Cognitive and Non-Cognitive Outcomes Associated with Student Engagement in a Novel Brain Chemoarchitecture Mapping Course-Based Undergraduate Research Experience.

Course-based undergraduate research experiences (CUREs) engage emerging scholars in the authentic process of scientific discovery, and foster their development of content knowledge, motivation, and persistence in the science, technology, engineering, and mathematics (STEM) disciplines. Importantly, authentic research courses simultaneously offer investigators unique access to an extended population of students who receive education and mentoring in conducting scientifically relevant investigations and who are thus able to contribute effort toward big-data projects. While this paradigm benefits fields in neuroscience, such as atlas-based brain mapping of nerve cells at the tissue level, there are few documented cases of such laboratory courses offered in the domain. Here, we describe a curriculum designed to address this deficit, evaluate the scientific merit of novel student-produced brain atlas maps of immunohistochemically-identified nerve cell populations for the rat brain, and assess shifts in science identity, attitudes, and science communication skills of students engaged in the introductory-level Brain Mapping and Connectomics (BM&C) CURE. BM&C students reported gains in research and science process skills following participation in the course. Furthermore, BM&C students experienced a greater sense of science identity, including a greater likelihood to discuss course activities with non-class members compared to their non-CURE counterparts. Importantly, evaluation of student-generated brain atlas maps indicated that the course enabled students to produce scientifically valid products and make new discoveries to advance the field of neuroanatomy. Together, these findings support the efficacy of the BM&C course in addressing the relatively esoteric demands of chemoarchitectural brain mapping.

Cryptic diversity in Rhampholeon boulengeri (Sauria: Chamaeleonidae), a pygmy chameleon from the Albertine Rift biodiversity hotspot.

Several biogeographic barriers in the Central African highlands have reduced gene flow among populations of many terrestrial species in predictable ways. Yet, a comprehensive understanding of mechanisms underlying species divergence in the Afrotropics can be obscured by unrecognized levels of cryptic diversity, particularly in widespread species. We implemented a multilocus phylogeographic approach to examine diversity within the widely distributed Central African pygmy chameleon, Rhampholeon boulengeri. Gene-tree analyses coupled with a comparative coalescent-based species delimitation framework revealed R. boulengeri as a complex of at least six genetically distinct species. The spatiotemporal speciation patterns for these cryptic species conform to general biogeographic hypotheses supporting vicariance as the main factor behind patterns of divergence in the Albertine Rift, a biodiversity hotspot in Central Africa. However, we found that parapatric species and sister species inhabited adjacent habitats, but were found in largely non-overlapping elevational ranges in the Albertine Rift, suggesting that differentiation in elevation was also an important mode of divergence. The phylogeographic patterns recovered for the genus-level phylogeny provide additional evidence for speciation by isolation in forest refugia, and dating estimates indicated that the Miocene was a significant period for this diversification. Our results highlight the importance of investigating cryptic diversity in widespread species to improve understanding of diversification patterns in environmentally diverse regions such as the montane Afrotropics.

Contextual fear retrieval-induced Fos expression across early development in the rat: An analysis using established nervous system nomenclature ontology.

The neural circuits underlying the acquisition, retention and retrieval of contextual fear conditioning have been well characterized in the adult animal. A growing body of work in younger rodents indicates that context-mediated fear expression may vary across development. However, it remains unclear how this expression may be defined across the full range of key developmental ages. Nor is it fully clear whether the structure of the adult context fear network generalizes to earlier ages. In this study, we compared context fear retrieval-induced behavior and neuroanatomically constrained immediate early-gene expression across infant (P19), early and late juvenile (P24 and P35), and adult (P90) male Long-Evans rats. We focused our analysis on neuroanatomically defined subregions and nuclei of the basolateral complex of the amygdala (BLA complex), dorsal and ventral portions of the hippocampus and the subregions of the medial prefrontal cortex as defined by the nomenclature of the Swanson (2004) adult rat brain atlas. Relative to controls and across all ages tested, there were greater numbers of Fos immunoreactive (Fos-ir) neurons in the posterior part of the basolateral amygdalar nuclei (BLAp) following context fear retrieval that correlated statistically with the expression of freezing. However, Fos-ir within regions having known connections with the BLA complex was differentially constrained by developmental age: early juvenile, but not adult rats exhibited an increase of context fear-dependent Fos-ir neurons in prelimbic and infralimbic areas, while adult, but not juvenile rats displayed increases in Fos-ir neurons within the ventral CA1 hippocampus. These results suggest that juvenile and adult rodents may recruit developmentally unique pathways in the acquisition and retrieval of contextual fear. This study extends prior work by providing a broader set of developmental ages and a rigorously defined neuroanatomical ontology within which the contextual fear network can be studied further.

Microarray analysis of aging-associated immune system alterations in the rostral ventrolateral medulla of F344 rats.

The rostral ventrolateral medulla (RVLM) is an area of the brain stem that contains diverse neural substrates that are involved in systems critical for physiological function. There is evidence that aging affects some neural substrates within the RVLM, although age-related changes in RVLM molecular mechanisms are not well established. The goal of the present study was to characterize the transcriptomic profile of the aging RVLM and to test the hypothesis that aging is associated with altered gene expression in the RVLM, with an emphasis on immune system associated gene transcripts. RVLM tissue punches from young, middle-aged, and aged F344 rats were analyzed with Agilent's whole rat genome microarray. The RVLM gene expression profile varied with age, and an association between chronological age and specific RVLM gene expression patterns was observed [P < 0.05, false discovery rate (FDR) < 0.3]. Functional analysis of RVLM microarray data via gene ontology profiling and pathway analysis identified upregulation of genes associated with immune- and stress-related responses and downregulation of genes associated with lipid biosynthesis and neurotransmission in aged compared with middle-aged and young rats. Differentially expressed genes associated with the complement system and microglial cells were further validated by quantitative PCR with separate RVLM samples (P < 0.05, FDR < 0.1). The present results have identified age-related changes in the transcriptomic profile of the RVLM, modifications that may provide the molecular backdrop for understanding age-dependent changes in physiological regulation.

Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gßγ-microtubule interaction.

BACKGROUND: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. RESULTS: Here, we report that Gßγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gßγ with MTs and stimulated MT assembly. While Gßγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gßγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gßγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gßγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gßγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gßγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gßγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gßγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells. CONCLUSIONS: Altogether, our results demonstrate that ßγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement.

Sympathetic innervation of interscapular brown adipose tissue is not a predominant mediator of OT-elicited reductions of body weight gain and adiposity in male diet-induced obese rats.

Recent studies indicate that central administration of oxytocin (OT) reduces body weight (BW) in high fat diet-induced obese (DIO) rodents by reducing energy intake and increasing energy expenditure (EE). Previous studies in our lab have shown that administration of OT into the fourth ventricle (4V; hindbrain) elicits weight loss and stimulates interscapular brown adipose tissue temperature (TIBAT) in DIO rats. We hypothesized that OT-elicited stimulation of sympathetic nervous system (SNS) activation of IBAT contributes to its ability to activate BAT and reduce BW in DIO rats. To test this, we determined the effect of disrupting SNS activation of IBAT on OT-elicited stimulation of TIBAT and reduction of BW in DIO rats. We first confirmed that bilateral surgical SNS denervation to IBAT was successful based on having achieved ≥ 60% reduction in IBAT norepinephrine (NE) content from DIO rats. NE content was selectively reduced in IBAT by 94.7 ± 2.7, 96.8 ± 1.8 and 85.9 ± 6.1% (P<0.05) at 1, 6 and 7-weeks post-denervation, respectively, and was unchanged in liver or inguinal white adipose tissue. We then measured the impact of bilateral surgical SNS denervation to IBAT on the ability of acute 4V OT (1, 5 µg) to stimulate TIBAT in DIO rats. We found that the high dose of 4V OT (5 µg) stimulated TIBAT similarly between sham and denervated rats (P=NS) and that the effects of 4V OT to stimulate TIBAT did not require beta-3 adrenergic receptor signaling. We subsequently measured the effect of bilateral surgical denervation of IBAT on the effect of chronic 4V OT (16 nmol/day) or vehicle infusion to reduce BW, adiposity and energy intake in DIO rats. Chronic 4V OT reduced BW gain by -7.2 ± 9.6 g and -14.1 ± 8.8 g in sham and denervated rats (P<0.05 vs vehicle treatment), respectively, and this effect was similar between groups (P=NS). These effects were associated with reductions in adiposity and energy intake (P<0.05). Collectively, these findings support the hypothesis that sympathetic innervation of IBAT is not required for central OT to increase BAT thermogenesis and reduce BW gain and adiposity in male DIO rats.

Withdrawal from repeated nicotine vapor exposure increases somatic signs of physical dependence, anxiety-like behavior, and brain reward thresholds in adult male rats.

Nicotine vapor consumption via electronic nicotine delivery systems has increased over the last decade. While prior work has shed light on the health effects of nicotine vapor inhalation, its unique effects on the brain and behavior have not been thoroughly explored. In this study we assessed markers of withdrawal following 14 days of nicotine vapor exposure. For Experiment 1, 21 adult male rats were exposed to ambient air or 6, 12, or 24 mg/mL nicotine vapor for 14 consecutive days. Following exposure on day 14, rats were injected with the nicotinic receptor antagonist mecamylamine (3.0 mg/mL) and assessed for somatic withdrawal signs and anxiety-like behavior in the elevated plus maze. For Experiment 2, 12 adult male rats were tested for intracranial self-stimulation (ICSS) immediately following exposure to vehicle vapor (50%/50%, vegetable glycerin/propylene glycol) or 24 mg/mL nicotine vapor, for 14 consecutive days. ICSS behavior was assessed for an additional 14 days, following cessation of repeated vapor exposure. Results reveal that rats with repeated nicotine vapor exposure display an increase in behavioral indicators of withdrawal following injection of mecamylamine (precipitated withdrawal). Additionally, increases in ICSS stimulation thresholds, indicative of reduced brain reward sensitivity, persist following cessation of repeated nicotine vapor exposure (spontaneous withdrawal). These data suggest that repeated e-cigarette use leads to nicotine dependence and withdrawal that affects behavior and brain reward function. Further characterization of the health effects of nicotine vapor is necessary to improve treatment strategies for nicotine use disorder and public health policies related to novel nicotine delivery systems.

Glycemic Challenge Is Associated with the Rapid Cellular Activation of the Locus Ceruleus and Nucleus of Solitary Tract: Circumscribed Spatial Analysis of Phosphorylated MAP Kinase Immunoreactivity.

Rodent studies indicate that impaired glucose utilization or hypoglycemia is associated with the cellular activation of neurons in the medulla (Winslow, 1733) (MY), believed to control feeding behavior and glucose counterregulation. However, such activation has been tracked primarily within hours of the challenge, rather than sooner, and has been poorly mapped within standardized brain atlases. Here, we report that, within 15 min of receiving 2-deoxy-d-glucose (2-DG; 250 mg/kg, i.v.), which can trigger glucoprivic feeding behavior, marked elevations were observed in the numbers of rhombic brain (His, 1893) (RB) neuronal cell profiles immunoreactive for the cellular activation marker(s), phosphorylated p44/42 MAP kinases (phospho-ERK1/2), and that some of these profiles were also catecholaminergic. We mapped their distributions within an open-access rat brain atlas and found that 2-DG-treated rats (compared to their saline-treated controls) displayed greater numbers of phospho-ERK1/2+ neurons in the locus ceruleus (Wenzel and Wenzel, 1812) (LC) and the nucleus of solitary tract (>1840) (NTS). Thus, the 2-DG-activation of certain RB neurons is more rapid than perhaps previously realized, engaging neurons that serve multiple functional systems and which are of varying cellular phenotypes. Mapping these populations within standardized brain atlas maps streamlines their targeting and/or comparable mapping in preclinical rodent models of disease.

MAP kinases couple hindbrain-derived catecholamine signals to hypothalamic adrenocortical control mechanisms during glycemia-related challenges.

Physiological responses to hypoglycemia, hyperinsulinemia, and hyperglycemia include a critical adrenocortical component that is initiated by hypothalamic control of the anterior pituitary and adrenal cortex. These adrenocortical responses ensure appropriate long-term glucocorticoid-mediated modifications to metabolism. Despite the importance of these mechanisms to disease processes, how hypothalamic afferent pathways engage the intracellular mechanisms that initiate adrenocortical responses to glycemia-related challenges are unknown. This study explores these mechanisms using network- and cellular-level interventions in in vivo and ex vivo rat preparations. Results show that a hindbrain-originating catecholamine afferent system selectively engages a MAP kinase pathway in rat paraventricular hypothalamic CRH (corticotropin-releasing hormone) neuroendocrine neurons shortly after vascular insulin and 2-deoxyglucose challenges. In turn, this MAP kinase pathway can control both neuroendocrine neuronal firing rate and the state of CREB phosphorylation in a reduced ex vivo paraventricular hypothalamic preparation, making this signaling pathway an ideal candidate for coordinating CRH synthesis and release. These results establish the first clear structural and functional relationships linking neurons in known nutrient-sensing regions with intracellular mechanisms in hypothalamic CRH neuroendocrine neurons that initiate the adrenocortical response to various glycemia-related challenges.

A historical perspective on training students to create standardized maps of novel brain structure: Newly-uncovered resonances between past and present research-based neuroanatomy curricula.

Recent efforts to reform postsecondary STEM education in the U.S. have resulted in the creation of course-based undergraduate research experiences (CUREs), which, among other outcomes, have successfully retained freshmen in their chosen STEM majors and provided them with a greater sense of identity as scientists by enabling them to experience how research is conducted in a laboratory setting. In 2014, we launched our own laboratory-based CURE, Brain Mapping & Connectomics (BMC). Now in its seventh year, BMC trains University of Texas at El Paso (UTEP) undergraduates to identify and label neuron populations in the rat brain, analyze their cytoarchitecture, and draw their detailed chemoarchitecture onto standardized rat brain atlas maps in stereotaxic space. Significantly, some BMC students produce atlas drawings derived from their coursework or from further independent study after the course that are being presented and/or published in the scientific literature. These maps should prove useful to neuroscientists seeking to experimentally target elusive neuron populations. Here, we review the procedures taught in BMC that have empowered students to learn about the scientific process. We contextualize our efforts with those similarly carried out over a century ago to reform U.S. medical education. Notably, we have uncovered historical records that highlight interesting resonances between our curriculum and that created at the Johns Hopkins University Medical School (JHUMS) in the 1890s. Although the two programs are over a century apart and were created for students of differing career levels, many aspects between them are strikingly similar, including the unique atlas-based brain mapping methods they encouraged students to learn. A notable example of these efforts was the brain atlas maps published by Florence Sabin, a JHUMS student who later became the first woman to be elected to the U.S. National Academy of Sciences. We conclude by discussing how the revitalization of century-old methods and their dissemination to the next generation of scientists in BMC not only provides student benefit and academic development, but also acts to preserve what are increasingly becoming "lost arts" critical for advancing neuroscience - brain histology, cytoarchitectonics, and atlas-based mapping of novel brain structure.

Female rats display greater nicotine withdrawal-induced cellular activation of a central portion of the interpeduncular nucleus versus males: A study of Fos immunoreactivity within provisionally assigned interpeduncular subnuclei.

BACKGROUND: The interpeduncular nucleus (>1840) (IPN) has been shown to modulate the behavioral effects of nicotine withdrawal in male rodents. To date, the contribution of this brain structure to sex differences in withdrawal is largely unexplored. METHODS: This study compared neuronal activation, as reported by observable Fos expression in the IPN of nicotine-dependent female and male rats experiencing withdrawal. We provisionally localized the Fos-expressing cells to certain IPN subnuclei within Swanson's standardized brain atlas (2018). Adult female and male rats were prepared with a pump that delivered nicotine (3.2 mg/kg/day; base) continuously. Controls received a sham surgery. Fourteen days later, the rats received administration of saline or the nicotinic receptor antagonist, mecamylamine (3.0 mg/kg; salt), and physical signs and anxiety-like behavior were assessed. The rats were then euthanized and brain sections containing the IPN were processed for Fos immunofluorescence to infer the possible IPN subnuclei displaying differential activation between sexes. RESULTS: Both female and male rats displayed withdrawal-induced Fos expression within the IPN. Compared to males, female rats displayed greater numbers of withdrawal-induced Fos-positive cells within a circumscribed portion of the IPN that may fall within the cytoarchitectural boundaries of the central subnucleus (>1840) (IPNc). The withdrawal-induced activation of the IPN was correlated with negative affective states in females, but not males. CONCLUSION: These data suggest that a centrally located group of IPN cells, presumably situated partly or completely within the IPNc, play a role in modulating sex differences in negative affective states produced by withdrawal.

Overexpression of corticotropin-releasing factor in the nucleus accumbens enhances the reinforcing effects of nicotine in intact female versus male and ovariectomized female rats.

This study assessed the role of stress systems in the nucleus accumbens (NAc) in promoting sex differences in the reinforcing effects of nicotine. Intravenous self-administration (IVSA) of various doses of nicotine was compared following overexpression of corticotropin-releasing factor (CRF) in the NAc of female and male rats. Ovariectomized (OVX) females were also included to assess the role of ovarian hormones in promoting nicotine reinforcement. Rats received intra-NAc administration of an adeno-associated vector that overexpressed CRF (AAV2/5-CRF) or green fluorescent protein (AAV2/5-GFP). All rats were then given extended access (23 h/day) to an inactive and an active lever that delivered nicotine. Separate groups of rats received intra-NAc AAV2/5-CRF and saline IVSA. Rats were also allowed to nose-poke for food and water during IVSA testing. At the end of the study, the NAc was dissected and rt-qPCR methods were used to estimate CRF overexpression and changes in CRF receptors (CRFr1, CRFr2) and the CRF receptor internalizing protein, ß-arrestin2 (Arrb2). Overexpression of CRF in the NAc increased nicotine IVSA to a larger extent in intact female versus male and OVX females. Food intake was increased to a larger extent in intact and OVX females as compared to males. The increase in CRF gene expression was similar across all groups; however, in females, overexpression of CRF resulted in a larger increase in CRFr1 and CRFr2 relative to males. In males, overexpression of CRF produced a larger increase in Arrb2 than females, suggesting greater CRF receptor internalization. Our results suggest that stress systems in the NAc promote the reinforcing effectiveness of nicotine in female rats in an ovarian hormone-dependent manner.

Distributions of hypothalamic neuron populations coexpressing tyrosine hydroxylase and the vesicular GABA transporter in the mouse.

The hypothalamus contains catecholaminergic neurons marked by the expression of tyrosine hydroxylase (TH). As multiple chemical messengers coexist in each neuron, we determined if hypothalamic TH-immunoreactive (ir) neurons express vesicular glutamate or GABA transporters. We used Cre/loxP recombination to express enhanced GFP (EGFP) in neurons expressing the vesicular glutamate (vGLUT2) or GABA transporter (vGAT), then determined whether TH-ir neurons colocalized with native EGFPVglut2 - or EGFPVgat -fluorescence, respectively. EGFPVglut2 neurons were not TH-ir. However, discrete TH-ir signals colocalized with EGFPVgat neurons, which we validated by in situ hybridization for Vgat mRNA. To contextualize the observed pattern of colocalization between TH-ir and EGFPVgat , we first performed Nissl-based parcellation and plane-of-section analysis, and then mapped the distribution of TH-ir EGFPVgat neurons onto atlas templates from the Allen Reference Atlas (ARA) for the mouse brain. TH-ir EGFPVgat neurons were distributed throughout the rostrocaudal extent of the hypothalamus. Within the ARA ontology of gray matter regions, TH-ir neurons localized primarily to the periventricular hypothalamic zone, periventricular hypothalamic region, and lateral hypothalamic zone. There was a strong presence of EGFPVgat fluorescence in TH-ir neurons across all brain regions, but the most striking colocalization was found in a circumscribed portion of the zona incerta (ZI)-a region assigned to the hypothalamus in the ARA-where every TH-ir neuron expressed EGFPVgat . Neurochemical characterization of these ZI neurons revealed that they display immunoreactivity for dopamine but not dopamine ß-hydroxylase. Collectively, these findings indicate the existence of a novel mouse hypothalamic population that may signal through the release of GABA and/or dopamine.

Catecholaminergic control of mitogen-activated protein kinase signaling in paraventricular neuroendocrine neurons in vivo and in vitro: a proposed role during glycemic challenges.

Paraventricular hypothalamic (PVH) corticotropin-releasing hormone (CRH) neuroendocrine neurons mount neurosecretory and transcriptional responses to glycemic challenges [intravenous 2-deoxyglucose (2-DG) or insulin]. Although these responses require signals from intact afferents originating from hindbrain CA (catecholaminergic) neurons, the identity of these signals and the mechanisms by which they are transduced by PVH neurons during glycemic challenge remain unclear. Here, we tested whether the prototypical catecholamine, norepinephrine (NE), can reproduce PVH neuroendocrine responses to glycemic challenge. Because these responses include phosphorylation of p44/42 mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinases 1/2 (ERK1/2)], we also determined whether NE activates ERK1/2 in PVH neurons and, if so, by what mechanism. We show that systemic insulin and 2-DG, and PVH-targeted NE microinjections, rapidly elevated PVH phospho-ERK1/2 levels. NE increased Crh and c-fos expression, together with circulating ACTH/corticosterone. However, because injections also increased c-Fos mRNA in other brain regions, we used hypothalamic slices maintained in vitro to clarify whether NE activates PVH neurons without contribution of inputs from distal regions. In slices, bath-applied NE triggered robust phospho-ERK1/2 immunoreactivity in PVH (including CRH) neurons, which attenuated markedly in the presence of the alpha1 adrenoceptor antagonist, prazosin, or the MAP kinase kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene). Therefore, at a systems level, local PVH delivery of NE is sufficient to account for hindbrain activation of CRH neuroendocrine neurons during glycemic challenge. At a cellular level, these data provide the first demonstration that MAP kinase signaling cascades (MEK-->ERK) are intracellular transducers of noradrenergic signals in CRH neurons, and implicate this transduction mechanism as an important component of central neuroendocrine responses during glycemic challenge.

Computer Vision Evidence Supporting Craniometric Alignment of Rat Brain Atlases to Streamline Expert-Guided, First-Order Migration of Hypothalamic Spatial Datasets Related to Behavioral Control.

The rat has arguably the most widely studied brain among all animals, with numerous reference atlases for rat brain having been published since 1946. For example, many neuroscientists have used the atlases of Paxinos and Watson (PW, first published in 1982) or Swanson (S, first published in 1992) as guides to probe or map specific rat brain structures and their connections. Despite nearly three decades of contemporaneous publication, no independent attempt has been made to establish a basic framework that allows data mapped in PW to be placed in register with S, or vice versa. Such data migration would allow scientists to accurately contextualize neuroanatomical data mapped exclusively in only one atlas with data mapped in the other. Here, we provide a tool that allows levels from any of the seven published editions of atlases comprising three distinct PW reference spaces to be aligned to atlas levels from any of the four published editions representing S reference space. This alignment is based on registration of the anteroposterior stereotaxic coordinate (z) measured from the skull landmark, Bregma (ß). Atlas level alignments performed along the z axis using one-dimensional Cleveland dot plots were in general agreement with alignments obtained independently using a custom-made computer vision application that utilized the scale-invariant feature transform (SIFT) and Random Sample Consensus (RANSAC) operation to compare regions of interest in photomicrographs of Nissl-stained tissue sections from the PW and S reference spaces. We show that z-aligned point source data (unpublished hypothalamic microinjection sites) can be migrated from PW to S space to a first-order approximation in the mediolateral and dorsoventral dimensions using anisotropic scaling of the vector-formatted atlas templates, together with expert-guided relocation of obvious outliers in the migrated datasets. The migrated data can be contextualized with other datasets mapped in S space, including neuronal cell bodies, axons, and chemoarchitecture; to generate data-constrained hypotheses difficult to formulate otherwise. The alignment strategies provided in this study constitute a basic starting point for first-order, user-guided data migration between PW and S reference spaces along three dimensions that is potentially extensible to other spatial reference systems for the rat brain.

Mapping Molecular Datasets Back to the Brain Regions They are Extracted from: Remembering the Native Countries of Hypothalamic Expatriates and Refugees.

This article focuses on approaches to link transcriptomic, proteomic, and peptidomic datasets mined from brain tissue to the original locations within the brain that they are derived from using digital atlas mapping techniques. We use, as an example, the transcriptomic, proteomic and peptidomic analyses conducted in the mammalian hypothalamus. Following a brief historical overview, we highlight studies that have mined biochemical and molecular information from the hypothalamus and then lay out a strategy for how these data can be linked spatially to the mapped locations in a canonical brain atlas where the data come from, thereby allowing researchers to integrate these data with other datasets across multiple scales. A key methodology that enables atlas-based mapping of extracted datasets-laser-capture microdissection-is discussed in detail, with a view of how this technology is a bridge between systems biology and systems neuroscience.

A role for the forebrain in mediating time-of-day differences in glucocorticoid counterregulatory responses to hypoglycemia in rats.

The time of day influences the magnitude of ACTH and corticosterone responses to hypoglycemia. However, little is known about the mechanisms that impart these time-of-day differences on neuroendocrine CRH neurons in the hypothalamic paraventricular nucleus (PVH). Rats received 0-3 U/kg insulin (or 0.9% saline) to achieve a range of glucose nadir concentrations. Brains were processed to identify phosphorylated ERK1/2 (phospho-ERK1/2)-immunoreactive cells in the PVH and hindbrain and CRH heteronuclear RNA in the PVH. Hypoglycemia did not stimulate ACTH and corticosterone responses in animals unless a glucose concentration of approximately 3.15 mM or below was reached. Critically the glycemic thresholds required to stimulate ACTH and corticosterone release in the morning and night were indistinguishable. Yet glucose concentrations below the estimated glycemic threshold correlated with a greater increase in corticosterone, ACTH, and phospho-ERK1/2-immunoreactive neurons in the PVH at night, compared with morning. In these same animals, the number of phospho-ERK1/2-immunoreactive neurons in the medial part of the nucleus of the solitary tract was unchanged at both times of day. These data collectively support a model whereby changes in forebrain mechanisms alter the sensitivity of neuroendocrine CRH to the hypoglycemia-related information conveyed by ascending catecholaminergic afferents. Circadian clock-driven processes together with glucose-sensing elements in the forebrain would seem to be strong contenders for mediating these effects.

NeuroScholar's electronic laboratory notebook and its application to neuroendocrinology.

Scientists continually relate information from the published literature to their current research. The challenge of this essential and time-consuming activity increases as the body of scientific literature continues to grow. In an attempt to lessen the challenge, we have developed an Electronic Laboratory Notebook (ELN) application. Our ELN functions as a component of another application we have developed, an open-source knowledge management system for the neuroscientific literature called NeuroScholar (http://www. neuroscholar. org/). Scanned notebook pages, images, and data files are entered into the ELN, where they can be annotated, organized, and linked to similarly annotated excerpts from the published literature within Neuroscholar. Associations between these knowledge constructs are created within a dynamic node-and-edge user interface. To produce an interactive, adaptable knowledge base. We demonstrate the ELN's utility by using it to organize data and literature related to our studies of the neuroendocrine hypothalamic paraventricular nucleus (PVH). We also discuss how the ELN could be applied to model other neuroendocrine systems; as an example we look at the role of PVH stressor-responsive neurons in the context of their involvement in the suppression of reproductive function. We present this application to the community as open-source software and invite contributions to its development.

Activation in neural networks controlling ingestive behaviors: what does it mean, and how do we map and measure it?

Over the past thirty years many of different methods have been developed that use markers to track or image the activity of the neurons within the central networks that control ingestive behaviors. The ultimate goal of these experiments is to identify the location of neurons that participate in the response to an identified stimulus, and more widely to define the structure and function of the networks that control specific aspects of ingestive behavior. Some of these markers depend upon the rapid accumulation of proteins, while others reflect altered energy metabolism as neurons change their firing rates. These methods are widely used in behavioral neuroscience, but the way results are interpreted within the context of defining neural networks is constrained by how we answer the following questions. How well can the structure of the behavior be documented? What do we know about the processes that lead to the accumulation of the marker? What is the function of the marker within the neuron? How closely in time does the marker accumulation track the stimulus? How long does the marker persist after the stimulus is removed? We will review how these questions can be addressed with regard to ingestive and related behaviors. We will also discuss the importance of plotting the location of labeled cells using standardized atlases to facilitate the presentation and comparison of data between experiments and laboratories. Finally, we emphasize the importance of comprehensive and accurate mapping for using newly emerging technologies in neuroinfomatics.

coliBASE: an online database for Escherichia coli, Shigella and Salmonella comparative genomics.

We have constructed coliBASE, a database for Escherichia coli, Shigella and Salmonella comparative genomics available online at http://colibase. bham.ac.uk. Unlike other E.coli databases, which focus on the laboratory model strain K12, coliBASE is intended to reflect the full diversity of E.coli and its relatives. The database contains comparative data including whole genome alignments and lists of putative orthologous genes, together with numerous analytical tools and links to existing online resources. The data are stored in a relational database, accessible by a number of user-friendly search methods and graphical browsers. The database schema is generic and can easily be applied to other bacterial genomes. Two such databases, CampyDB (for the analysis of Campylobacter spp.) and ClostriDB (for Clostridium spp.) are also available at http://campy.bham.ac.uk and http://clostri. bham.ac.uk, respectively. An example of the power of E.coli comparative analyses such as those available through coliBASE is presented.