Disrupted inhibitory plasticity and homeostasis in Fragile X syndrome.

Fragile X Syndrome (FXS) is a neurodevelopmental disorder instigated by the absence of a key translation regulating protein, Fragile X Mental Retardation Protein (FMRP). The loss of FMRP in the CNS leads to abnormal synaptic development, disruption of critical periods of plasticity, and an overall deficiency in proper sensory circuit coding leading to hyperexcitable sensory networks. However, little is known about how this hyperexcitable environment affects inhibitory synaptic plasticity. Here, we show that in vivo layer 2/3 of the primary somatosensory cortex of the Fmr1 KO mouse exhibits basal hyperexcitability and an increase in neuronal firing rate suppression during whisker activation. This aligns with our in vitro data that indicate an increase in GABAergic spontaneous activity, a faulty mGluR-mediated inhibitory input and impaired inhibitory plasticity processes. Specifically, we find that mGluR activation sensitivity is overall diminished in the Fmr1 KO mouse leading to both a decreased spontaneous inhibitory postsynaptic input to principal cells and a disrupted form of inhibitory long-term depression (I-LTD). These data suggest an adaptive mechanism that acts to homeostatically counterbalance the cortical hyperexcitability observed in FXS.

Generation of mice lacking DUF1220 protein domains: effects on fecundity and hyperactivity.

Sequences encoding DUF1220 protein domains show the most extreme human lineage-specific copy number increase of any coding region in the genome and have been linked to human brain evolution. In addition, DUF1220 copy number (dosage) has been implicated in influencing brain size within the human species, both in normal populations and in individuals associated with brain size pathologies (1q21-associated microcephaly and macrocephaly). More recently, increasing dosage of a subtype of DUF1220 has been linked with increasing severity of the primary symptoms of autism. Despite these intriguing associations, a function for these domains has not been described. As a first step in addressing this question, we have developed the first transgenic model of DUF1220 function by removing the single DUF1220 domain (the ancestral form) encoded in the mouse genome. In a hypothesis generating exercise, these mice were evaluated by 197 different phenotype measurements. While resulting DUF1220-minus (KO) mice show no obvious anatomical peculiarities, they exhibit a significantly reduced fecundity (χ(2) = 19.1, df = 2, p = 7.0 × 10(-5)). Further extensive phenotypic analyses suggest hyperactivity (p < 0.05) of DUF1220 mice and changes in gene expression levels of brain associated with distinct neurological functions and disease. Other changes that met statistical significance include an increase in plasma glucose concentration (as measured by area under the curve, AUC 0-30 and AUC 30-120) in male mutants, fasting glucose levels, reduce sodium levels in male mutants, increased levels of the liver functional indicator ALAT/GPT in males, levels of alkaline phosphatase (also an indicator of liver function), mean R and SR amplitude by electrocardiography, elevated IgG3 levels, a reduced ratio of CD4:CD8 cells, and a reduced frequency of T cells; though it should be noted that many of these differences are quite small and require further examination. The linking of DUF1220 loss to a hyperactive phenotype is consistent with separate findings in which DUF1220 over expression results in a down-regulation of mitochondrial function, and potentially suggests a role in developmental metabolism. Finally, the substantially reduced fecundity we observe associated with KO mice argues that the ancestral DUF1220 domain provides an important biological functionthat is critical to survivability and reproductive success.

Decision-Making Time Cells in Hippocampal Dorsal CA1.

Sequential neural dynamics encoded by "time cells" play a crucial role in hippocampal function. However, the role of hippocampal sequential neural dynamics in associative learning is an open question. In this manuscript, we used two-photon Ca2+ imaging of dorsal CA1 pyramidal neurons in head-fixed mice performing a go-no-go associative learning task. We found that pyramidal cells responded differentially to the rewarded or unrewarded stimuli. The stimuli were decoded accurately from the activity of the neuronal ensemble, and accuracy increased substantially as the animal learned to differentiate the stimuli. Decoding the stimulus from individual pyramidal cells that responded differentially revealed that decision-making took place at discrete times after stimulus presentation. Lick prediction decoded from the ensemble activity of cells in dCA1 correlated linearly with lick behavior indicating that sequential activity of pyramidal cells in dCA1 constitutes a temporal memory map used for decision-making in associative learning.

Characterization and application of a coating of starch extracted from avocado (Persea americana L. cv. Hass) seeds as an alternative to reduce acrylamide content in French fries.

The starch extracted from avocado (Persea americana L. cv. Hass) seeds was characterized and used in the preparation of an edible coating to reduce the oil uptake and acrylamide content in French fries. Starch characterization was carried out using Differential Scanning Calorimetry, Fourier transform infrared spectrophotometry, gelatinization, and scanning electron microscopy. Uncoated (UFF) and coated (CFF) French fries were compared and evaluated for moisture, water activity (Aw), fat, color, firmness, acrylamide content, and sensorial analysis. The extracted starch presented a high crystalline structure and good stability to mechanical work and heat treatments. The CFF French fries showed significantly higher Aw, color parameter a*, but lower luminosity and acrylamide content than UFF samples. Similarly, the CFF samples tended to decrease the fat content, although without statistical differences. Avocado seed starch can be an economical and technically feasible alternative to the food industry as an effective coating to reduce acrylamide content in French fries.

Unifying casualty distributions within and across conflicts.

The distribution of whole war sizes and the distribution of event sizes within individual wars, can both be well approximated by power laws where size is measured by the number of fatalities. However the power-law exponent value for whole wars has a substantially smaller magnitude - and hence a flatter distribution - than for individual wars. We provide detailed numerical evidence that confirms that these numerically different power-law exponent values are interrelated in a simple way by the effect of aggregating fatalities from individual events within wars to whole wars. We offer intuition for this finding and hence strengthen the case for a unified description and understanding of human conflict across scales.

Odor stimuli trigger influx of calcium into olfactory neurons of the channel catfish.

Olfactory transduction is thought to be mediated by a G protein-coupled increase in intracellular adenosine 3',5'-monophosphate (cAMP) that triggers the opening of cAMP-gated cation channels and results in depolarization of the plasma membrane of olfactory neurons. In olfactory neurons isolated from the channel catfish, Ictalurus punctatus, stimulation with olfactory stimuli (amino acids) elicits an influx of calcium that leads to a rapid increase in intracellular calcium. In addition, in a reconstitution assay a plasma membrane calcium channel has been identified that is gated by inositol-1,4,5-trisphosphate (IP3), which could mediate this calcium influx. Together with previous studies indicating that stimulation with olfactory stimuli leads to stimulation of phosphoinositide turnover in olfactory cilia, these data suggest that an influx of calcium triggered by odor stimulation of phosphoinositide turnover may be an alternate or additional mechanism of olfactory transduction.

Sobredosis de acetaminofén y comportamientos relacionados con suicidio / Acetaminophen overdose and suicide-related behaviors / Overdose de paracetamol e comportamentos suicidas

Objetivo: este trabajo busca caracterizar el comportamiento relacionado con el suicidio en la población admitida al Hospital San Vicente Fundación, Rionegro, con sobredosis de acetaminofén entre enero 2019 y diciembre 2020 y detectar factores asociados con la dosis tóxica. Metodología: análisis descriptivo con información obtenida de historias clínicas. Resultados: 63 individuos presentaron ingestión aguda de dosis tóxica de acetaminofén como comportamiento relacionado con suicidio. Cuarenta y tres eran mujeres, 60% tenía antecedente de enfermedad psiquiátrica, 35% reportó al menos un intento suicida previo y 22% consumieron 25g o más. La lesión hepática aguda se asoció con una dosis tóxica. Conclusiones: evidenciamos una alta prevalencia de antecedente de enfermedad psi­quiátrica y comportamiento relacionado con suicidio y casi un tercio de los pacientes ingirió dosis mayores al umbral de riesgo para falla hepática. Además, la impulsividad e ingesta en casa sugiere que políticas públicas restrictivas pueden no impactar en la reducción de estos eventos en la población.

Objective: this work seeks to characterize the behavior related to suicide in the po­pulation admitted to the Hospital San Vicente Fundación, Rionegro, with an overdose of acetaminophen between January 2019 and December 2020, and to identify factors associated with the toxic dose. Methodology: descriptive analysis with information obtained from medical records. Results: 63 individuals presented acute ingestion of a toxic dose of acetaminophen as behavior related to suicide. Forty-three were women, 60% had a history of psychiatric illness, 35% reported at least one previous suicide attempt, and 22% consumed 25g or more. Acute liver injury was associated with a toxic dose. Conclusions: we evidenced a high prevalence of a history of psychiatric illness and beha­vior related to suicide; almost a third of the patients ingested doses greater than the risk threshold for liver failure. In addition, impulsiveness and eating at home suggests that res­trictive public policies may not have an impact on reducing these events in the population.

Objetivo: Este trabalho busca caracterizar o comportamento relacionado ao suicídio na população internada no Hospital San Vicente Fundación, Rionegro, com overdose de acetaminofeno entre janeiro de 2019 e dezembro de 2020 e detectar fatores associados à dose tóxica. Metodologia: análise descritiva com informações obtidas dos prontuários. Resultados: 63 indivíduos apresentaram ingestão aguda de dose tóxica de paracetamol como comportamento relacionado ao suicídio. Quarenta e três eram mulheres, 60% tinham histórico de doença psiquiátrica, 35% relataram pelo menos uma tentativa de suicídio anterior e 22% consumiram 25g ou mais. A lesão hepática aguda foi associada a uma dose tóxica. Conclusões: evidenciamos alta prevalência de história de doença psiquiátrica e com-portamento relacionado ao suicídio e quase um terço dos pacientes ingeriu doses superiores ao limiar de risco para insuficiência hepática. Além disso, a impulsividade e a alimentação em casa sugerem que políticas públicas restritivas podem não ter impacto na redução desses eventos na população.

Perfil epidemiológico de pacientes intoxicados con acetaminofén con intenciones suicidas / Epidemiological profile of patients intoxicated with acetaminophen for suicidal purposes / Perfil epidemiológico de pacientes intoxicados com acetaminofen (Paracetamol) com intenções suicidas

Objetivo: este estudio busca describir los individuos evaluados por sobredosis de acetaminofén entre 2019 y 2020 en un centro de referencia de trasplante hepático en Colombia. Metodología: estudio derivado del análisis secundario de historias clínicas entre el 1.º de enero de 2019 y el 31 de diciembre de 2020. Los criterios de inclusión abarcan individuos con ingestión aguda y voluntaria de dosis tóxicas de acetaminofén (>4 g/día). Resultados: sesenta y tres casos, 68% mujeres, 67% menores de 18 años y 54% estudiantes. Reportó historia personal de enfermedad psiquiátrica el 60% y el 35% al menos un intento de suicidio previo. La mediana de dosis de acetaminofén fue 15g, 46% refirieron co-ingesta de otras sustancias y 13% estaba bajo efecto de sustancias psicoactivas. El 57% tenía la intención clara de suicidarse, así como 81% vomitó antes de acudir al servicio de urgencias, 22% recibió medidas de descontaminación y 10% no recibió N - acetilcisteína. Quince individuos desarrollaron lesión hepática aguda, nueve con criterios de severidad. Conclusiones: la población era predominantemente joven, la historia de enfermedad psiquiátrica fue muy prevalente y la mayoría refirieron un evento vital que explicara el comportamiento impulsivo de consumo. Ninguno desarrolló criterios para trasplante hepático, lo cual podría explicarse por la edad de los individuos, los episodios de vómito temprano, y la ausencia de enfermedad hepática crónica o de consumo de sustancias hepatotóxicas.

Objective: this study aims to describe patients with overdose intake of acetaminophen between 2019 and 2020 at a reference center for liver transplantation in Colombia. Methodology: study derived from a secondary analysis of the clinical records between January 1st, 2019, to December 31st, 2020. Inclusion criteria were individuals with voluntary acute ingestion of toxic doses of acetaminophen (>4 g/day). Results: sixty-three cases, 68% women, 67% <18-year-old, and 54% students. 60% had personal history of psychiatric illness and 35% reported at least one previous suicide attempt. The median dose of acetaminophen was 15g, 46% referred to co-ingestion with other substances and 13% were under the effect of any psychoactive substance. 57% had a clear intention of suicide. 81% vomited before the arrival to the emergency room, 22% received decontamination intervention with gastric lavage or activated charcoal, and 10% did not receive any dose of N-Acetylcysteine. Fifteen individuals developed an acute liver injury, nine with severity criteria. Conclusions: the population was predominantly young, the personal history of psychiatric disease was highly prevalent, and most of the cases referred a vital event that explains the impulsive behavior in acetaminophen consumption. None developed criteria for liver transplantation, and this could be explained by the young age of the individuals, the episodes of early vomiting, and the absence of chronic liver disease or hepatotoxic substance consumption.

Objetivo:este estudo busca descrever os indivíduos avaliados por sobredose de acetaminofen entre 2019 e 2020 num centro de referência de transplante hepático na Colômbia. Metodologia: estudo derivado da análise secundário de histórias clínicas entre o dia 1.º de janeiro de 2019 e 31 de dezembro de 2020. Os critérios de inclusão abrangem indivíduos com ingestão aguda e voluntária de dose tóxicas de acetaminofen (>4 g/dia).Resultados:sessenta e três casos, 68% mulheres, 67% menores de 18 anos e 54% estudantes. Reportou história pessoal de doença psiquiátrica, 60% e 35% pelo menos uma tentativa de suicídio prévio. A média de dose de acetaminofen foi de 15g, 46% referiram com ingestão de outras sustâncias e 13% estava sob efeito de sustâncias psicoativas. 57% tinham a intenção clara de suicidar-se, assim como 81% vomitou antes de acudir ao serviço de urgências, 22% receberam medidas de descontaminação e 10% não recebeu N - acetilcisteína. Quinze indivíduos desenvolveram lesão hepática aguda, nove com critérios de severidade. Conclusões: a população era predominantemente jovem, a história de doençapsiquiátrica foi muito prevalente e a maioria referiram um evento vital que explicasse o comportamento impulsivo de consumo. Nenhum desenvolveu critérios para transplantehepático, o qual se poderia explicar pela idade dos indivíduos, os episódios de vómito precoce, e a ausência de doença hepática crónica ou de consumo de sustâncias hepatotóxicas.

Experiencias psicóticas e ideación suicida en adolescentes / Psychotic experiences and suicidal behavior in adolescents / Experiências psicóticas e ideação suicida em adolescentes

Objetivo: Determinar la asociación entre experiencias psicóticas y trastornos mentales no psicóticos en población general. Metodología: Estudio de prevalencia analítico con fuente de información secundaria en población general de 15 a 65 años seleccionados de modo aleatorio por muestreo probabilístico. El instrumento de recolección de la información fue el CIDI (por su sigla en inglés), que realiza diagnóstico de 23 trastornos mentales según criterios DSM-IV (por su sigla en inglés). Se evaluaron variables sociodemográficas, clínicas psiquiátricas y seis experiencias psicóticas (alucinaciones e ideas delirantes) como variable principal. El estudio primario fue aprobado por el Comité de Investigación y de Ética Universitario. Se consideró significancia estadística valor de p<0.05. Se calcularon como medidas de asociación OR con su IC95%. Resultados: Se evaluaron 2 072 personas, el 1.85% presentó experiencias psicóticas, de los cuales el 53.3 % eran mujeres, y el 44.4 %, adolescentes. Hubo asociación entre experiencias psicóticas e ideación suicida (OR 3.51, IC95% 1.49-8.29]), ser adolescente (OR 2.29, IC95% 1.24-4.22]) y pertenecer al régimen contributivo de salud (OR 0.34, IC95% 0.18-0.63). Conclusiones: Los jóvenes y aquellas personas con ideación suicida tuvieron mayor probabilidad de reportar experiencias psicóticas. Dentro del intento global por comprender mejor para actuar de forma más efectiva frente al comportamiento suicida especialmente en la población más joven, una mejor comprensión del papel que desempeñan las experiencias psicóticas (EP) puede ser de ayuda.

Objective: To determine the association between psychotic experiences and non-psychotic mental disorders in the general population. Methodology: Analytical prevalence study with secondary information source applied in the general population aged 15 to 65 years old, randomly selected by probabilistic sampling. The instrument for collecting the information was the Composite International Diagnosis Interview (CIDI), which diagnoses 23 mental disorders according to the DSM-IV criteria (Diagnostic and Statistical Manual of Mental Disorders). Sociodemographic and psychiatric clinical variables were assessed, but the main variable evaluated consisted in six psychotic experiences (hallucinations and delusions). The primary study was approved by the University Research and Ethics Committee. The p-value <0.05 was the threshold for statistical significance. OR and its 95% CI were calculated as association measures. Results: 2072 people were evaluated, 1.85% presented psychotic experiences, of which 53.3% were women and 44.4% were adolescents. An association was found between psychotic experiences and suicidal ideation (OR 3.51, 95% CI [1.49-8.29]), being an adolescent (OR 2.29, 95% CI [1.24-4.22]) and belonging to the contributory health system (OR 0.34, 95% CI [0.18-0.63]).Conclusions: Young people and those with suicidal ideation were more likely to report psychotic experiences. Within the overall attempt to better understand in order to act more effectively in the face of suicidal behavior, especially in the younger population, a better understanding of the role of psychotic experiences (PE) can be of help

Objetivo: Determinar a associação entre experiências psicóticas e transtornos mentais não psicóticos na população em geral. Metodologia: Estudo analítico de prevalência com fonte secundária de informação na população geral de 15 a 65 anos, selecionada aleatoriamente por amostragem probabi-lística. O instrumento de coleta de informações foi o CIDI (por sua sigla em inglês), que faz o diagnóstico de 23 transtornos mentais de acordo com os critérios do DSM-IV (por sua sigla em inglês). Variáveis sociodemográficas, clínicas psiquiátricas e seis experiências psicóticas (alucinações e delírios) foram avaliadas como a variável principal. O estudo principal foi aprovado pelo Comitê de Ética e Pesquisa da Universidade. A significância estatística foi considerada um valor de p <0,05. Eles foram calculados como medidas de associação OR com seu IC 95%. Resultados: foram avaliadas 2.072 pessoas, 1,85% apresentaram experiências psicóticas, sendo 53,3% mulheres e 44,4% adolescentes. Houve associação entre experiências psi-cóticas e ideação suicida (OR 3,51, IC 95% 1,49-8,29]), ser adolescente (OR 2,29, IC 95% 1,24-4,22]) e pertencer ao regime contributivo de saúde (OR 0,34, 95 % CI 0,18-0,63). Conclusões: Jovens e aqueles com ideação suicida foram mais propensos a relatar ex-periências psicóticas. Na tentativa geral de compreender melhor como agir de maneira mais eficaz diante do comportamento suicida, especialmente na população mais jovem, pode ser útil uma melhor compreensão do papel das experiências psicóticas (TP).

Olfactory fingerprints for major histocompatibility complex-determined body odors.

Recognition of individual body odors is analogous to human face recognition in that it provides information about identity. Individual body odors determined by differences at the major histocompatibility complex (MHC or H-2) have been shown to influence mate choice, pregnancy block, and maternal behavior in mice. Unfortunately, the mechanism and extent of the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) involvement in the discrimination of animals according to H-2-type has remained ambiguous. Here we study the neuronal activation patterns evoked in the MOB in different individuals on exposure to these complex, biologically meaningful sensory stimuli. We demonstrate that body odors from H-2 disparate mice evoke overlapping but distinct maps of neuronal activation in the MOB. The spatial patterns of odor-evoked activity are sufficient to be used like fingerprints to predict H-2 identity using a novel computer algorithm. These results provide functional evidence for discrimination of H-2-determined body odors in the MOB, but do not preclude a role for the AOB. These data further our understanding of the neural strategies used to decode socially relevant odors.

Odorant-regulated Ca2+ gradients in rat olfactory neurons.

Olfactory neurons respond to odors with a change in conductance that mediates an influx of cations including Ca2+. The concomitant increase in [Cai] has been postulated to play a role in the adaptation to maintained odorant stimulation (Kurahashi, T., and T. Shibuya. 1990. Brain Research. 515:261-268. Kramer, R. H., and S. A. Siegelbaum. 1992. Neuron. 9:897-906. Zufall, F., G. M. Shepherd, and S. Firestein. 1991. Proceedings of the Royal Society of London, B. 246:225-230.) We have imaged the distribution of [Cai] in rat olfactory neurons (RON) using the Ca2+ indicator fura-2. A large percentage of the RON (42%, n = 35) responded to odorants with an increase in [Cai]. About half of the responding neurons displayed an increase in [Cai] at the apical end of the cell, but not at the soma. Moreover, in those cells that responded to odors with a standing [Cai] gradient, the gradient could be maintained for long periods of time (minutes) provided that the cells were continuously stimulated. In contrast, K(+)-induced depolarization elicited a more homogeneous increase in [Cai]. The spatially inhomogeneous increase in [Cai] elicited by odorants in some cells has important implications for the role of Ca2+ in adaptation because channels and enzymes regulated by Ca2+ will be affected differently depending on their location.

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

pH homeostasis in promyelocytic leukemic HL60 cells.

By measuring the membrane potential using the influx of the lipophilic cation tetraphenylphosphonium and intracellular pH using 2,7-biscarboxy-ethyl-5(6)-carboxyfluorescein and the distribution of the weak acid 5,5-dimethyl-2,4-oxazolidinedione, we have determined that intracellular pH is 0.9-1.1 pH units above electrochemical equilibrium in undifferentiated HL60 cells, indicating that these cells actively extrude proton equivalents. The Na/H exchanger is not the system responsible for keeping the pH above the electrochemical equilibrium, since adding inhibitors of this transport system (dimethylamiloride and ethylisopropylamiloride) or removing the extracellular sodium has no effect on intracellular pH. In contrast, the addition of the Cl/HCO3 exchange inhibitors H2 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) or pentachlorophenol (PCP) causes a drop in intracellular pH, and the removal of extracellular chloride in the presence of bicarbonate leads to a large intracellular alkalinization, which indicates a role for the anion exchanger in pH homeostasis in these cells. In addition, we find that the intracellular chloride concentration is about one order of magnitude above electrochemical equilibrium. We conclude that an H2DIDS and PCP inhibitable system, probably the Cl/HCO3 exchanger, is at least partially responsible for keeping intracellular pH above electrochemical equilibrium in HL60 cells under resting conditions. We also find no change in intracellular pH when cells differentiate along the granulocytic pathway (having been induced by the addition of dimethylsulfoxide or of retinoic acid), which indicates that changes in intracellular pH are not causally related to cell differentiation.

A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.

Variability of position of the P2 glomerulus within a map of the mouse olfactory bulb.

Olfactory receptor neurons (ORNs) that express a common odorant receptor molecule target specific glomeruli in the olfactory bulb. We systematically assessed the location of the olfactory glomeruli that receive input from ORNs expressing P2 receptors in the P2-internal ribosome entry site-tau-lacZ mouse. We present a new mapping method that includes an Internet-accessible computer program for generating two- and three-dimensional maps of the glomerular sheet in the olfactory bulbs of mice. Cylindrical coordinates were used to define glomerular location: The coordinates were given as the anteroposterior (AP) distance parallel to the long axis of the bulb (rostrocaudal; RC) and angular measurements with origin defined by the remnant ependymal layer in the center of the granule cell layer in the bulb. Using this method, we can apply rigorous statistical methods to give objective estimates of position and variability. At the 95% confidence interval, the lateral P2 glomerulus lies at coordinates 1,008 microm +/- 306 microm AP x 146 degrees +/- 12 degrees, and the medial P2 glomerulus lies at 1,828 microm +/- 196 microm AP x 204 degrees +/- 8 degrees. We estimate that these coordinates encompass a domain containing 29 and 37 of the 1,800 glomeruli ( approximately 2%) for the lateral and medial glomeruli, respectively. Furthermore, the data reported here demonstrate that the rostrocaudal position of small P2 glomeruli is three times more variable than that of large glomeruli.

Mature olfactory receptor neurons express connexin 43.

The expression of the gap junction subunit connexin 43 was studied in the olfactory epithelium of adult mice. In agreement with conclusions from previous immunohistochemical studies, we observed expression of mRNA encoding for connexin 43 in layers of the epithelium containing nuclei belonging to sustentacular cells. However, we also observed expression of connexin 43 mRNA in the layers containing nuclei belonging to mature olfactory receptor neurons (ORNs), immature ORNs, and basal cells. Connexin 43 mRNA expression was low in dorsomedial regions of the nasal cavity but higher ventrally. This differential regional distribution was consistent with expression in a transgenic mouse of a LacZ reporter gene driven by the proximal 6.5 kb of the connexin 43 promoter. LacZ was expressed in cells colabeled with antibody against olfactory marker protein (OMP), corroborating that mature ORNs express connexin 43. LacZ staining also was observed in sustentacular and basal cells and in immature ORNs. Double-label studies with antibodies against connexin 43 and OMP and expression of connexin 43 in the epithelium of bulbectomized mice were also consistent with expression of connexin 43 in mature ORNs. This is the first report of expression of a connexin subunit in mature ORNs. Our findings of connexin subunits in mature ORNs raise the novel possibility that gap junctions may play a fundamental role in information processing in the olfactory epithelium.

NADPH diaphorase staining suggests localization of nitric oxide synthase within mature vertebrate olfactory neurons.

Nitric oxide, a simple gas which serves as a neurotransmitter in the CNS, has been proposed to serve as an interneuronal second messenger in olfactory transduction. However, the role of nitric oxide in olfaction has been questioned by experiments in which nitric oxide synthase, the enzyme that generates nitric oxide, could not be localized to the olfactory epithelium. We have localized nitric oxide synthase to the olfactory neurons in adult rat and catfish olfactory epithelia using a modified nicotinamide adenine dinucleotide phosphate diaphorase technique. In the rat, staining was also found in cells with morphology reminiscent of microvillar olfactory cells. In contrast, the respiratory epithelium and the sustentacular cells in the olfactory epithelium displayed no staining. The nicotinamide adenine dinucleotide phosphate diaphorase reaction, which has been shown to co-localize with immunohistochemical staining for nitric oxide synthase in the brain, was stimulated by addition of the nitric oxide synthase substrate L-arginine, and was inhibited by the nitric oxide synthase inhibitor L-NG-nitro arginine, indicating that staining was specific for nitric oxide synthase. Unilateral bulbectomy, which causes degeneration of mature olfactory neurons on the bulbectomized size, markedly reduced nicotinamide adenine dinucleotide phosphate diaphorase staining. These observations were substantiated by biochemical assays for nitric oxide synthase by monitoring the production of [3H]-L-citrulline from [3H]-L-arginine. This is the first demonstration of specific NADPH diaphorase staining of mature olfactory neurons in rat and catfish olfactory epithelial suggesting the presence of nitric oxide synthase in these cells. Our histological and biochemical findings, in conjunction with data from other research, are supportive of a role for nitric oxide synthase in olfactory function.

Modulation of odor-induced increases in [Ca(2+)](i) by inhibitors of protein kinases A and C in rat and human olfactory receptor neurons.

Protein kinases A and C have been postulated to exert multiple effects on different elements of signal transduction pathways in olfactory receptor neurons. However, little is known about the modulation of olfactory responses by protein kinases in intact olfactory receptor neurons. To further elucidate the details of the modulation of odorant responsiveness by these protein kinases, we investigated the action of two protein kinase inhibitors: H89, an inhibitor of protein kinase A, and N-myristoylated EGF receptor, an inhibitor of protein kinase C, on odorant responsiveness in intact olfactory neurons. We isolated individual olfactory neurons from the adult human and rat olfactory epithelium and measured responses of the isolated cells to odorants or biochemical activators that have been shown to initiate cyclic AMP or inositol 1,4,5-trisphospate production in biochemical preparations. We employed calcium imaging techniques to measure odor-elicited changes in intracellular calcium that occur over several seconds. In human olfactory receptor neurons, the protein kinase A and C inhibitors affected the responses to different sets of odorants. In rats, however, the protein kinase C inhibitor affected responses to all odorants, while the protein kinase A inhibitor had no effect. In both species, the effect of inhibition of protein kinases was to enhance the elevation and block termination of intracellular calcium levels elicited by odorants. Our results show that protein kinases A and C may modulate odorant responses of olfactory neurons by regulating calcium fluxes that occur several seconds after odorant stimulation. The effects of protein kinase C inhibition are different in rat and human olfactory neurons, indicating that species differences are an important consideration when applying data from animal studies to apply to humans.

G alpha 9/G alpha 11: immunolocalization in the olfactory epithelium of the rat (Rattus rattus) and the channel catfish (Ictalurus punctatus).

The immunohistochemical localization of G alpha 9/G alpha 11 was studied in the olfactory and respiratory epithelium of two representative vertebrates, the rat and the channel catfish. Localization in the rat was found at the apical surface of cells in the epithelium and within nerve tracts in the lamina propria. Immunostaining of neuronal cilia and supporting cell microvilli was confirmed by electron microscopy. Immunoreactivity on the ipsilateral neuroepithelium was abolished five weeks following unilateral bulbectomy. An emergence of patchy immunoreactivity was found, however, after fifteen weeks. In catfish, G alpha 9/G alpha 11 antigenicity was found at the apical surface of cells within the olfactory epithelium, at supranuclear regions within some cell bodies and in basal nerve tracts of the olfactory rosette. Immunoreactivity was removed with unilateral bulbectomy. Specific labelling in both rat and catfish was eliminated by preincubation of the G alpha 9/G alpha 11 antibodies with the cognate peptide. Proteins were extracted from olfactory tissues of both species and solubilized. Using western blotting, bands corresponding in apparent molecular weight to a 38,000 mol. wt protein were found. These data demonstrate the presence of G alpha 9/G alpha 11 in the olfactory tissues of these vertebrates and suggest a role in olfaction for this class of G-protein.

Induction of differentiation of human olfactory neuroblastoma cells into odorant-responsive cells.

Olfactory neuroblastoma is a rare malignancy of the olfactory mucosa that may be derived from the olfactory epithelium. To characterize this tumor, we cultured olfactory neuroblastoma cells in the presence or absence of growth factors (transforming growth factor alpha and basic fibroblast growth factor) known to affect olfactory tissue and assessed their responsiveness to known odorants by measuring changes in intracellular calcium. Untreated cells did not respond to odorants. Basic fibroblast growth factor treatment had cytotoxic effects, and treated cells did not respond to odorants. Transforming growth factor alpha treatment resulted in the induction of odor responsiveness in these cells. Cells responded to odorants at 100 nM to 100 microM concentrations and responded with both increases and decreases in intracellular calcium. Increases in intracellular calcium were mediated by a calcium influx and were reversibly blocked by compounds known to inhibit second messenger pathways in olfactory receptor neurons. The calcium responses of the olfactory neuroblastoma cells were thus specific to the odorants and similar to those found in olfactory receptor neurons. The results support the notion that olfactory neuroblastoma cells may be of olfactory origin and thus they can be used as a model cell line to study human olfaction.