Corrigendum: The olfactory tract: Basis for future evolution in response to rapidly changing ecological niches.

[This corrects the article DOI: 10.3389/fnana.2022.831602.].

The Olfactory Organ Is a Unique Site for Neutrophils in the Brain.

In the vertebrate olfactory tract new neurons are continuously produced throughout life. It is widely believed that neurogenesis contributes to learning and memory and can be regulated by immune signaling molecules. Proteins originally identified in the immune system have subsequently been localized to the developing and adult nervous system. Previously, we have shown that olfactory imprinting, a specific type of long-term memory, is correlated with a transcriptional response in the olfactory organs that include up-regulation of genes associated with the immune system. To better understand the immune architecture of the olfactory organs we made use of cell-specific fluorescent reporter lines in dissected, intact adult brains of zebrafish to examine the association of the olfactory sensory neurons with neutrophils and blood-lymphatic vasculature. Surprisingly, the olfactory organs contained the only neutrophil populations observed in the brain; these neutrophils were localized in the neural epithelia and were associated with the extensive blood vasculature of the olfactory organs. Damage to the olfactory epithelia resulted in a rapid increase of neutrophils both within the olfactory organs as well as the central nervous system. Analysis of cell division during and after damage showed an increase in BrdU labeling in the neural epithelia and a subset of the neutrophils. Our results reveal a unique population of neutrophils in the olfactory organs that are associated with both the olfactory epithelia and the lymphatic vasculature suggesting a dual olfactory-immune function for this unique sensory system.

The U.S. Armed Services Blood Program support to U.S. Central Command 2014-2021: Transformation of combat trauma resuscitation through blood product innovation and expansion of blood availability far forward.

BACKGROUND: The United States Armed Services Blood Program (ASBP) faced complex blood supply challenges during two decades of military operations in the U.S. Central Command (CENTCOM) and through an adaptive, responsive, and agile system, gained valuable insights on blood product usage in combat casualty care. STUDY DESIGN AND METHODS: A retrospective review of blood product introduction and utilization trends was compiled from ASBP data collected during CENTCOM operations from 2014 through 2021. RESULTS: During the study period, several blood products were introduced to the CENTCOM area of operations including Low Titer O Whole Blood (LTOWB), Cold-Stored Platelets (CSP), Liquid Plasma (LP), and French Freeze Dried Plasma (FDP) manufactured from U.S. sourced donor plasma, all while expanding Walking Blood Bank capabilities. There was a gradual substitution of component therapy for whole blood; blood utilization peaked in 2017. Transfusion of Fresh Whole Blood (FWB) from Walking Blood Banks decreased as fully pre-tested LTOWB was supplied by the ASBP. LTOWB was initially supplied in citrate-phosphate-dextrose (CPD) anticoagulant (21-day shelf life) but was largely replaced with LTOWB in citrate-phosphate-dextrose-adenine (CPDA-1) anticoagulant (35-day shelf life) by 2019. Implementation of prehospital transfusion and expansion of surgical and resuscitation teams led to an increase in the number of sites receiving blood. DISCUSSION: ASBP introduced new products to its inventory in order to meet changing blood product demands driven by changes in the Joint Trauma System Clinical Practice Guidelines and operational demands. These products were adopted into clinical practice with a resultant evolution in transfusion strategies.

The Olfactory Tract: Basis for Future Evolution in Response to Rapidly Changing Ecological Niches.

Within the forebrain the olfactory sensory system is unique from other sensory systems both in the projections of the olfactory tract and the ongoing neurogenic potential, characteristics conserved across vertebrates. Olfaction plays a crucial role in behaviors such as mate choice, food selection, homing, escape from predators, among others. The olfactory forebrain is intimately associated with the limbic system, the region of the brain involved in learning, memory, and emotions through interactions with the endocrine system and the autonomic nervous system. Previously thought to lack a limbic system, we now know that teleost fishes process emotions, have exceptional memories, and readily learn, behaviors that are often associated with olfactory cues. The association of neuromodulatory hormones, and more recently, the immune system, with odor cues underlies behaviors essential for maintenance and adaptation within natural ecological niches. Increasingly anthropogenic perturbations affecting ecosystems are impacting teleost fishes worldwide. Here we examine the role of the olfactory tract as the neural basis for the integration of environmental cues and resulting behaviors necessary for the regulation of biotic interactions that allow for future adaptation as the climate spins out of control.

Gonadotropin Releasing Hormone (Gnrh) Triggers Neurogenesis in the Hypothalamus of Adult Zebrafish.

Recently, it has been shown in adult mammals that the hypothalamus can generate new cells in response to metabolic changes, and tanycytes, putative descendants of radial glia, can give rise to neurons. Previously we have shown in vitro that neurospheres generated from the hypothalamus of adult zebrafish show increased neurogenesis in response to exogenously applied hormones. To determine whether adult zebrafish have a hormone-responsive tanycyte-like population in the hypothalamus, we characterized proliferative domains within this region. Here we show that the parvocellular nucleus of the preoptic region (POA) labels with neurogenic/tanycyte markers vimentin, GFAP/Zrf1, and Sox2, but these cells are generally non-proliferative. In contrast, Sox2+ proliferative cells in the ventral POA did not express vimentin and GFAP/Zrf1. A subset of the Sox2+ cells co-localized with Fezf2:GFP, a transcription factor important for neuroendocrine cell specification. Exogenous treatments of GnRH and testosterone were assayed in vivo. While the testosterone-treated animals showed no significant changes in proliferation, the GnRH-treated animals showed significant increases in the number of BrdU-labeled cells and Sox2+ cells. Thus, cells in the proliferative domains of the zebrafish POA do not express radial glia (tanycyte) markers vimentin and GFAP/Zrf1, and yet, are responsive to exogenously applied GnRH treatment.

Dissecting the neural divide: a continuous neurectoderm gives rise to the olfactory placode and bulb.

The olfactory epithelia arise from morphologically identifiable structures called olfactory placodes. Sensory placodes are generally described as being induced from the ectoderm suggesting that their development is separate from the coordinated cell movements generating the central nervous system. Previously, we have shown that the olfactory placodes arise from a large field of cells bordering the telencephalic precursors in the neural plate, and that cell movements, not cell division, underlie olfactory placode morphogenesis. Subsequently by image analysis, cells were tracked as they moved in the continuous sheet of neurectoderm giving rise to the peripheral (olfactory organs) and central (olfactory bulbs) nervous system (Torres-Paz and Whitlock, 2014). These studies lead to a model whereby the olfactory epithelia develop from within the border of the neural late and are a neural tube derivative, similar to the retina of the eye (Torres-Paz and Whitlock, 2014; Whitlock, 2008). Here we show that randomly generated clones of cells extend across the morphologically differentiated olfactory placodes/olfactory bulbs, and test the hypothesis that these structures are patterned by a different level of distal-less (dlx) gene expression subdividing the anterior neurectoderm into OP precursors (high Dlx expression) and OB precursors (lower Dlx expression). Manipulation of DLX protein and RNA levels resulted in morphological changes in the size of the olfactory epithelia and olfactory bulb. Thus, the olfactory epithelia and bulbs arise from a common neurectodermal region and develop in concert through coordinated morphological movements.

The Olfactory Organ Is Populated by Neutrophils and Macrophages During Early Development.

The immune system of vertebrates is characterized by innate and adaptive immunity that function together to form the natural defense system of the organism. During development innate immunity is the first to become functional and is mediated primarily by phagocytic cells, including macrophages, neutrophils, and dendritic cells. In the olfactory sensory system, the same sensory neurons in contact with the external environment have their first synapse within the central nervous system. This unique architecture presents a potential gateway for the entry of damaging or infectious agents to the nervous system. Here we used zebrafish as a model system to examine the development of the olfactory organ and to determine whether it shares immune characteristics of a host defense niche described in other tissues. During early development, both neutrophils and macrophages appear coincident with the generation of the primitive immune cells. The appearance of neutrophils and macrophages in the olfactory organs occurs as the blood and lymphatic vascular system is forming in the same region. Making use of the neurogenic properties of the olfactory organ we show that damage to the olfactory sensory neurons in larval zebrafish triggers a rapid immune response by local and non-local neutrophils. In contrast, macrophages, although present in greater numbers, mount a slower response to damage. We anticipate our findings will open new avenues of research into the role of the olfactory-immune response during normal neurogenesis and damage-induced regeneration and contribute to our understanding of the formation of a potential host defense immune niche in the peripheral nervous system.

Neuroendocrinology of reproduction: Is gonadotropin-releasing hormone (GnRH) dispensable?

Gonadotropin releasing hormone (GnRH) is a highly conserved neuroendocrine decapeptide that is essential for the onset of puberty and the maintenance of the reproductive state. First identified in mammals, the GnRH signaling pathway is found in all classes of vertebrates; homologues of GnRH have also been identified in invertebrates. In addition to its role as a hypothalamic releasing hormone, GnRH has multiple functions including modulating neural activity within specific regions of the brain. These various functions are mediated by multiple isoforms, which are expressed at diverse locations within the central nervous system. Here we discuss the GnRH signaling pathways in light of new reports that reveal that some vertebrate genomes lack GnRH1. Not only do other isoforms of GnRH not compensate for this gene loss, but elements upstream of GnRH1, including kisspeptins, appear to also be dispensable. We discuss routes that may compensate for the loss of the GnRH1 pathway.

Changes in Olfactory Receptor Expression Are Correlated With Odor Exposure During Early Development in the zebrafish (Danio rerio).

We have previously shown that exposure to phenyl ethyl alcohol (PEA) causes an increase in the expression of the transcription factor otx2 in the olfactory epithelium (OE) of juvenile zebrafish, and this change is correlated with the formation of an odor memory of PEA. Here, we show that the changes in otx2 expression are specific to ßPEA: exposure to αPEA did not affect otx2 expression. We identified 34 olfactory receptors (ORs) representing 16 families on 4 different chromosomes as candidates for direct regulation of OR expression via Otx2. Subsequent in silico analysis uncovered Hnf3b binding sites closely associated with Otx2 binding sites in the regions flanking the ORs. Analysis by quantitative polymerase chain reaction and RNA-seq of OR expression in developing zebrafish exposed to different isoforms of PEA showed that a subset of ORs containing both Otx2/Hnf3b binding sites were downregulated only in ßPEA-exposed juveniles and this change persisted through adult life. Localization of OR expression by in situ hybridization indicates the downregulation occurs at the level of RNA and not the number of cells expressing a given receptor. Finally, analysis of immediate early gene expression in the OE did not reveal changes in c-fos expression in response to either αPEA or ßPEA.

Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH) neurons.

Gonadotropin-releasing hormone (GnRH) is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.

The loss of scents: do defects in olfactory sensory neuron development underlie human disease?

The olfactory system is a fascinating and beguiling sensory system: olfactory sensory neurons detect odors underlying behaviors essential for mate choice, food selection, and escape from predators, among others. These sensory neurons are unique in that they have dendrites contacting the outside world, yet their first synapse lies in the central nervous system. The information entering the central nervous system is used to create odor memories that play a profound role in recognition of individuals, places, and appropriate foods. Here, the structure of the olfactory epithelium is given as an overview to discuss the origin of the olfactory placode, the plasticity of the olfactory sensory neurons, and finally the origins of the gonadotropin-releasing hormone neuroendocrine cells. For the purposes of this review, the development of the peripheral sensory system will be analyzed, incorporating recently published studies highlighting the potential novelties in development mechanisms. Specifically, an emerging model where the olfactory epithelium and olfactory bulb develop simultaneously from a continuous neurectoderm patterned at the end of gastrulation, and the multiple origins of the gonadotropin-releasing hormone neuroendocrine cells associated with the olfactory sensory system development will be presented. Advances in the understanding of the basic mechanisms underlying olfactory sensory system development allows for a more thorough understanding of the potential causes of human disease.

Zebrafish invade Valparaiso: third meeting and symposium of the Latin American zebrafish network.

Zebrafish are an excellent model system for research and teaching. Because of their relatively low maintenance costs, beautiful and bountiful embryos, and tool box of molecular genetic technique, zebrafish are ideal for countries with smaller research budgets and less well-developed science infrastructure. For these reasons, zebrafish are growing in popularity as a model system for research in Latin America. In response to this growing need, we held the Third Latin American Zebrafish Network (LAZEN) Course and Symposium in Valparaiso, Chile, in April 4-13, 2014. The course covered a wide variety of topics from fish husbandry to outreach and ended with a symposium hosting excellent scientists from Latin America and beyond.

Zebrafish Invade Valparaiso: Third Meeting and Symposium of the Latin American Zebrafish Network.

Abstract Zebrafish are an excellent model system for research and teaching. Because of their relatively low maintenance costs, beautiful and bountiful embryos, and tool box of molecular genetic technique, zebrafish are ideal for countries with smaller research budgets and less well-developed science infrastructure. For these reasons, zebrafish are growing in popularity as a model system for research in Latin America. In response to this growing need, we held the Third Latin American Zebrafish Network (LAZEN) Course and Symposium in Valparaiso, Chile, in April 4-13, 2014. The course covered a wide variety of topics from fish husbandry to outreach and ended with a symposium hosting excellent scientists from Latin America and beyond.

Olfactory sensory system develops from coordinated movements within the neural plate.

BACKGROUND: The peripheral olfactory sensory system arises from morphologically identifiable structures called placodes. Placodes are relatively late developing structures, evident only well after the initiation of somitogenesis. Placodes are generally described as being induced from the ectoderm suggesting that their development is separate from the coordinated cell movements generating the central nervous system. RESULTS: With the advent of modern techniques it is possible to follow the development of the neurectoderm giving rise to the anterior neural tube, including the olfactory placodes. The cell movements giving rise to the optic cup are coordinated with those generating the olfactory placodes and adjacent telencephalon. The formation of the basal lamina separating the placode from the neural tube is coincident with the anterior migration of cranial neural crest. CONCLUSIONS: Olfactory placodes are transient morphological structures arising from a continuous sheet of neurectoderm that gives rise to the peripheral and central nervous system. This field of cells is specified at the end of gastrulation and not secondarily induced from ectoderm. The separation of olfactory placodes and telencephalon occurs through complex cell movements within the developing neural plate similar to that observed for the developing optic cup.

Quantitative analysis of cell migration using optical flow.

Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations.

The Armed Services Blood Program: blood support to combat casualty care 2001 to 2011.

BACKGROUND: The Armed Services Blood Program (ASBP) provides the farthest-reaching blood supply in the world. This article provides statistics and a review of blood operations in support of combat casualty care during the last 10 years. It also outlines changes in blood doctrine in support of combat casualty care. METHODS: This is a descriptive overview and review of blood product use and transfusions used by ASBP personnel to support combat operations in Iraq and Afghanistan between October 2001 and November 2011. RESULTS: The ASBP initiated major changes in blood availability and age of blood in theater. In support of data published by physicians in theater, showing improved patient survival when a higher ratio of fresh frozen plasma and red blood cells (RBCs) is achieved, plus the use of platelets, the ASBP increased availability of plasma and established platelet collection facilities in theater. New capabilities included emergency collection of apheresis platelets in the battlefield, availability and transfusion of deglycerolized red cells, rapid diagnostic donor screening, and a new modular blood detachment. Forward surgical facilities that were at one time limited to a blood inventory consisting of RBCs now have a complete arsenal of products at their fingertips that may include fresher RBCs, fresh frozen plasma, cryoprecipitate, and platelets. A number of clinical practice guidelines are in place to address these processes. Changes in blood doctrine were made to support new combat casualty care and damage-control resuscitation initiatives. CONCLUSION: Despite the challenges of war in two theaters of operation, a number of improvements and changes to blood policy have been developed during the last 10 years to support combat casualty care. The nature of medical care in combat operations will continue to be dynamic and constantly evolving. The ASBP needs to be prepared to meet future challenges. LEVEL OF EVIDENCE: Epidemiologic study, level IV.

Close association of olfactory placode precursors and cranial neural crest cells does not predestine cell mixing.

Vertebrate sensory organs originate from both cranial neural crest cells (CNCCs) and placodes. Previously, we have shown that the olfactory placode (OP) forms from a large field of cells extending caudally to the premigratory neural crest domain, and that OPs form through cell movements and not cell division. Concurrent with OP formation, CNCCs migrate rostrally to populate the frontal mass. However, little is known about the interactions between CNCCs and the placodes that form the olfactory sensory system. Previous reports suggest that the OP can generate cell types more typical of neural crest lineages such as neuroendocrine cells and glia, thus marking the OP as an unusual sensory placode. One possible explanation for this exception is that the neural crest origin of glia and neurons has been overlooked due to the intimate association of these two fields during migration. Using molecular markers and live imaging, we followed the development of OP precursors and of dorsally migrating CNCCs in zebrafish embryos. We generated a six4b:mCherry line (OP precursors) that, with a sox10:EGFP line (CNCCs), was used to follow cell migration. Our analyses showed that CNCCs associate with and eventually surround the forming OP with limited cell mixing occurring during this process.

Food and conspecific chemical cues modify visual behavior of zebrafish, Danio rerio.

Animals use the different qualities of olfactory and visual sensory information to make decisions. Ethological and electrophysiological evidence suggests that there is cross-modal priming between these sensory systems in fish. We present the first experimental study showing that ecologically relevant chemical mixtures alter visual behavior, using adult male and female zebrafish, Danio rerio. Neutral-density filters were used to attenuate the light reaching the tank to an initial light intensity of 2.3×10(16) photons/s/m2. Fish were exposed to food cue and to alarm cue. The light intensity was then increased by the removal of one layer of filter (nominal absorbance 0.3) every minute until, after 10 minutes, the light level was 15.5×10(16) photons/s/m2. Adult male and female zebrafish responded to a moving visual stimulus at lower light levels if they had been first exposed to food cue, or to conspecific alarm cue. These results suggest the need for more integrative studies of sensory biology.

Zebrafish preference for light or dark is dependent on ambient light levels and olfactory stimulation.

Zebrafish have been shown to have preference for light or dark environments depending on the ambient light level and the presence or absence of food odor. We used a cylindrical tank, half of which was surrounded by a black surface and the other half by white, to elicit a choice from individual wild-type, adult zebrafish. One treatment group was exposed to food odor and the other to water (as a control) at the beginning of the trial. During 10-min trials, the light level was increased each minute over a fivefold range in steps from 1.34 × 10(17) photons/s/m(2) at the beginning to a final light level of 8.31 × 10(17) photons/s/m(2). We demonstrate that the preference of the zebrafish for the light or dark half of the cylinder is dependent upon ambient light levels as well as olfactory stimulation. These results provide a potential explanation for the contradictory observations that, when given a choice, adult zebrafish prefer brighter light environments (Gerlai et al., 2000) or darker light environments (Serra et al., 1999). Thus, we present data useful in designing more powerful and reliable behavioral assays for use with zebrafish as well as further information about the effect of olfactory stimulation on zebrafish visual behavior.