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SummaryLifestyle induces long lasting effects on brain and cognition, with some interventions like stress including transgenerational inheritance mediated by epigenetic mechanisms. Physical exercise is one lifestyle intervention driving robust improvements of cognition, including intergenerational transmission to the litter. However, little is known about whether exercise effects are transgenerationally transmitted. Here we analyzed adult hippocampal neurogenesis (AHN) and behavioral phenotype of sedentary adult male mice of F2 generation of exercised grandfathers (F0). Both F1 and F2 were sedentary, while F0 performed moderate exercise. We found F2 mice from exercised F0 acquired and recalled both spatial and non-spatial information better than F2 from sedentary F0. Contextual fear conditioning was not affected, together with no differences in AHN markers. Hippocampal smallRNAseq analysis revealed 35 significant differentially expressed (sDE) microRNAs (miRNAs) associated to relevant brain function families. Moreover, 11 of the 35 miRNAs target gene sets were found also enriched in F0 and F1, as well as target genes of 6 of them were differentially expressed also in F0 or F1. One of these 6 is miRNA-144, that together with miRNA-298 were found inversely correlated to cognitive index in F2. These results demonstrate that transgenerational transmission of the effects of exercise on specific cognitive tasks persists after two generations, even though some cellular changes induced in F1 vanish in F2. Thus, they suggest moderate exercise training has longer-lasting effects than previously thought, probably mediated by a small group of miRNAs acting across generations, and this is worth taking into account in public health programs.

Maladaptive coping behaviors are probably involved in post-traumatic stress disorders (PTSD), but underlying mechanisms are incompletely understood. We now report that mice lacking functional insulin-like growth factor I (IGF-I) receptors in orexin neurons of the lateral hypothalamus (Firoc mice) are unresponsive to the anxiolytic actions of IGF-I and develop PTSD-like behavior that is ameliorated by inhibition of orexin neurons. Conversely, systemic IGF-I treatment ameliorated PTSD-like behavior in a wild-type mouse model of PTSD (PTSD mice). Further, systemic IGF-I modified the GABA/Glutamate synaptic structure in orexin neurons of naïve wild-type mice by increasing the dephosphorylation of GABA(B) receptor subunit through inhibition of AMP-kinase (AMPK). Significantly, pharmacological inhibition of AMPK mimicked IGF-I, normalizing fear behavior in PTSD mice. Thus, we suggest that IGF-I enables coping behaviors by balancing E/I input onto orexin neurons in a context-dependent manner. These observations provide a novel therapeutic approach to PTSD through modulation of AMPK., We are thankful to M. Garcia and R. Cañadas for technical support. This work was funded by a grant from Ciberned, and from SAF2016-76462, and PID2019-104376RB-I00 (AEI/FEDER; MICINN). JAZ-V acknowledges the financial support of the National Council of Science, Technology and Technological Innovation (CONCYTEC, Perú) through the National Fund for Scientific and Technological Development (FONDECYT, Perú).

Insulin-like growth factor-I (IGF-I) signaling plays a key role in learning and memory processes. While the effects of IGF-I on neurons have been studied extensively, the involvement of astrocytes in IGF-I signaling and the consequences on synaptic plasticity and animal behavior remain unknown. We have found that IGF-I induces long-term potentiation (LTP) of the postsynaptic potentials that is caused by a long-term depression of inhibitory synaptic transmission in mice. We have demonstrated that this long-lasting decrease in the inhibitory synaptic transmission is evoked by astrocytic activation through its IGF-I receptors (IGF-IRs). We show that LTP not only increases the output of pyramidal neurons, but also favors the NMDAR-dependent LTP, resulting in the crucial information processing at the barrel cortex since specific deletion of IGF-IR in cortical astrocytes impairs the whisker discrimination task. Our work reveals a novel mechanism and functional consequences of IGF-I signaling on cortical inhibitory synaptic plasticity and animal behavior, revealing that astrocytes are key elements in these processes., This work was supported by Grants BFU2016-80802-P from Agencia Estatal de Investigación Spain/Fondo Europeo de Desarrollo Regional, and from the European Union [Ministerio de Economía y Competitividad (MINECO)] to D.F.d.S.; Grants R01-NS-097312 and R01-DA-048822 from National Institutes of Health/National Institute of Neurological Disorders and Stroke to A.A.; and grants from Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Grant SAF2016-76462-C2-1-P from MINECO to I.T.-A. J.A.Z.-V. was supported by the National Council of Science, Technology and Technological Innovation (CONCYTEC, Perú) through the National Fund for Scientific and Technological Development (FONDECYT, Perú). J.F. received a postdoctoral fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Grants #2017/14742-0 and #2019/03368-5). We thank the University of Minnesota Viral Vector and Cloning Core for production of some of the viral vectors used in this study; and Dr. G. Perea and Dr. Washington Buño for helpful comments.

Uncoupling of metabolism and circadian activity is associated with an increased risk of a wide spectrum of pathologies. Recently, insulin and the closely related insulin-like growth factor I (IGF-I) were shown to entrain feeding patterns with circadian rhythms. Both hormones act centrally to modulate peripheral glucose metabolism; however, whereas central targets of insulin actions are intensely scrutinized, those mediating the actions of IGF-I remain less defined. We recently showed that IGF-I targets orexin neurons in the lateral hypothalamus, and now we evaluated whether IGF-I modulates orexin neurons to align circadian rhythms with metabolism. Mice with disrupted IGF-IR activity in orexin neurons (Firoc mice) showed sexually dimorphic alterations in daily glucose rhythms and feeding activity patterns which preceded the appearance of metabolic disturbances. Thus, Firoc males developed hyperglycemia and glucose intolerance, while females developed obesity. Since IGF-I directly modulates orexin levels and hepatic expression of KLF genes involved in circadian and metabolic entrainment in an orexin-dependent manner, it seems that IGF-I entrains metabolism and circadian rhythms by modulating the activity of orexin neurons., This research was funded by a grant from Ciberned (Instituto de Salud Carlos III, Spain), an Inter-CIBER project (PIE14/00061), part of the projects PID2019-104376RB-I00 funded by MCIN/AEI/10.13039/ 501100011033; and a grant by Comunidad de Madrid through the European Social Fund (ESF)-financed program NEUROMETAB-CM (B2017/BMD-3700) to ITA. Sperber, Jacob was funded by a Fulbright grant.

Changes in the transcription factor (TF) expression are critical for brain development, and they may also underlie neurodevelopmental disorders. Indeed, T-box brain1 (Tbr1) is a TF crucial for the formation of neocortical layer VI, and mutations and microdeletions in that gene are associated with malformations in the human cerebral cortex, alterations that accompany autism spectrum disorder (ASD). Interestingly, Tbr1 upregulation has also been related to the occurrence of ASD-like symptoms, although limited studies have addressed the effect of increased Tbr1 levels during neocortical development. Here, we analysed the impact of Tbr1 misexpression in mouse neural progenitor cells (NPCs) at embryonic day 14.5 (E14.5), when they mainly generate neuronal layers II–IV. By E18.5, cells accumulated in the intermediate zone and in the deep cortical layers, whereas they became less abundant in the upper cortical layers. In accordance with this, the proportion of Sox5+ cells in layers V–VI increased, while that of Cux1+ cells in layers II–IV decreased. On postnatal day 7, fewer defects in migration were evident, although a higher proportion of Sox5+ cells were seen in the upper and deep layers. The abnormal neuronal migration could be partially due to the altered multipolar-bipolar neuron morphologies induced by Tbr1 misexpression, which also reduced dendrite growth and branching, and disrupted the corpus callosum. Our results indicate that Tbr1 misexpression in cortical NPCs delays or disrupts neuronal migration, neuronal specification, dendrite development and the formation of the callosal tract. Hence, genetic changes that provoke ectopic Tbr1 upregulation during development could provoke cortical brain malformations.

Sleep disturbances are common during aging. Compared to young animals, old mice show altered sleep structure, with changes in both slow and fast electrocorticographic (ECoG) activity and fewer transitions between sleep and wake stages. Insulin-like growth factor I (IGF-I), which is involved in adaptive changes during aging, was previously shown to increase ECoG activity in young mice and monkeys. Furthermore, IGF-I shapes sleep architecture by modulating the activity of mouse orexin neurons in the lateral hypothalamus (LH). We now report that both ECoG activation and excitation of orexin neurons by systemic IGF-I are abrogated in old mice. Moreover, orthodromical responses of LH neurons are facilitated by either systemic or local IGF-I in young mice, but not in old ones. As orexin neurons of old mice show dysregulated IGF-I receptor (IGF-IR) expression, suggesting disturbed IGF-I sensitivity, we treated old mice with AIK3a305, a novel IGF-IR sensitizer, and observed restored responses to IGF-I and rejuvenation of sleep patterns. Thus, disturbed sleep structure in aging mice may be related to impaired IGF-I signaling onto orexin neurons, reflecting a broader loss of IGF-I activity in the aged mouse brain., This work was funded by a grant from Ciberned and is part of the project SAF2016-76462 funded by MCIN/AEI/https://doi.org/10.13039/501100011033. J.A. ZegarraValdivia acknowledges the fnancial support of the National Council of Science, Technology and Technological Innovation (CONCYTEC, Perú) through the National Fund for Scientifc and Technological Development (FONDECYT, Perú). J. Fernandes received a post-doc fellowship from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: # 2017/14742–0; # 2019/03368–5).

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IRdeficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1¿ and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling., We are thankful to M.Garcia and R. Cañadas for technical support. This work was funded by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) (Instituto de Salud CarlosIII, Spain) to I.T.A., A.G., and T.I.; an Inter-CIBER project (PIE14/00061) to I.T.A.that forms part of the projects PID2019-104376RB-I00 (I.T.A.) and RTI2018-094887-B-I00 (M.N.) funded by MCIN/AEI/10.13039/501100011033; a grant from Junta de Andalucia Consejería de Economía y Conocimiento (P18-RT-2233 to A.G.) cofinanced by Programa Operativo FEDER 2014–2020; a grant from Instituto de Salud Carlos III Spain (cofinanced by FEDER funds from the European Union; PI21/00915 to A.G.); Grant PID2020-115218RB-I00 to T.I. funded by Ministerio de Ciencia e Innovación/Agencia Española de Investigación (MCIN/AEI/10.13039/501100011033); and a grant from Comunidad de Madrid through the European Social Fund (ESF)–financed programme Neurometabolismo-Comunidad de Madrid (NEUROMETAB-CM) (B2017/BMD-3700 to I.T.A.and T.I.). M.N. was also supported by the Spanish Ministry of Science and Innovation (Ramón y Cajal RYC-2016-20414). J.P.-U. was contracted by CIBERNED.

Insulin (IR) and insulin-like growth factor I (IGF-IR) receptors share structural homology and can form hybrid heterodimers. While different observations suggest that hybrid receptors are important in physiology and pathology, little is known about their function in the brain. To gain further insight into the role of IR/IGF-IR hybrids in this organ, we analyzed their cellular distribution in the mouse brain. We combined proximity ligation assays (PLA) for IR and IGF-IR, a technique that detects close protein-protein interactions, with immunocytochemistry for brain cell markers to identify IR/IGF-IR hybrids in the major types of brain cells. Intriguingly, while all the types of brain cells analyzed co-express both receptors, only neurons, astroglia, and microglia show readily detectable IR/IGF-IR hybrids. Hybrid PLA signal was negligible in brain endothelial cells and was absent in oligodendrocytes. Hybrids were comparatively more abundant in neurons and peaked after brain development was completed. Cell-specific expression and greater abundance in the adult brain suggests specialized actions of IR/IGF-IR hybrids in this organ, particularly in neurons., This work was funded by grants SAF2013-40710-R (MINECO,Spain), S2017/BMD-3700 (NEUROMETAB-CM) from Comunidad de Madrid co-financed with the Structural Funds of the European Union,and by Inter-CIBER (PIE14/00061).

Uncoupling of metabolism and circadian activity is associated with an increased risk of various pathologies, including neurodegeneration. Recently, insulin and the closely related insulin-like growth factor I (IGF-I) were shown to entrain feeding patterns with circadian rhythms. Moreover, both hormones act centrally to modulate peripheral glucose metabolism; however, whereas central targets of insulin actions are intensely scrutinized, those mediating the actions of IGF-I remain undefined. We analyzed whether IGF-I targets orexin neurons in the lateral hypothalamus, as these neurons are involved in circadian rhythms and energy allocation, and are modulated by IGF-I. Mice with disrupted IGF-IR activity in orexin neurons show phase shifts in circadian feeding behavior, loss of circadian orexin expression, and gradually develop sex-dependent metabolic alterations. In addition, modulation by IGF-I of hepatic KLF transcription factors involved in peripheral glucose metabolism is mediated by orexin neurons. Thus, IGF-I entrains energy metabolism and circadian rhythms through hypothalamic orexin neurons.

Obesity is a risk factor for Alzheimer’s disease (AD), but underlying mechanisms are not clear. We analyzed peripheral clearance of amyloid β (Aβ) in overweight mice because its systemic elimination may impact on brain Aβ load, a major landmark of AD pathology. Overweight mice showed increased peripheral Aβ clearance by the liver, the major site of elimination of systemic Aβ, but unaltered brain Aβ levels. Since circulating insulin-like growth factor I (IGF-I) modulates brain Aβ clearance, and is increased in serum of overweight mice, we determined whether it affects peripheral Aβ clearance. We found that Aβ uptake by hepatocytes is stimulated by IGF-I. Moreover, mice with low serum IGF-I levels show reduced peripheral Aβ clearance. In the brain, IGF-I favored association of its receptor (IGF-IR) with Aβ precursor protein (APP), and at the same time stimulated non-amyloidogenic processing of APP in astrocytes, as indicated by an increased sAPPα/sAPPβ ratio after IGF-I treatment. Since serum IGF-I enters into the brain in an activity-dependent manner, we analyzed in overweight mice the effect of brain activation by environmental enrichment (EE) on brain IGF-IR phosphorylation and its association to APP, as a readout of IGF-I activity. After EE, significantly less activation of brain IGF-IR phosphorylation and APP/IGF-IR association was found in overweight mice as compared to lean controls. Collectively, these results indicate that diet influences peripheral clearance of Aβ without affecting brain Aβ load. Increased serum IGF-I likely contributes to enhanced peripheral Aβ clearance in overweight mice, without affecting brain Aβ clearance probably because its brain entrance is reduced.

Knowledge of mechanisms involved in vulnerability/resilience to stress disorders is crucial for prevention and treatment schemes. We previously documented that insulin-like growth factor I (IGF-I) is associated to vulnerability to stress both in mice and humans. Since hypothalamic orexin neurons express IGF-I receptors and are involved in responses to stress, we analyzed their role in the modulatory actions of IGF-I on stress. Anxiolytic actions of IGF-I after exposure to a predator were absent in mice lacking IGF-I receptors in orexin neurons (Firoc mice). Based on these observations we speculated that Firoc mice may be prone to develop fear-related disturbances, including post-traumatic stress disorder (PTSD)-like symptoms when confronted to fear learning, a process that is postulated to be altered in PTSD. Firoc mice submitted to fear conditioning showed increased freezing responses, suggesting aberrant fear learning. Exaggerated freezing was accompanied by increased levels of orexin, together with enhanced c-fos staining of these neurons –an indicator of increased cell activity, and of noradrenergic neurons of the locus coeruleus nucleus, a region downstream of orexinergic activation. After fear conditioning, Firoc mice developed PTSD-like behavioral traits such as prolonged context-dependent fear and post-stress anhedonia. Since abnormal fear learning was ameliorated by chemogenetic (DREADD) inhibition of orexin neurons, reduced IGF-I input to orexin neurons in Firoc mice seems to enhance their excitability to fear-related inputs. Collectively, these results suggest that IGF-I input to orexin neurons is an important determinant of vulnerability to stress disorders, which provides additional targets for therapy of these high social impact conditions.

Although sleep disturbances are common co-morbidities of metabolic diseases, the underlying processes linking both are not yet fully defined. Changes in the duration of sleep are paralleled by changes in the levels of insulin-like growth factor-I (IGF-I), an anabolic hormone that shows a circadian pattern in the circulation and activity-dependent entrance in the brain. However, the specific role, if any, of IGF-I in this universal homeostatic process remains poorly understood. We now report that the activity of orexin neurons, a discrete cell population in the lateral hypothalamus that is involved in the circadian sleep/wake cycle and arousal, is modulated by IGF-I. Furthermore, mice with blunted IGF-I receptor activity in orexin neurons have lower levels of orexin in the hypothalamus, show altered electro-corticographic patterns with predominant slow wave activity, and reduced onset-sleep latency. Collectively, these results extend the role in the brain of this pleiotropic growth factor to shaping sleep architecture through the regulation of orexin neurons. We speculate that poor sleep quality associated to diverse conditions may be related to disturbed brain IGF-I input to orexin neurons., This work was funded by a grant from Ciberned, and from SAF2016-76462 (AEI/FEDER; MINECO). JA Zegarra-Valdivia acknowledges the financial support of the National Council of Science, Technology and Technological Innovation (CONCYTEC, Perú) through the National Fund for Scientific and Technological Development (FONDECYT, Perú). We are thankful to M. Garcia for technical support.

Obesity is a risk factor for Alzheimer&rsquo, s disease (AD), but underlying mechanisms are not clear. We analyzed peripheral clearance of amyloid &beta, (A&beta, ) in overweight mice because its systemic elimination may impact brain A&beta, load, a major landmark of AD pathology. We also analyzed whether circulating insulin-like growth factor I (IGF-I) intervenes in the effects of overweight as this growth factor modulates brain A&beta, clearance and is increased in the serum of overweight mice. Overweight mice showed increased A&beta, accumulation by the liver, the major site of elimination of systemic A&beta, but unaltered brain A&beta, levels. We also found that A&beta, accumulation by hepatocytes is stimulated by IGF-I, and that mice with low serum IGF-I levels show reduced liver A&beta, accumulation&mdash, ameliorated by IGF-I administration, and unchanged brain A&beta, levels. In the brain, IGF-I favored the association of its receptor (IGF-IR) with the A&beta, precursor protein (APP), and at the same time, stimulated non-amyloidogenic processing of APP in astrocytes, as indicated by an increased sAPP&alpha, /sAPP&beta, ratio after IGF-I treatment. Since serum IGF-I enters into the brain in an activity-dependent manner, we analyzed in overweight mice the effect of brain activation by environmental enrichment (EE) on brain IGF-IR phosphorylation and its association to APP, as a readout of IGF-I activity. After EE, significantly reduced brain IGF-IR phosphorylation and APP/IGF-IR association were found in overweight mice as compared to lean controls. Collectively, these results indicate that a high-fat diet influences peripheral clearance of A&beta, without affecting brain A&beta, load. Increased serum IGF-I likely contributes to enhanced peripheral A&beta, clearance in overweight mice, without affecting brain A&beta, load probably because its brain entrance is reduced.

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long-term cultures, these cells develop neurochemical and synaptic markers of mature-like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin-I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH- and GAD-immunoreactive indicating that they are dopaminergic-GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine D1 receptor agonist SKF-38393 induced c-Fos in TH(+) cells and dopamine release was detected in these cultures under basal and KCl-evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH-positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.

The neurons in the olfactory bulb originate from molecularly defined and spatially distinct proliferative regions. Glutamatergic projection neurons are generated during the embryonic period in the local ventricular zone of the olfactory bulb, a territory in the dorsal telencephalon in which the transcription factor Pax6 is expressed. Some cells in this zone also express Tbr1, a marker of glutamatergic neurons. By contrast, embryonic olfactory bulb interneurons are derived from Gsx2 expressing cells in the dorsal lateral ganglionic eminence of the ventral telencephalon, and from progenitors outside the dorsal lateral ganglionic eminence, including the olfactory bulb neuroepithelium. Postnatally, interneurons arise from the subventricular zone of the lateral ventricle, although the rostral migratory stream and the olfactory bulb also appear to serve as a source of neurons. Transcription factors are crucial to generate all classes of neurons and glia in the olfactory bulb, both during development and adulthood. In this article, we discuss and propose models on how the spatial and temporal regulation of transcription factor expression controls the self-renewal, proliferation and cell fate of neural stem cells and progenitors, which finally leads to the generation of distinct functional subtypes of neurons in the developing and adult olfactory bulb.

A study was performed to evaluate porcine torovirus (PToV) seroprevalence and infection in three multi-site farms from the North-eastern region of Spain. Serum samples from 120 piglets and faecal samples from 36 piglets were longitudinally collected at 1, 3, 7, 11 and 15 weeks of age. Serum samples from their dams (n = 30) were also taken 1-week post-farrowing. PToV antibodies in serum were monitored by ELISA, while viral infection was assessed by real-time RT-PCR in faeces. A high seroprevalence (about 100%) was observed in animals older than 11 weeks and in adult sows. Moreover, all 1-week-old animals were seropositive, indicating maternal antibody transference through colostrum. The antibody titers declined to close to or below the ELISA cut-off value by the age of weaning (3 weeks of age). Development of a significant antibody response to PToV occurred before 7 weeks of age in about 50% of piglets, and the remaining animals developed the response by weeks 11 or 15. These results indicate that PToV infection occurred soon after weaning. Although the prevalence of infection in suckling piglets varied among the studied farms, PToV prevalences in 7 and 11-week-old pigs were between 50–67% and 58–75%, respectively, in all farms. Sequencing results indicated that more than one PToV strains were circulating in the studied farms. Present data suggest that PToV was endemic on the studied farms, and provide new insights on the epidemiology of PToV.

Toroviruses are emergent viruses, belonging to the Nidovirales order, that remain mostly ignored, despite they are able to infect different species of domestic animals and humans, causing enteric diseases and diarrhea. Thus far, only five variants of porcine torovirus (PToV) have been identified. In this report we describe the identification and partial characterization of a new strain of porcine torovirus (PToV-BRES) that was detected by RT-PCR in a swine faecal specimen from a farm in Brescia (Italy). The complete genes coding for the nucleocapsid (N), hemagglutinin-esterase (HE) and membrane (M) proteins were amplified, and sequence analysis showed that PToV-BRES is a new PToV strain that, based on the HE gene sequence, is phylogenetically related to P4 strain, that was up to now the only member of a distinct PToV lineage. The nucleocapsid protein from PToV-BRES was expressed in insect cells as a his-tagged protein, purified by affinity chromatography and used to develop an ELISA method to detect antibodies against PToV. This assay was evaluated using a serum collection including 45 samples from three commercial farms from Spain. High antibody prevalence against PToV was observed in the three farms, both in adult animals and in piglets, which could suggest that PToV might be endemic in Spanish porcine population. The ELISA method developed in this work could be useful in future epidemiological surveys about toroviruses.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α+ in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103+ DCs and the human CD141+ DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8a. This CD8α+ MHC II+ DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8+ DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DClike subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved.

Tetraspanins are a family of membrane-organizing proteins, characterized by the presence of four highly conserved transmembrane regions that mediate diverse physiological functions. In the current study, we have identified two novel tetraspanin members in rainbow trout (Oncorhynchus mykiss), homologs to mammalian CD9 and CD63. Both genes were expressed in muscle, skin, gills, hindgut, gonad, liver, spleen, head kidney, thymus and peripheral blood leukocytes. Throughout the early life cycle stages, CD9 mRNA levels significantly increased after first feeding, whereas CD63 transcription remained constant during all the developmental stages analyzed. In response to an experimental bath infection with viral hemorrhagic septicemia virus (VHSV), CD9 transcription was down-regulated in the gills, while CD63 mRNA levels were down-regulated in the head kidney. Instead, when the virus was intraperitoneally injected, the transcription of both genes was significantly up-regulated in peritoneal cells at several days post-infection. Additionally, both genes were transcriptionally up-regulated in the muscle of trout injected with a VHSV DNA vaccine. To gain insight on the relation of these tetraspanins with B cell activity we determined their constitutive expression in naive IgM+ populations from different sources and observed that both molecules were being transcribed by IgM+ cells in different tissues. Furthermore, CD9 transcription was significantly down-regulated in splenic IgM+ cells in response to in vitro VHSV exposure. Our results provide insights on the potential role of these tetraspanins on teleost B cell and antiviral immunity. © 2015 Elsevier Ltd.

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyer's patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem) CCR7 were recorded in the gill (7.5 6 2% CCR7+ cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed

The immune response of the adipose tissue (AT) has been neglected in most animal models until recently, when the observations made in human and mice linking obesity to chronic inflammation and diabetes highlighted an important immune component of this tissue. In the current study, we have immunologically characterized the AT for the first time in teleosts. We have analyzed the capacity of rainbow trout (Oncorhynchus mykiss) AT to produce different immune mediators and we have identified the presence of local populations of B lymphocytes expressing IgM, IgD or IgT, CD8α+ cells and cells expressing major histocompatibility complex II (MHC-II). Because trout AT retained antigens from the peritoneal cavity, we analyzed the effects of intraperitoneal infection with viral hemorrhagic septicemia virus (VHSV) on AT functionality. A wide range of secreted immune factors were modulated within the AT in response to VHSV. Furthermore, the viral infection provoked a significant decrease in the number of IgM+ cells which, along with an increased secretion of IgM in the tissue, suggested a differentiation of B cells into plasmablasts. The virus also increased the number of CD8α+ cells in the AT. Finally, when a fat-enriched diet was fed to the fish, a significant modulation of immune gene expression in the AT was also observed. Thus, we have demonstrated for the first time in teleost that the AT functions as a relevant immune tissue; responsive to peritoneal viral infections and that this immune response can be modulated by the fat-content in the diet. © 2014 Pignatelli et al.

The transcription factor Nurr1 is expressed in the mouse olfactory bulb (OB), although it remains unknown whether it influences the generation of dopaminergic neurons (DA) (DA neurons) in cells isolated from this brain region. We found that expressing Nurr1 in proliferating olfactory bulb stem cells (OBSCs) produces a marked inhibition of cell proliferation and the generation of immature neurons immunoreactive for tyrosine hydroxylase (TH) concomitant with marked upregulations of Th, Dat, Gad, and Fgfr2 transcripts. In long-term cultures, these cells develop neurochemical and synaptic markers of mature-like mesencephalic DA neurons, expressing GIRK2, VMAT2, DAT, calretinin, calbindin, synapsin-I, and SV2. Concurring with the increase in both Th and Gad expression, a subpopulation of induced cells was both TH- and GAD- immunoreactive indicating that they are dopaminergic- GABAergic neurons. Indeed, these cells could mature to express VGAT, suggesting they can uptake and store GABA in vesicles. Remarkably, the dopamine Dl receptor agonist SKF-38393 induced c-Fos in TH(+) cells and dopamine release was detected in these cultures under basal and KCl-evoked conditions. By contrast, cotransducing the Neurogenin2 and Nurr1 transcription factors produced a significant decrease in the number of TH- positive neurons. Our results indicate that Nurr1 overexpression in OBSCs induces the formation of two populations of mature dopaminergic neurons with features of the ventral mesencephalon or of the OB, capable of responding to functional dopaminergic stimuli and of releasing dopamine. They also suggest that the accumulation of Fgfr2 by Nurr1 in OBSCs may be involved in the generation of DA neurons.

Two major classes of B lymphocytes have been described to date in rainbow trout IgM+ and IgT+ cells. IgM+ cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM+ cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM+ lymphocytes from different rainbow trout tissues. IgM+ populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcRβ. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM + populations. The relevant differences in transcriptional patterns observed for each of these IgM+ populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM + B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM+ cells suggests an important role for these cells in innate immunity. © 2013 Abós et al.

Hemagglutinin-esterases (HE) are viral envelope proteins present in some members from the toro-, corona- and orthomyxovirus families, all related with enteric and/or respiratory tract infections. HE proteins mediate reversible binding to sialic acid receptor determinants, very abundant glycan residues in the enteric and respiratory tracts. The role of the HE protein during the torovirus infection cycle remains unknown, although it is believed to be important in the natural infection process. The phylogenetic analysis of HE coding sequences from porcine torovirus (PToV) field strains revealed the existence of two distinct HE lineages. In a previous study, PToV virus strains with HE proteins from the two lineages were found coexisting in a pig herd, and they were even obtained from the same animal at two consecutive sampling time points. In this work, we report antigenic differences between the two HE lineages, and discuss the possible implications that the coexistence of viruses belonging to both lineages might have on the spread and sustainment of PToV infection in the farms., This work was supported by grants AGL2010-15495 and CONSOLIDER-PORCIVIR CSD2006-00007 from the Spanish Ministry of Science and Innovation. Jaime Pignatelli and Julio Alonso-Padilla were both recipients of contracts financed with founding from the CONSOLIDER-PORCIVIR research project.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleost has only been briefly addressed and the identification of a specific DC subset in teleost remained elusive due to the lack of specific antibodies. In mice, CD8 DCs from lymphoid tissues have the capacity of cross-presenting extracellular antigens to T cells through MHC I, similarly to tissue derived CD103 DCs and the human CD141 DC population. In the current study, we have identified in trout a subpopulation of leukocytes of large size and high complexity co-expressing MHC II, CD8CD103 and CD141. These cells, mostly present in mucosal tissues, exhibit functional characteristics of DCs and thus provide the first evidence of a specific DC-like subtype in teleost and support the hypothesis of a common origin for all mammalian antigen cross-presenting cells. In the current study, we have compared the functional and phenotypical characteristics of these DCs in different mucosal tissues (skin, gills and intestine), analyzing their phagocytic ability, their T cell activating capacity, their responsiveness to TLR ligands and the expression of DC-specific markers. Interestingly, this DC subtype exhibited specific phenotypes and functionalities in skin, gills and intestine, suggesting important differences in their activation state depending on their location. These results shed light on how a specific subset of mucosa-resident DCs triggers local responses in the different fish mucosal tissues.

Background Torovirus infections have been associated with gastroenteritis and diarrhea in horses, cows, pigs and humans, especially in young animals and in children. Although asymptomatic in a large percentage of cases, however toroviruses may pose a potential threat to worsen disease outcome in concurrent infections with other enteric pathogens. Previous studies based on the analysis of limited numbers of samples indicated high seroprevalences against porcine torovirus (PToV) in various European countries. The aim of this work was to perform a seroepidemiological survey of PToV in Spanish farms in order to define the seroprevalence against this virus. Results Serum samples (n = 2664) from pigs of different ages were collected from 100 Spanish farms coming from 10 regions that concentrate 96.1% of the 3392 farms with 80 or more sows censused in Spain. Samples were screened by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant PToV nucleocapsid protein as antigen. The analysis of the whole serum collection yielded a total of 95.7% (2550/2664) seropositive samples. The highest prevalence (99.6%, 1382/1388) and ELISA values (average O.D. ± standard deviation) were observed in the sows (1.03±0.36) and the lowest prevalence (59.4%, 98/165) and anti-PToV IgG levels (0.45±0.16) were found amongst 3-week-old piglets. Both ELISA reactivity values and seroprevalence percentages rose quickly with piglet’s age from 3 to 11 weeks of age; the seroprevalence was 99.3% (2254/2270) when only the samples from sows and pigs over 11-weeks of age were considered. Antibodies against PToV were detected in all analyzed farms. Conclusions This report describes the results of the largest torovirus seroepidemiological survey in farmed swine performed so far. Overall, the seroprevalence against PToV in animals older than 11 weeks of age was >99%, indicating that this virus is endemic in pig herds from Spain., This work was supported by grants from the Spanish Ministry of Science and Technology AGL2010-15495 to D.R. and CONSOLIDER-PORCIVIR CSD2006-00007 to DR and JC. JAP and SPC were recipients of contracts financed with funding from the CONSOLIDER-PORCIVIR research project.

The neurons in the olfactory bulb originate from molecularly defined and spatially distinct proliferative regions. Glutamatergic projection neurons are generated during the embryonic period in the local ventricular zone of the olfactory bulb, a territory in the dorsal telencephalon in which the transcription factor Pax6 is expressed. Some cells in this zone also express Tbr1, a marker of glutamatergic neurons. By contrast, embryonic olfactory bulb interneurons are derived from Gsx2 expressing cells in the dorsal lateral ganglionic eminence of the ventral telencephalon, and from progenitors outside the dorsal lateral ganglionic eminence, including the olfactory bulb neuroepithelium. Postnatally, interneurons arise from the subventricular zone of the lateral ventricle, although the rostral migratory stream and the olfactory bulb also appear to serve as a source of neurons. Transcription factors are crucial to generate all classes of neurons and glia in the olfactory bulb, both during development and adulthood. In this article, we discuss and propose models on how the spatial and temporal regulation of transcription factor expression controls the self-renewal, proliferation and cell fate of neural stem cells and progenitors, which finally leads to the generation of distinct functional subtypes of neurons in the developing and adult olfactory bulb.