromachined shape memory polymer probe, which provides a vehicle to self-deploy an electrode at suitably slow rates and which can provide sufficient force to penetrate the brain. The deployment rate and composition of shape memory polymer probes can be tailored by polymer chemistry and actuator design. We conclude that it is feasible to fabricate shape memory polymer-based electrodes that would slowly self-implant compliant conductors into the brain, and both decrease initial trauma resulting from implantation and enhance long-term biocompatibility for long-term neuronal measurement and stimulation.

Aberrant expression of the pore-forming KATP channel subunit Kir6.2 in hippocampal reactive astrocytes in the 3xTg-AD mouse model and human Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by beta-amyloid (Aß) deposition, neurofibrillary tangles and cognitive decline. Recent pharmacologic studies have found that ATP-sensitive potassium (KATP) channels may play a role in AD and could be a potential therapeutic target. Interestingly, these channels are found in both neurons and astrocytes. One of the hallmarks associated with AD is reactive gliosis and a change in astrocytic function has been identified in several neuropathological conditions including AD. Thus the goal of this study was to examine whether the pore-forming subunits of KATP channels, Kir6.1 and Kir6.2, are altered in the hippocampus in a cell type-specific manner of the 3xTg-AD mouse model of AD and in human AD tissue obtained from the Chinese brain bank. Specifically, in old 3xTg-AD mice, and age-matched controls, we examined glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), Kir6.1 and Kir6.2 in hippocampal region CA1 with a combination of immunoblotting and immunohistochemistry (IHC). A time point was selected when memory impairment and histopathological changes have been reported to occur in 3xTg-AD mice. In human AD and age-matched control tissue IHC experiments were performed using GFAP and Kir6.2. In the hippocampus of 3xTg-AD mice, compared to wild-type controls, Western blots showed a significant increase in GFAP indicating astrogliosis. Further, there was an increase in Kir6.2, but not Kir6.1 in the plasma membrane fraction. IHC examination of hippocampal region CA1 in 3xTg-AD sections revealed an increase in Kir6.2 immunoreactivity (IR) in astrocytes as identified by GFAP and GS. In human AD tissue similar data were obtained. There was an increase in GFAP-IR in the stratum oriens (SO) and alveus (ALV) of CA1 concomitant with an increase in Kir6.2-IR in cells with an astrocytic-like morphology. Dual immunofluorescence revealed a dramatic increase in co-localization of Kir6.2-IR and GFAP-IR. Taken together, these data demonstrate that increased Kir6.2 is seen in reactive astrocytes in old 3xTg-AD mice and human AD tissue. These changes could dramatically alter astrocytic function and subsequently contribute to AD phenotype in either a compensatory or pathophysiological manner.

In vivo penetration mechanics and mechanical properties of mouse brain tissue at micrometer scales.

Substantial advancement in the understanding of the neuronal basis of behavior and the treatment of neurological disorders has been achieved via the implantation of various devices into the brain. To design and optimize the next generation of neuronal implants while striving to minimize tissue damage, it is necessary to understand the mechanics of probe insertion at relevant length scales. Unfortunately, a broad-based understanding of brain-implant interactions at the necessary micrometer scales is largely missing. This paper presents a generalizable description of the micrometer-scale penetration mechanics and material properties of mouse brain tissue in vivo. Cylindrical stainless steel probes were inserted into the cerebral cortex and olfactory bulb of mice. The effects of probe size, probe geometry, insertion rate, insertion location, animal age, and the presence of the dura and pia on the resulting forces were measured continuously throughout probe insertion and removal. Material properties (modulus, cutting force, and frictional force) were extracted using mechanical analysis. The use of rigid, incompressible, cylindrical probes allows for a general understanding of how probe design and insertion methods influence the penetration mechanics of brain tissue in vivo that can be applied to the quantitative design of most future implantable devices.

GABAergic modulation of primary gustatory afferent synaptic efficacy.

Modulation of synaptic transmission at the primary sensory afferent synapse is well documented for the somatosensory and olfactory systems. The present study was undertaken to test whether GABA impacts on transmission of gustatory information at the primary afferent synapse. In goldfish, the vagal gustatory input terminates in a laminated structure, the vagal lobes, whose sensory layers are homologous to the mammalian nucleus of the solitary tract. We relied on immunoreactivity for the GABA-transporter, GAT-1, to determine the distribution of GABAergic synapses in the vagal lobe. Immunocytochemistry showed dense, punctate GAT-1 immunoreactivity coincident with the layers of termination of primary afferent fibers. The laminar nature and polarized dendritic structure of the vagal lobe make it amenable to an in vitro slice preparation to study early synaptic events in the transmission of gustatory input. Electrical stimulation of the gustatory nerves in vitro produces synaptic field potentials (fEPSPs) predominantly mediated by ionotropic glutamate receptors. Bath application of either the GABA(A) receptor agonist muscimol or the GABA(B) receptor agonist baclofen caused a nearly complete suppression of the primary fEPSP. Coapplication of the appropriate GABA(A) or GABA(B) receptor antagonist bicuculline or CGP-55845 significantly reversed the effects of the agonists. These data indicate that GABAergic terminals situated in proximity to primary gustatory afferent terminals can modulate primary afferent input via both GABA(A) and GABA(B) receptors. The mechanism of action of GABA(B) receptors suggests a presynaptic locus of action for that receptor.