Arshad M. Khan

Elucidation of the anatomy of a satiety network: Focus on connectivity of the parabrachial nucleus in the adult rat.

We hypothesized that brain regions showing neuronal activation after refeeding comprise major nodes in a satiety network, and tested this hypothesis with two sets of experiments. Detailed c‐Fos mapping comparing fasted and refed rats was performed to identify candidate nodes of the satiety network. In addition to well‐known feeding‐related brain regions such as the arcuate, dorsomedial, and paraventricular hypothalamic nuclei, lateral hypothalamic area, parabrachial nucleus (PB), nucleus of the solitary tract and central amygdalar nucleus, other refeeding activated regions were also identified, such as the parastrial and parasubthalamic nuclei. To begin to understand the connectivity of the satiety network, the interconnectivity of PB with other refeeding‐activated neuronal groups was studied following administration of anterograde or retrograde tracers into the PB. After allowing for tracer transport time, the animals were fasted and then refed before sacrifice. Refeeding‐activated neurons that project to the PB were found in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamic area; arcuate, paraventricular, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; parasubthalamic nucleus; central amygdalar nucleus; area postrema; and nucleus of the solitary tract. Axons originating from the PB were observed to closely associate with refeeding‐activated neurons in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamus; paraventricular, arcuate, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; central amygdalar nucleus; parasubthalamic nucleus; ventral posterior thalamic nucleus; area postrema; and nucleus of the solitary tract. These data indicate that the PB has bidirectional connections with most refeeding‐activated neuronal groups, suggesting that short‐loop feedback circuits exist in this satiety network. J. Comp. Neurol. 524:2803–2827, 2016.

Rescuing Perishable Neuroanatomical Information from a Threatened Biodiversity Hotspot: Remote Field Methods for Brain Tissue Preservation Validated by Cytoarchitectonic Analysis, Immunohistochemistry, and X-Ray Microcomputed Tomography

Biodiversity hotspots, which harbor more endemic species than elsewhere on Earth, are increasingly threatened. There is a need to accelerate collection efforts in these regions before threatened or endangered species become extinct. The diverse geographical, ecological, genetic, morphological, and behavioral data generated from the on-site collection of an individual specimen are useful for many scientific purposes. However, traditional methods for specimen preparation in the field do not permit researchers to retrieve neuroanatomical data, disregarding potentially useful data for increasing our understanding of brain diversity. These data have helped clarify brain evolution, deciphered relationships between structure and function, and revealed constraints and selective pressures that provide context about the evolution of complex behavior. Here, we report our field-testing of two commonly used laboratory-based techniques for brain preservation while on a collecting expedition in the Congo Basin and Albertine Rift, two poorly known regions associated with the Eastern Afromontane biodiversity hotspot. First, we found that transcardial perfusion fixation and long-term brain storage, conducted in remote field conditions with no access to cold storage laboratory equipment, had no observable impact on cytoarchitectural features of lizard brain tissue when compared to lizard brain tissue processed under laboratory conditions. Second, field-perfused brain tissue subjected to prolonged post-fixation remained readily compatible with subsequent immunohistochemical detection of neural antigens, with immunostaining that was comparable to that of laboratory-perfused brain tissue. Third, immersion-fixation of lizard brains, prepared under identical environmental conditions, was readily compatible with subsequent iodine-enhanced X-ray microcomputed tomography, which facilitated the non-destructive imaging of the intact brain within its skull. In summary, we have validated multiple approaches to preserving intact lizard brains in remote field conditions with limited access to supplies and a high degree of environmental exposure. This protocol should serve as a malleable framework for researchers attempting to rescue perishable and irreplaceable morphological and molecular data from regions of disappearing biodiversity. Our approach can be harnessed to extend the numbers of species being actively studied by the neuroscience community, by reducing some of the difficulty associated with acquiring brains of animal species that are not readily available in captivity.

Identifying links in the chain: The dynamic coupling of catecholamines, peptide synthesis, and peptide release in hypothalamic neuroendocrine neurons

Compared to neurons that communicate using synapses, some neuroendocrine neurons release relatively large quantities of peptide into the vasculature to control neuroendocrine function. Maintaining adequate amounts of peptide for release through controlled biosynthesis is therefore critical for their function. But how neuroendocrine-or in fact, any neuropeptide-neurons link appropriate levels of peptide biosynthesis with the action potentials that drive peptide release is unknown. Here, we review possible mechanisms in paraventricular hypothalamic CRH neuroendocrine neurons to coordinate these processes in response to catecholaminergic inputs. We show that CRH synthesis and release mechanisms are not invariably linked as CRH neurons are activated. Instead, coupling mechanisms exist in the premotor network that provides their synaptic inputs and in their intracellular signal transduction mechanisms, where transmitter-regulated phosphorylation of p44/42 mitogen-activated protein kinases (ERK1/2) may play a prominent role. These versatile and dynamic coupling mechanisms provide a way to link peptide biosynthesis and release.

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.

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 Agilents 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.

Tracking the Coupling of External Signals to Intracellular Programs Controlling Peptide Synthesis and Release in Hypothalamic Neuroendocrine Neurons

Corticotropin-releasing hormone (CRH) neuroendocrine neurons of the paraventricular hypothalamic nucleus constitute the final common pathway for the hypothalamo–pituitary–adrenal axis. These neurons trigger an important cascade of hormone releasing events that allow the organism to respond adaptively to stress, including the release of CRH from the hypothalamus, adrenocorticotropic hormone from the anterior pituitary gland, and cortisol (corticosterone in rats) from the adrenal cortex. Despite the central place of CRH neuroendocrine neurons in this hierarchy, the precise manner by which stimuli engage the brain to drive these neurons remains unclear. In this chapter, we describe experiments establishing functional linkages among a peripheral stressor (glycemic challenge), hindbrain-originating neural circuits, and intracellular programs that control the synthesis and release of CRH neuropeptide. A model is presented based on these studies that offers a testable framework upon which to build, and extensions of these findings to larger brain networks are discussed.

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.

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

&NA; 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.