Extinction training suppresses activity of fear memory ensembles across the hippocampus and alters transcriptomes of fear-encoding cells.

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells-the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

Extinction Training Suppresses Activity of Fear Memory Ensembles Across the Hippocampus and Alters Transcriptomes of Fear-Encoding Cells.

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells - the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

Cluster analysis of bovine respiratory disease (BRD)-associated pathogens shows the existence of two epidemiological patterns in BRD outbreaks.

A hierarchical cluster analysis was used to classify outbreaks of bovine respiratory disease (BRD; n = 156) in natural groups according to the detection of nine pathogens (parainfluenza 3 virus (PI-3), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BHV-1), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Pathogens were detected by individual q-PCRs. Two clusters were identified. Cluster 1 was characterized by a relatively high frequency (40-72%) of four BRD-associated viruses, supporting their primary involvement in BRD. Cluster 2 was characterized by frequencies of PI-3, BRSV, or BVDV below 10% each. P. multocida and M. haemolytica were detected with high frequencies in both clusters (P > 0.05), while M. bovis and H. somni showed a significantly higher frequency in cluster 1and 2, respectively. Outbreaks in cluster 1 were associated with preweaning calves younger than 5 months (OR 2.2; 95% CI 1.1-4.5) and with cold months, whereas cluster 2 was associated with fattening calves older than 5 months after arrival to feedlots and without any seasonality. Thus, in addition to the classic epidemiological BRD pattern characterized by the primary involvement of viruses occurring preferably during winter and affecting young calves, there is a second pattern in which viruses would be less relevant, affecting mainly calves older than 5 months at any time of the year. This study allows a better understanding of the BRD epidemiology, which can be useful when implementing management and prophylaxis measures for a better control of this disease.

Vesicular glutamate transporter 2-containing neurons of the centrally-projecting Edinger-Westphal nucleus regulate alcohol drinking and body temperature.

Previous studies in rodents have repeatedly demonstrated that the centrally-projecting Edinger-Westphal nucleus (EWcp) is highly sensitive to alcohol and is also involved in regulating alcohol intake and body temperature. Historically, the EWcp has been known as the main site of Urocortin 1 (Ucn1) expression, a corticotropin-releasing factor-related peptide, in the brain. However, the EWcp also contains other populations of neurons, including neurons that express the vesicular glutamate transporter 2 (Vglut2). Here we transduced the EWcp with adeno-associated viruses (AAVs) encoding Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to test the role of the EWcp in alcohol drinking and in the regulation of body temperature. Activation of the EWcp with excitatory DREADDs inhibited alcohol intake in a 2-bottle choice procedure in male C57BL/6J mice, whereas inhibition of the EWcp with DREADDs had no effect. Surprisingly, analysis of DREADD expression indicated Ucn1-containing neurons of the EWcp did not express DREADDs. In contrast, AAVs transduced non-Ucn1-containing EWcp neurons. Subsequent experiments showed that the inhibitory effect of EWcp activation on alcohol intake was also present in male Ucn1 KO mice, suggesting that a Ucn1-devoid population of EWcp regulates alcohol intake. A final set of chemogenetic experiments showed that activation of Vglut2-expressing EWcp neurons inhibited alcohol intake and induced hypothermia in male and female mice. These studies expand on previous literature by indicating that a glutamatergic, Ucn1-devoid subpopulation of the EWcp regulates alcohol consumption and body temperature.

Involvement of Centrally Projecting Edinger-Westphal Nucleus Neuropeptides in Actions of Addictive Drugs.

The centrally-projecting Edinger-Westphal nucleus (EWcp) is a brain region distinct from the preganglionic Edinger-Westphal nucleus (EWpg). In contrast to the EWpg, the EWcp does not send projections to the ciliary ganglion and appears not to regulate oculomotor function. Instead, evidence is accumulating that the EWcp is extremely sensitive to alcohol and several other drugs of abuse. Studies using surgical, genetic knockout, and shRNA approaches further implicate the EWcp in the regulation of alcohol sensitivity and self-administration. The EWcp is also known as the site of preferential expression of urocortin 1, a peptide of the corticotropin-releasing factor family. However, neuroanatomical data indicate that the EWcp is not a monotypic brain region and consists of several distinct subpopulations of neurons. It is most likely that these subpopulations of the EWcp are differentially involved in the regulation of actions of addictive drugs. This review summarizes and analyzes the current literature of the EWcp's involvement in actions of drugs of abuse in male and female subjects in light of the accumulating evidence of complexities of this brain region.

Effects of pharmacological inhibition of the centrally-projecting Edinger-Westphal nucleus on ethanol-induced conditioned place preference and body temperature.

Alcohol use disorder is a chronic disease characterized in part by repeated relapsing events. Exposure to environmental stimuli or cues that have previously been associated with the effects of alcohol can promote relapse through the triggering of craving for alcohol. Therefore, identifying and characterizing neuronal populations that may regulate these associations is of the upmost importance. Previous studies have implicated the centrally-projecting Edinger Westphal nucleus (EWcp) in this process, as the EWcp is both sensitive to, and can regulate alcohol intake. To date however, it is unclear if the EWcp is involved in the formation or expression of these alcohol-cue associations. As such, the present studies examined the involvement of the EWcp in male DBA/2J mice in the acquisition and expression of place preference for an alcohol-paired cue using the conditioned place preference (CPP) procedure. Pharmacological inhibition of the EWcp via the GABAA and GABAB receptor agonists muscimol and baclofen did not affect either the acquisition or the expression of CPP. Follow up studies did find however, that pharmacological inhibition of the EWcp increased body temperature and prevented alcohol-induced increases in c-Fos expression in the EWcp. When considered in light of previous studies, the present results indicate that the EWcp may be involved in the regulation of alcohol self-administration, and not conditioned alcohol-seeking. Additionally, the present studies provide further evidence for the involvement of the EWcp in thermoregulation and help elucidate the molecular mechanisms by which alcohol increases c-Fos in the EWcp.

Rewarding and aversive doses of caffeine alter activity but not conditioned place preference induced by ethanol in DBA/2J mice.

Increases in the consumption of ethanol and caffeine have been attributed to increased subjective feelings of intoxication and pleasure from the combination. Previous studies have shown that caffeine can be rewarding at low doses and aversive at high doses, although these findings are at times inconsistent between studies using comparable doses. Similarly, studies investigating the rewarding effects of ethanol and caffeine combinations have yielded mixed results. To address this issue, the present experiments were designed to investigate the rewarding effects of caffeine, as well as of caffeine + ethanol combinations. Male DBA/2J mice were exposed to an unbiased conditioned place preference (CPP) procedure with various doses of caffeine (1, 3, 10, 30 mg/kg) and ethanol (1, 2 g/kg), as well as various conditioning trial durations (5, 30, 60 min). Caffeine dose-dependently increased locomotor activity during conditioning, and produced a biphasic effect on place conditioning. Specifically, a low dose of caffeine (3 mg/kg) produced place preference, while a high dose (30 mg/kg) produced place aversion. When combined with alcohol, caffeine dose-dependently increased ethanol's stimulatory effect. However, the addition of caffeine had no effect on ethanol place preference, as there were no differences in the strength of place preference between mice conditioned with ethanol alone, and mice conditioned with any combination of ethanol and caffeine. These studies add evidence for caffeine's biphasic effects while also emphasizing the importance of considering temporal and methodological parameters when using Pavlovian conditioning procedures to study drug combinations.

Diet, dietary selectivity and density of South American grey fox, Lycalopex griseus, in Central Chile.

The South American grey fox Lycalopex griseus is a canid widely distributed in southern South America; however, some aspects of its biology are still poorly known. We studied the diet and density of L. griseus in the Lago Peñuelas Biosphere Reserve, in Central Chile. The trophic niche breadth was B = 6.16 (Bsta = 0.47) and prey diversity was H' = 2.46 (Hmax ' = 3.17, J' = 0.78). The highest proportions of prey consumed in the diet were Oryctolagus cuniculus (52.21%) and other mammals (32.78%). We compared these results with a latitudinal gradient of diet results for this species in Chile. L. griseus eats mostly mammals (>90% of total prey), consuming the rodent Phyllotis darwini and reptiles in the northern zone; Oryctolagus cuniculus, Octodon degus and Abrocoma bennetti in the central zone; Abrothrix spp. and lagomorphs in the southern zone; and Lepus capensis and Ovis aries in the austral zone. The estimated density of L. griseus in Lago Peñuelas NR was 1.3 foxes/km2 .

The Microtubule Regulatory Protein Stathmin Is Required to Maintain the Integrity of Axonal Microtubules in Drosophila.

Axonal transport, a form of long-distance, bi-directional intracellular transport that occurs between the cell body and synaptic terminal, is critical in maintaining the function and viability of neurons. We have identified a requirement for the stathmin (stai) gene in the maintenance of axonal microtubules and regulation of axonal transport in Drosophila. The stai gene encodes a cytosolic phosphoprotein that regulates microtubule dynamics by partitioning tubulin dimers between pools of soluble tubulin and polymerized microtubules, and by directly binding to microtubules and promoting depolymerization. Analysis of stai function in Drosophila, which has a single stai gene, circumvents potential complications with studies performed in vertebrate systems in which mutant phenotypes may be compensated by genetic redundancy of other members of the stai gene family. This has allowed us to identify an essential function for stai in the maintenance of the integrity of axonal microtubules. In addition to the severe disruption in the abundance and architecture of microtubules in the axons of stai mutant Drosophila, we also observe additional neurological phenotypes associated with loss of stai function including a posterior paralysis and tail-flip phenotype in third instar larvae, aberrant accumulation of transported membranous organelles in stai deficient axons, a progressive bang-sensitive response to mechanical stimulation reminiscent of the class of Drosophila mutants used to model human epileptic seizures, and a reduced adult lifespan. Reductions in the levels of Kinesin-1, the primary anterograde motor in axonal transport, enhance these phenotypes. Collectively, our results indicate that stai has an important role in neuronal function, likely through the maintenance of microtubule integrity in the axons of nerves of the peripheral nervous system necessary to support and sustain long-distance axonal transport.

Seroprevalencia del virus de larinotraqueitis infecciosa bovina en ganado lechero del valle de Lima / Seroprevalence of infectious bovine larinotraqueitis virus in dairy cattle in the valley of Lima

The seroprevalence of bovine herpes virus type 1 (BHV-1), the causative agent of Infections Bovine Rhinotracheitis (IBR) was determined in non-vaccinated bovines older than 6 months of age and reared in 12 dairy farms in the valley of Lima, Peru. Blood samples were collected from 395 animals and assayed for antibodies to BHV-1 by using virus neutralization test. Antibodies against BHV-1 were detected in 26 more less 0.47% (143/395) of the animals with titers between 2 and >256. Sixty seven per cent (8/12) of herds had seropositive animals. The higher prevalence was found in herds with more than 300 animals and in herds located in the north and south of Lima, and in those animals older than 2 years. These results confirmed the wide distribution of BHV-1 in the Lima valley, despite of the absence of clinical signs. Since farmers have indicated to have observed respiratory diseases in young animals, the BHV-1 would be associated to the bovine respiratory complex in calves.

El presente estudio tuvo como objetivo determinar la seroprevalencia del virus herpes bovino tipo 1 (VHB-1), agente causal de la Rinotraqueitis Infecciosa Bovina (RIB), en bovinos mayores de 6 meses de edad, procedentes de 12 hatos lecheros del valle de Lima y sin historia de vacunación. Se tomó muestras de sangre en 395 hembras para la detección de anticuerpo neutralizantes en suero mediante la prueba de neutralización viral. El 36 más menos 0.47% (143/395) de los animales presentaron anticuerpos neutralizantes contra el VHB-1 con títulos entre 2 a >256. El 67% (8/12) de los hatos muestreados tuvieron animales serorreactores. Las mayores prevalencias se presentaron en hatos con >300 animales, en hatos ubicados en el norte y sur del valle de Lima y en animales mayores de 2 años de edad. Estos resultados confirman que el VHB-1 está difundido en el valle de Lima a pesar que no se obtuvo evidencias clínicas de la IRB. Sin embargo, considerando que los ganadores manifestaron observar problemas respiratorios en animales jóvenes, esto podría ser indicativo de que el virus estaría asociado al complejo respiratorio bovino.

Prevalencia del virus de la diarrea viral bovina y animales portadores del virus en un hato lechero del Valle del Mantaro / Prevalence of bovine viral diarrhea virus and persintently infected in a diary herd at the Mantaro Valley

The objective of this study was to determine the prevalence of BVDV and persistently infected animals in a dairy herd located at the Mantaro valley in Junin, Peru. For this purpose, blood samples were taken from all animals older than 6 months (n=127) for detection of antibodies against BVDV by viral neutralization test (VN). Six months later, blood samples with and without heparine were taken from animals that scored negative for antibodies against BVDV, for the detection of BVDV antibodies and by VN and antigen capture ELISA tests. The 70,9 more and less 7,8% (90/127) of the animals had antibodies against BVDV. The highest prevalence (94,2%) was detected in cows older than two years followed by heifers (46,3%) and calves older than 6 months of age (35,3%). The antibody titers ranged from 2 to > 256. Six months later, 90.2% (55/61) of the animals with no antibodies against BVDV in the first sampling plus those animals that has reached sin month of age and were not sampled earlier, remained negative. The prevalence of persistently infected animals was 1,8% (1/55) in the group of antibody-negative animals and 0,74 (0,04-2,3) (1/135) for the population as a whole. The high prevalence of BVDV in the herd suggests this PI animal could have been an important source of viral transmission in the cattle herd as it is mentioned in the literature.

El objetivo del presente trabajo fue determinar la prevalencia del virus de la diarrea viral bovina (VDVB) y de animales persistentemente infectados (PI) con el VDBV en un hato lechero del valle del Mantaro. Se obtuvieron muestras de suero de los animales mayores a 6 meses de edad (n=127) para la detección de anticuerpos neutralizantes contra el VDVB mediante la prueba de neutralización viral. Seis meses después se colectó una segunda muestra de sangre con y sin anticoagulante para la detección de anticuerpos y antígeno a los animales que resultaron negativos a anticuerpos en el primer muestreo y a los terneros que hasta la fecha habían cumplido 6 meses de edad. El 70,9 más menos 7,8% (90/127) de los animales tuvo anticuerpos neutralizantes contra el VDVB. El virus tuvo mayor prevalencia en el grupo de vacas (94,2%) seguido por vaquillas (46,3%) y terneras mayores a 6 meses (35,3%). Los títulos de anticuerpos contra el VDVB tuvieron un rango de 2 a >256. Seis meses después, el 90,2% (55/61) de los animales negativos a anticuerpos, incluidos los terneros que habían alcanzado 6 meses de edad y que no fueron muestreados en la primera vez, permanecieron sin anticuerpos contra VDVB. La prevalencia de animales PI en el grupo de animales negativos a anticuerpos fue de 1,8% (1/55) significando para toda la población del hato 0,74 con intervalos entre 0,04 a 2,3% (1/135), la alta prevalencia del VDVB en el hato pudo deberse a la presencia del animal PI como se indica en la literatura, puesto que estos animales son los principales diseminadores del virus.

Terneros persistentemente infectados con el virus de la diarrea bovina en dos hatos lecheros de la provincia de Arequipa / Persistently infected calves with bovine virus diarrhea virus in two dairy herds in Arequipa

The objective of the present study was to identify persistently infected (PI) calves with bovine viral diarrhea virus (BVDV) in two dairy farms located in the Santa Rita (Farm A) and Vitor (Farm B) valleys in the province of Arequipa, Peru. Blood samples with EDTA were collected from calves of farm A (n=36) and B (n=95) soon after birth and before the ingestion of colostrums for the detection of BVDB by capture antigen ELISA test. One PO calf was detected in farm A. the prevalence of PI calves in farm A was 2,78% (1/36) and 0,76 with the intervals between 0,024 to 3,5% (1/131) for the total of calves studied. These results indicate that the PI animal could be detected soon after birth and before the intake of colostrums reducing the risk of spreading the infection to susceptible animals and increasing the cost of maintenance of those calves for six months or more.

El presente estudio tuvo como objetivo identificar terneros persistentemente infectados (PI) con el virus de la diarrea viral bovina (VDVB), en dos establos lecheros de crianza intensiva ubicados en las irrigaciones de Santa Rita de Siguas (Establo A) y Vitor (Establo B) de la cuenca lechera de Arequipa, Perú. Se obtuvieron muestras de sangre con anticoagulante (EDTA) de terneros de los establos A (n=36) y B (n=95) poco después de nacer y antes que tomen el calostro para la detección del VDVB mediante la prueba de ELISA de captura de antígeno. Un ternero con infección congénita o persistente fue identificado en el establo A, significando una prevalencia de 2,78% (1/36) de terneros PI en ese establo y 0,76 con intervalos entre 0,024 a 3,5 (1/131) de terneros PI del total de muestreados. Los resultados indican que los teneros PI pueden ser detectados antes que tomen el calostro evitando la difusión de virus en el hato y el costo adicional de mantenimiento de los terneros por 6 o más meses, y que finalmente deben ser beneficiados.

Detección de animales portadores del virus de la diarrea viral bovina en un establo vacunado y en otro sin vacunar del valle de Lima / Detection of persistently infected animls with bovine viral diarrhea virus in a vaccinated and a non-vaccinated farm in the Lima valley

The objective of this study was to identify persistently infected animals (PI) in two dairy farms with reproductive problems and increased neonatal mortality. Farm A had a vaccination programme against Bovine Viral Diarrhea Virus (BVDV) and farm B did not vaccinate. Both farms were located in the valley of Lima, Peru. Blood samples were taken from 6-24 month old heifers in farm A (n=105) and B (n=103) to detect antibodies against BVDV through virus neutralization test. Additional blood samples were taken from those heifers without antibodies against BVDV for virus isolation in cell culture system. In addition, antibodies against BVDV were evaluated before vaccination and 15 and 45 days post vaccination in a group of farm A. antibodies against BVDV were not detected in heifers of farm B. in farm A, 67.62 more less 8.95% (71/105) of vaccinated animals had antibodies whereas 32,4 more less 8,95% (34/105) did not have. Among those without antibodies, 11,76 more less 10,83% (4/34) were identified as PI animals, which means a 3,81 more less 3,66% (4/105) of prevalence of PI animals in farm A. some of the animals of these group seroconverted to BVDV, others remained negative but they were not PI at 45 days post vaccination. The presence of PI animals in farm A that uses an inactivated vaccine for BVDV control evidenced failure of BVD control. The absence of antibodies against BVDV in farm B, suggests that BVD control is possible without vaccination but with a good management and biosecurity programme.

El presente trabajo tuvo por objetivo detectar la prevalencia de animales portadores en un grupo de vaquillonas de 6 a 24 meses de edad procedentes de dos establos lecheros de crianza intensiva de Lima con problemas reproductivos e incrementada mortalidad neonatal. El establo A tenía un programa de vacunación contra la diarrea viral bovina (DVB) y el establo (B) sin programa de vacunación contra la DVB. Se obtuvieron muestras de sangre de los animales de los establos A (n=105) y B (n=103) para la detección de anticuerpos contra la VDVB mediante la prueba de neutralización viral. Adicionalmente se obtuvieron muestras de sangre de los animales negativos a anticuerpos contra el VDVB para la detección del virus mediante las pruebas de aislamiento viral en cultivo celular e inmunoperoxidasa. No se detectaron anticuerpos contra el VDVB en los animales del establo B. Unicamente el 67,32 más menos 8.95% (71/105) de los animales vacunados del establo A presentó anticuerpos contra el VDVB en tanto que no se detectaron en el 32,38 más menos 8.95% (34/105). De los 34 animales seronegativos, el 11,76 más menos 10,83 (4/34) fue positivo al VDVB mediante el aislamiento viral confirmándose que estos animales fueron portadores del virus. Esto representó una prevalencia de 3,81 más menos 3,66% (4/105) de animales con títulos de anticuerpos en rangos de a > 256 antes de la primera vacunación. Así mismo, los animales vacunados tuvieron amplia variación en los títulos de anticuerpos e inclusive hubieron animales que hasta los 45 días post vacunación no seroconvirtieron pero tampoco fueron animales portadores. La presencia de animales con infección persistente en el establo A sugiere fallas en la estrategia de control de la DVB, como deficiencias en medidas de bioseguridad, fallan en la identificación y beneficio de los animales portadores, y deficiencias en el manejo de la vacuna.

Estudio serológico de durina y muermo equino / Serological Study dourine and equine glanders

Durine and Glanders are cronic and progressive infectious diseases of horses, donkeys and mules, and human beings in case of glanders. These diseases are caused by a parasite, Trypanosoma equiperdum (T. equiperdum) and a bacteria Burkholderia mallei (B. mallei), respectively. In Peru there are no information about the clinical and serological evidence of durine and glanders. The objective of this study was to detect antibodies against T. equiperdum and B. mallei in serum samples from horses (n=505) of normal appearance, and from different areas of Peru but mainly from Lima by using the complement fixation test. No antibodies against T. equiperdum and B. mallei were detected in the serum samples tested. These results suggest that both diseases are absent or the prevalence are below 1 percent in the studied Peruvian horses.