Circadian rhythms are the outward manifestation of an internal timing system that measures time in 24-hr increments. The mammalian circadian system is hierarchical, with a pacemaker in the suprachiasmatic nucleus (SCN) synchronizing cell-autonomous oscillators in peripheral tissues. Much of what we know about rhythmicity in peripheral tissues comes from studies monitoring bioluminescence rhythms in PERIOD2::LUCIFERASE knock-in mice. A limitation with this model is that rhythmicity cannot be monitored in specific cells due to widespread reporter expression. To address this shortcoming, we generated a mouse that expresses luciferase from the Dbp locus only after Cre-mediated recombination. I validated this conditional mouse to provide a tool for monitoring circadian rhythms in a tissue/cell-specific manner. Crossing the conditional reporter mice with mice expressing Cre recombinase in various cell types allowed detection of rhythmic bioluminescence in the expected tissues, in vivo and ex vivo, as well as in slice cultures containing the SCN. The phase of bioluminescence rhythms from explants of mouse peripheral tissues indicated that DbpLuc/+ bioluminescence rhythms have an earlier phase than PER2::LUC/+ rhythms. Importantly, we confirmed that editing of the Dbp locus did not alter the period of circadian locomotor activity rhythms and did not alter liver Dbp RNA rhythms. Finally, the reporter mouse allows for monitoring rhythms in specific tissues in ambulatory mice. Thus, this mouse line is useful for studying circadian rhythms in a tissue/cell-type specific manner, which can be used to better monitor phase relationships between tissues at baseline and after environmental perturbations that disrupt circadian rhythms.

Neuroergonomics, the application of neuroscience to human factors and ergonomics, is an emerging science focusing on the human brain concerning performance at work and in everyday settings. The advent of portable neurophysiological methods, including electroencephalography (EEG), has enabled measurements of real-time brain activity during physical tasks without restricting body movements. However, the EEG signatures of different physical exertion activity levels that involve the musculoskeletal system in everyday settings remain poorly understood. Furthermore, the assessment of functional connectivity among different brain regions during different force exertion levels remains unclear. One approach to investigating the brain connectome is to model the underlying mechanism of the brain as a complex network. This study applied employed a graph-theoretical approach to characterize the topological properties of the functional brain network induced by predefined force exertion levels, namely extremely light (EL), light (L), somewhat hard (SWH), hard (H), and extremely hard (EH) in two frequency bands, i.e., alpha and beta. Twelve female participants performed an isometric force exertion task and rated their perception of physical comfort at different physical exertion levels. A CGX-Mobile-64 EEG was used for recording spontaneous brain electrical activity. After preprocessing the EEG data, a source localization method was applied to study the functional brain connectivity at the source level. Subsequently, the alpha and beta networks were constructed by calculating the coherence between all pairs of 84 brain regions of interests that were selected using Brodmann Areas. Graph -theoretical measures were then employed to quantify the topological properties of the functional brain networks at different levels of force exertions at each frequency band. During an 'extremely hard' exertion level, a small-world network was observed for the alpha coherence network, whereas an ordered network was observed for the beta coherence network. The results suggest that high-level force exertions are associated with brain networks characterized by a more significant clustering coefficient, more global and local efficiency, and shorter characteristic path length under alpha coherence. The above suggests that brain regions are communicating and cooperating to a more considerable degree when the muscle force exertions increase to meet physically challenging tasks. The exploration of the present study extends the current understanding of the neurophysiological basis of physical efforts with different force levels of human physical exertion to reduce work-related musculoskeletal disorders.

Ultrasound (US) has become a standard procedure used during pregnancy to document the health and development of a fetus. When ultrasound was first developed, some researchers urged caution, suggesting that the possibility of hazard should be kept under constant review. Given the routine application of fetal ultrasound imaging, any possibility of deleterious developmental effects resulting from its use is an important public health issue. Rats have a well characterized central nervous system whose neurochemical pathways and neuronal electrophysiology qualitatively correspond to those of humans. Because of this, we opted to use Wistar rats as an animal model to document effects from ultrasound exposure. We exposed one group of rats on prenatal days 15 and 20 for fifteen minutes. A control group was exposed subjected to similar conditions, however no ultrasound exposure was given. A third group was exposed for ten minutes each on post natal days (PND) 2 and 3 while a fourth control group was exposed to the same conditions as group three with no ultrasound exposure. The rats were then watched for developmental delays. When the rats reached the appropriate age, they were given a locomotor task to test for appropriate motor responses. Acoustic startle and prepulse inhibition tests were administered to test for sensorimotor gating, hearing, and motor response. Finally, a brainstem auditory evoke potential test was given to track auditory threshold and appropriate neural firing at various auditory nuclei. Postnatally US exposed rats showed a decreased acoustic startle response and prenatally exposed rats exhibited a speeding up in components of the brainstem auditory evoked potential test.

Progesterone regulates female reproduction in many ways, yet it is still unclear how signals are conveyed through nuclear and extranuclear receptors. The traditional notion was that progesterone binds classical progesterone receptors to alter gene transcription. This view has been challenged by the discovery of additional progesterone signaling molecules important for progesterone actions in non-neural cells. In granulosa cells, the progesterone receptor membrane component 1 (Pgrmc1) mediates progesterone effects by forming a receptor complex with binding partner, Serpine mRNA binding protein 1, but it is unknown whether these molecules function similarly in the brain. To begin to address these issues, I investigated the neural role of Pgrmc1 in female mouse brain, rat brain and in neural cells. By examining the neuroanatomical localization, hormonal regulation, and colocalization of Pgrmc1 within key neurons in the neural control of ovulation, Pgrmc1 emerged as a candidate signaling molecule likely to mediate progesterone functions. Furthermore, Pgrmc1 levels regulate the expression of several diverse genes and signaling pathways in neural cells. Taken together, these results demonstrate that Pgrmc1 function is likely to impact diverse neural functions.

Prototoxin proteins have been identified as members of the Ly6/uPAR super family whose three-finger motif resembles that of α-bungarotoxin. Though they are known to modify the function of nAChRs, their specificity is still unclear. Our lab identified three prototoxin proteins in the chicken ciliary ganglion: Ch3ly, Ch5ly, and Ch6ly. Ch6ly was later identified as prostate stem cell antigen (PSCA), and specifically decreased the amount of calcium influx through the homomeric α7 nAChR subtype. I then identifiedCh3ly and Ch5ly as LY6E and LYPD6B, respectively. I focused my attention onLYPD6B because of its expression in the brain. This dissertation tests whether LYPD6Bis a prototoxin protein that specifically co-localizes with and modifies the function of the heteromeric α3β4* nAChRs (the other nAChR subtype expressed in the chicken ciliary ganglia). In the first part of my dissertation I performed intracellular two-electrode voltage clamp on Xenopus oocytes co-expressing human LYPD6B and different stoichiometries of the α3β4* nAChR, these included two (α3)2(β4)3 withβ4−α3−β4−β4−α3 and β4−α3−β4−α3−β4 stoichiometries, two (α3)3(β4)2 with stoichiometries β4−α3−α3−β4−α3 and β4−α3−β4−α3−α3, two (α3β4)2(α5D)β4−α3−α5D−β4−α3 and β4−α3−β4−α3−α5D, and (α3β4)2(α5N) with stoichiometries β4−α3−α5N−β4−α3 and β4−α3−β4−α3−α5N. Concatemeric constructs are designed to link nAChR subunits, thus when translated it is done so as a single polypeptide. LYPD6Bincreased the acetylcholine (ACh) potency and desensitization rate, but decreased the maximum current response (Imax) for the (α3)3(β4)2 nAChR subtype. Yet, LYPD6Bonly decreased the Imax for the (α3β4)2α5 D-variant and not the N-variant (associated with increase nicotine consumption). For the second part of my dissertation, I determined if the expression of LYPD6B correlated with nAChRs in an activity dependent manner. Though LYPD6B mRNA expression correlates with nAChR subunit mRNA expression levels, it seemed to be independent of nAChR activity. To determine if fluorescent colocalization occurs between LYPD6B and a specific nAChR subtype, I genetically engineered LYPD6B to express a human influenza hemagglutinin (HA) epitope tag and cloned into a chicken retrovirus. LYPD6B was shown to co-localize only with the α3β4*heteromeric and not the homomeric α7 nAChRs, in a nAChR activity dependent manner. This study adds to the complexity of a prototoxin’s function by suggesting that the specificity is dependent on nAChR type and stoichiometry. It is the first in identifying a prototoxin protein, LYPD6B, which specifically modulates the function of the(α3)3(β4)2 and (α3β4)2(α5 D-variant) heteromeric nAChR subtypes. For the (α3β4)2(α5D-variant) nAChR subtype LYPD6B decreased the Imax. Such observation may be telling of a novel mechanism involved with nicotine dependence. For the(α3)3(β4)2 nAChR subtype LYPD6B increases its ACh sensitivity, desensitization rate, while decreasing Imax. Additionally, the co-localization of LYPD6B and α3β4* nAChRsin the lack of nAChR activity highlights the relevance of the functional effects α3β4*nAChRs exhibit due to LYPD6B. Such relevance may be the utilization of limiting Ach amounts.

Carbon dioxide (CO2) inhalation lowers brain pH and induces anxiety, fear, and panic responses in humans. In mice, CO2 produces freezing and avoidance behavior that has been suggested to depend on the amygdala. However, a recent study in humans with bilateral amygdala lesions revealed that CO2 can trigger fear and panic even in the absence of amygdalae, suggesting and important role for extra-amygdalar brain structures. Because the bed nucleus of the stria terminalis (BNST) contributes to fear- and anxiety-related behaviors and expresses acid sensing ion channel-1A (ASIC1A), we hypothesized that the BNST plays an important role in CO2-evoked fear-related behaviors in mice. We found that BNST lesions decreased both CO2-evoked freezing and CO2-conditioned place avoidance. In addition, we found that CO2 inhalation caused BNST acidosis, and that acidosis was sufficient to depolarize BNST neurons and induce freezing behavior; both responses depended on ASIC1A. Finally, disrupting Asic1a specifically in the BNST reduced CO2-evoked freezing whereas viral vector mediated expression of ASIC1A in the BNST of Asic1a-/- and Asic1a+/+ mice increased CO2-evoked freezing. Together, these findings identify the BNST as an extra-amygdalar fear circuit structure important in CO2-evoked fear-related behavior. Genetic disruption of the acid-sensing ion channel-1A (ASIC1A) in mice results in deficits in several fear- and anxiety-related behaviors. These deficits have been largely attributed to the loss of ASIC1A in neurons. However, recent studies have identified ASIC1A in several types of non-neuronal cells, including glia. To test the hypothesis that it is the loss of ASIC1A in neurons that results in the behavioral deficits seen in Asic1a-/- mice, we generated SynCre+Asic1aloxP/loxP mice, in which ASIC1A is disrupted specifically in neurons. To validate these mice, we confirmed by PCR that the Asic1a floxed allele was disrupted in brain, but not tail DNA. We further detected a reduction in ASIC1A protein in the SynCre+Asic1aloxP/loxP mice by western blotting and ASIC1A immunohistochemsitry. Further characterization of cre expression with a Rosa26 cre reporter mouse revealed that cre expression did not occur in all neurons, but verified that cre expression was neuron-specific. This neuron-specific knockout of ASIC1A led to behavioral deficits in several models of fear and anxiety, including cued and context fear conditioning, predator odor-evoked freezing and CO2-evoked freezing. Together, these findings suggest that it is ASIC1A in neurons that mediates these fear- and anxiety-related behaviors. Trimethylthiazoline (TMT), a predator odor isolated from fox feces, elicits freezing and avoidance responses in rodents. This TMT-evoked freezing behavior depends on the bed nucleus of the stria terminalis (BNST), a brain region thought to contribute to anxiety in both humans and mice. Because the acid-sensing ion channel-1A (ASIC1A) is robustly expressed in the BNST and has been previously implicated in TMT-evoked freezing, we hypothesized that the BNST might be a site of ASIC1A action in the TMT-evoked freezing response. Consistent with previous studies, we found that TMT-evoked freezing depended both on the olfactory bulb and on ASIC1A. Viral-mediated disruption of ASIC1A in the BNST reduced TMT-evoked freezing, whereas, viral mediated expression of ASIC1A in the BNST of Asic1a-/- mice increased TMT-evoked freezing. We further observed that TMT exposure induces a modest acidosis, likely due to TMT-induced respiratory suppression. However, this respiratory suppression was not unique to odors that evoke freezing, suggesting that it does not drive the TMT-evoked freezing response. Together, these findings suggest that the BNST is a key site of ASIC1A action in TMT-evoked freezing. Regulation of cerebral blood flow (CBF) is critical to insure that the brain has adequate resources to maintain normal function. One of the strongest regulators of CBF is carbon dioxide (CO2). CO2 and acidosis are thought to induce vasodilation and increase CBF by initiating nitric oxide (NO) synthesis, though the mechanism by which this occurs is unknown. Recent unpublished studies have suggested that the acid-sensing ion channel-1A (ASIC1A) plays a role in hypercapnia-induced vasodilation. Therefore, we hypothesized that CO2-induced NO production would depend on ASIC1A. We found that CO2 induced robust NO production in Asic1a+/+ but not Asic1a-/- mice. To test the role of neuronal ASIC1A in CO2-induced NO production, we generated SynCre+Asic1aloxP/loxP mice, in which ASIC1A is disrupted specifically in neurons. We found that CO2 did not induce significant NO production in the SynCre+Asic1aloxP/loxP mice, suggesting that it is ASIC1A in neurons that mediates this response. Together, these studies suggest that ASIC1A may mediate neurovascular coupling and regulate CBF.

Biological functions take place within tightly controlled parameters, including pH, which is managed in part through the ventilatory chemoreflex. This reflex is mediated by central respiratory chemoreceptors (CRCs) specialized to detect blood pH/CO2. Two neuronal populations are thought to mediate this response: the serotonergic (5-HT) neurons of the medullary raphé, and the Phox2b expressing neurons of the retrotrapezoid nucleus (RTN). These groups are both responsive to CO2 stimuli in vivo and in vitro. There are also apparent one-way connections from the raphé to the RTN, which is sensitive to 5-HT. Due to its complex innervation, study of RTN neurons while isolated from other cells, especially 5-HT neurons, has been limited. Here, we developed a culture model that simplifies this circuit, limiting cell types to those found in the rostral ventral medulla. This protocol yielded healthy RTN and 5-HT neurons in vitro, as well as other cell types from that area. Upon study with patch-clamp electrophysiology, cultured RTN neurons responded to CO2 and 5-HT in similar ways to what is reported for different RTN neuron preparations. Using this model, RTN neuron chemosensitivity was significantly decreased during application of 5-HT7 antagonists (SB258719, SB269970) and a 5-HT2A antagonist (MDL 11,939). The effect of 5-HT7 antagonists was recapitulated in slice recordings. Therefore, signaling at 5-HT7 and 5-HT2A receptors is necessary for RTN neuron chemosensitivity. Exogenous 5-HT application also increased RTN neuron firing rate without potentiating the response to CO2, most likely indicating that the necessary 5-HT stimulation must come from neurons that can alter their activity during acidosis. We conclude that RTN neuron chemosensitivity is largely driven by chemosensitive 5-HT neurons, and should be considered an integrative or relay center, rather than an independently chemosensitive one.

Due to a steady increase in the number of babies born prematurely over the past 20 years, prematurity (a birth occurring before 37 weeks gestation) has emerged as an important public health concern. Even with improved survival of these infants, they remain at risk for many unfavorable health outcomes. Most of those risks include cognitive and behavioral deficits that show up later in life, highlighting the importance of studying the development of the brain, in particular. The current study investigates brain development outcomes in the first years of life using: (1) structural magnetic resonance imaging (MRI) to study brain structure, and (2) three novel cognitive assessments of visual working memory, attention, and speed of processing information. Healthy 12-month-old infants were recruited through University of Iowa’s Neonatal Admissions Registry. An MRI imaging acquisition protocol was developed in order to scan infants during their naptime without sedation. Additionally, a new automatic approach to classifying areas of the brain was developed at the University of Iowa Department of Radiology for 12-month-old brain images. These novel cognitive assessments are based on infant eye movements (including how long it takes for an infant to react to certain stimuli and the direction of their looking). Results from this study support the use of these cognitive tasks to detect specific functional changes in performance based on gestational age. Therefore, these tasks may be potential early markers of risk in preterm populations, but continued investigations are necessary to fully elucidate early brain outcomes during this critical period of development.

The Transient Receptor Potential, Melastatin-like 7 (TRPM7) protein is composed of a long amino terminus, 6 trans-membrane domains, and a carboxy terminal α-kinase domain; TRPM7 tetramers form non-selective cation channels with unusual permeability to Mg2+. TRPM7 is thought to be expressed in all cell types, and studies conducted primarily on cultured cells have implicated TRPM7 in cellular functions that include cell adhesion, synaptic vesicle release, kidney cation balance, differentiation, survival, and cellular magnesium homeostasis. The full extent of its physiological functions in vivo remains elusive because mouse TRPM7 homozygous null mutants die at embryonic stages. By contrast, zebrafish homozygous for hypomorphic alleles of trpm7 survive for two weeks post fertilization, making it possible to study the physiological consequences of Trpm7 deficiency in a living organism. My work primarily utilizes homozygous animals carrying the trpm7b508 allele, which we suspect encodes a non-functional protein for three reasons. This protein variant is predicted to lack a kinase domain, patch clamp studies fail to detect current, and morpholino knockdown of Trpm7 yields a similar phenotype. Herein, we explore the mechanisms behind each of three phenotypes in trpm7b508 homozygous embryos, i.e., trpm7 mutants. First, we find that cell death of embryonic melanophores in trpm7 mutants is not by apoptosis, and it is dependent upon melanin synthesis and the ion channel Trpm2. Second, we show that paralysis of trpm7 mutants is rescued by surgical opening of the circulatory system to surrounding media, implying that paralysis results from an organismal ion imbalance. Third, we report a variety of findings supporting the model that abnormally low levels of spontaneous swimming in larval trpm7 mutants results from reduced dopamine signaling. We find that specific populations of catecholaminergic neurons are reduced in mutants relative to their unaffected siblings, and that mutants are sensitized to the neurotoxic effects of 1-Methyl-4-phenylpyridinium iodide (MPP+). Together, these results suggest that Trpm7 has a role in ameliorating the toxic effects of reactive oxygen species in certain populations of melanophores and neurons. These findings advance understanding of the function of TRPM7 during embryonic development, and may have relevance to the gene-environment interaction behind certain neurodegenerative conditions.

Poikilothermic and endothermic animals demonstrate febrile response to infection with bacteria or to injection with endogenous pyrogen extract of Prostaglandin E1. Febrile response is measured in endotherms as a relative change in metabolically achieved body temperature and in poikilotherms as an increase in selected temperatures relative to previously established preferred temperatures. Final preferendum change with environmental factors or associated physiological states.