Dietary restriction modulates ultradian rhythms and autocorrelation properties in mice behavior.

Animal behavior emerges from integration of many processes with different spatial and temporal scales. Dynamical behavioral patterns, including daily and ultradian rhythms and the dynamical microstructure of behavior (i.e., autocorrelations properties), can be differentially affected by external cues. Identifying these patterns is important for understanding how organisms adapt to their environment, yet unbiased methods to quantify dynamical changes over multiple temporal scales are lacking. Herein, we combine a wavelet approach with Detrended Fluctuation Analysis to identify behavioral patterns and evaluate changes over 42-days in mice subjected to different dietary restriction paradigms. We show that feeding restriction alters dynamical patterns: not only are daily rhythms modulated but also the presence, phase and/or strength of ~12h-rhythms, as well as the nature of autocorrelation properties of feed-intake and wheel running behaviors. These results highlight the underlying complexity of behavioral architecture and offer insights into the multi-scale impact of feeding habits on physiology.

Computational Approaches and Tools as Applied to the Study of Rhythms and Chaos in Biology.

The temporal dynamics in biological systems displays a wide range of behaviors, from periodic oscillations, as in rhythms, bursts, long-range (fractal) correlations, chaotic dynamics up to brown and white noise. Herein, we propose a comprehensive analytical strategy for identifying, representing, and analyzing biological time series, focusing on two strongly linked dynamics: periodic (oscillatory) rhythms and chaos. Understanding the underlying temporal dynamics of a system is of fundamental importance; however, it presents methodological challenges due to intrinsic characteristics, among them the presence of noise or trends, and distinct dynamics at different time scales given by molecular, dcellular, organ, and organism levels of organization. For example, in locomotion circadian and ultradian rhythms coexist with fractal dynamics at faster time scales. We propose and describe the use of a combined approach employing different analytical methodologies to synergize their strengths and mitigate their weaknesses. Specifically, we describe advantages and caveats to consider for applying probability distribution, autocorrelation analysis, phase space reconstruction, Lyapunov exponent estimation as well as different analyses such as harmonic, namely, power spectrum; continuous wavelet transforms; synchrosqueezing transform; and wavelet coherence. Computational harmonic analysis is proposed as an analytical framework for using different types of wavelet analyses. We show that when the correct wavelet analysis is applied, the complexity in the statistical properties, including temporal scales, present in time series of signals, can be unveiled and modeled. Our chapter showcase two specific examples where an in-depth analysis of rhythms and chaos is performed: (1) locomotor and food intake rhythms over a 42-day period of mice subjected to different feeding regimes; and (2) chaotic calcium dynamics in a computational model of mitochondrial function.

Análisis clínico e histopatológico de la prevalencia de enfermedades cardiacas en muerte súbita. Estudio en autopsias / Clinical and histopathological analysis of the prevalence of cardiac diseases in sudden death: a study based on autopsies

Introducción: un evento de muerte súbita supone un gran impacto para la sociedad, siendo importante su estudio para aportar conocimiento y fortalecer estrategias de promoción y prevención. Objetivo: determinar la prevalencia de los diagnósticos anatomopatológicos cardiacos definitivos y los hallazgos histopatológicos asociados con la muerte súbita de origen cardíaco en los pacientes sometidos a autopsias clínicas realizadas en el Hospital de San José de Bogotá DC, Colombia, durante el período 2015 a 2018. Métodos: estudio descriptivo de corte transversal retrospectivo en pacientes a quienes se les realizó autopsia con diagnóstico de muerte súbita de origen cardiovascular en el servicio de patología del Hospital de SanJosé, Bogotá DC, Colombia. Resultados: se incluyeron 178 autopsias con diagnóstico de muerte súbita cardíaca. El promedio global de edad fue 56.1 años (DE: 15.06) con una relación hombre-mujer de 3:1. El hallazgo macroscópico más frecuente fue cardiomegalia (promedio 428.1 g (DE 112.8), acompañado de la presencia de coronariopatía esclerótica (p=0.000) con obstrucción de la luz de las arterias coronarias mayores de 80% (p=0.037). Conclusiones: los diagnósticos anatomopatológicos definitivos prevalentes en la muerte súbita cardiaca fueron cardiopatía isquémica crónica complicada (65%) e infarto agudo de miocardio (16%), datos similares a los reportados a nivel mundial. La cardiomegalia es un hallazgo frecuente que debe valorarse en forma cuidadosa.

Introduction: a sudden death incident has a great impact on society and its study is important to provide understanding for strengthening promotion and prevention strategies. Objective: to determine the prevalence of definitive anatomopathological cardiac diagnoses and histopathological findings associated with sudden death due to a cardiovascular cause based on autopsies performed at Hospital de San José in Bogotá DC, Colombia, during the period between 2015 and 2018. Methods: retrospective descriptive cross-sectional study of cases of sudden cardiac death of cardiovascular origin diagnosed in autopsies performed at the pathology service of Hospital de San José, Bogotá DC, Colombia. Results: 178 autopsies with a diagnosis of sudden cardiac death were included. The overall mean age was 56.1 years (SD: 15.06). Male/female ratio was 3:1. The most frequent macroscopic finding was cardiomegaly (average 428.1 g (SD 112.8), with the presence of sclerotic coronary artery disease (p=0.000) with a blockage greater than 80% (p=0.037) in the major coronary arteries. Conclusions: the prevalent definitive anatomopathological diagnoses in SCD were complicated chronic ischemic heart disease (65%) and acute myocardial infarction (16%). Data obtained in this study is similar to that reported worldwide. Cardiomegaly is a frequent finding that should be carefully evaluated.

A single-cell tumor immune atlas for precision oncology.

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients, and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing published data sets of >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying colocalization patterns of immune, stromal, and cancer cells in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immunotherapy.

[Peritoneal drainage versus laparotomy for perforated necrotizing enterocolitis in preterm low birth weight infants.] / Drenaje peritoneal versus laparotomía como abordaje para enterocolitis necrosante perforada en recién nacidos pretérmino de bajo peso.

El desarrollo de enterocolitis necrosante, con la consecuente perforación intestinal, es frecuente en los recién nacidos pretérmino. El tratamiento estándar de la perforación intestinal es quirúrgico. Sin embargo, se sugiere que la inserción de un drenaje en el abdomen puede ser efectivo para tratar esta afección. Se resumen los resultados de una revisión sistemática Cochrane que compara la efectividad del drenaje peritoneal con la de la laparotomía en neonatos con enterocolitis necrosante perforada.Necrotizing enterocolitis is common in preterm newborns, with consequent intestinal perforation. The standard treatment for intestinal perforation is surgery. However, it is suggested that inserting a drain into the abdomen may be effective in treating this condition. This document summarizes the results of a Cochrane systematic review comparing the effectiveness of peritoneal drainage with laparotomy in neonates with perforated necrotizing enterocolitis.

Drenaje peritoneal versus laparotomía como abordaje para enterocolitis necrosante perforada en recién nacidos pretérmino de bajo peso / Peritoneal drainage versus laparotomy for perforated necrotizing enterocolitis in preterm low birth weight infants

Resumen El desarrollo de enterocolitis necrosante, con la consecuente perforación intestinal, es frecuente en los recién nacidos pretérmino. El tratamiento estándar de la perforación intestinal es quirúrgico. Sin embargo, se sugiere que la inserción de un drenaje en el abdomen puede ser efectivo para tratar esta afección. Se resumen los resultados de una revisión sistemática Cochrane que compara la efectividad del drenaje peritoneal con la de la laparotomía en neonatos con enterocolitis necrosante perforada.

Abstract Necrotizing enterocolitis is common in preterm newborns, with consequent intestinal perforation. The standard treatment for intestinal perforation is surgery. However, it is suggested that inserting a drain into the abdomen may be effective in treating this condition. This document summarizes the results of a Cochrane systematic review comparing the effectiveness of peritoneal drainage with laparotomy in neonates with perforated necrotizing enterocolitis.

Single human oocyte transcriptome analysis reveals distinct maturation stage-dependent pathways impacted by age.

Female fertility is inversely correlated with maternal age due to a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, however, the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown germinal vesicle stage (GV) and in vitro matured (IVM-MII) oocytes from women of varying reproductive age. First, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4445 and 324 putative marker genes, respectively. Furthermore, we identified genes for which transcript representation either progressively increased or decreased with age. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1219 genes) than in GV oocytes (596 genes). In particular, we found that transcripts of genes involved in chromosome segregation and RNA splicing significantly increased representation with age, while genes related to mitochondrial activity showed a lower representation. Gene regulatory network analysis facilitated the identification of potential upstream master regulators of the genes involved in those biological functions. Our analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript representation, particularly in IVM-MII oocytes, which might contribute to the age-related quality decline in human oocytes.

SPOTlight: seeded NMF regression to deconvolute spatial transcriptomics spots with single-cell transcriptomes.

Spatially resolved gene expression profiles are key to understand tissue organization and function. However, spatial transcriptomics (ST) profiling techniques lack single-cell resolution and require a combination with single-cell RNA sequencing (scRNA-seq) information to deconvolute the spatially indexed datasets. Leveraging the strengths of both data types, we developed SPOTlight, a computational tool that enables the integration of ST with scRNA-seq data to infer the location of cell types and states within a complex tissue. SPOTlight is centered around a seeded non-negative matrix factorization (NMF) regression, initialized using cell-type marker genes and non-negative least squares (NNLS) to subsequently deconvolute ST capture locations (spots). Simulating varying reference quantities and qualities, we confirmed high prediction accuracy also with shallowly sequenced or small-sized scRNA-seq reference datasets. SPOTlight deconvolution of the mouse brain correctly mapped subtle neuronal cell states of the cortical layers and the defined architecture of the hippocampus. In human pancreatic cancer, we successfully segmented patient sections and further fine-mapped normal and neoplastic cell states. Trained on an external single-cell pancreatic tumor references, we further charted the localization of clinical-relevant and tumor-specific immune cell states, an illustrative example of its flexible application spectrum and future potential in digital pathology.

Progressive Vaccinia Acquired through Zoonotic Transmission in a Patient with HIV/AIDS, Colombia.

In March 2015, a patient in Colombia with HIV/AIDS was hospitalized for disseminated ulcers after milking cows that had vesicular lesions on their udders. Vaccinia virus was detected, and the case met criteria for progressive vaccinia acquired by zoonotic transmission. Adherence to an optimized antiretroviral regimen resulted in recovery.

Translational thresholds in a core circadian clock model.

Organisms have evolved in a daily cyclic environment, developing circadian cell-autonomous clocks that temporally organize a wide range of biological processes. Translation is a highly regulated process mainly associated with the activity of microRNAs (miRNAs) at the translation initiation step that impacts on the molecular circadian clock dynamics. Recently, a molecular titration mechanism was proposed to explain the interactions between some miRNAs and their target mRNAs; new evidence also indicates that regulation by miRNA is a nonlinear process such that there is a threshold level of target mRNA below which protein production is drastically repressed. These observations led us to use a theoretical model of the circadian molecular clock to study the effect of miRNA-mediated translational thresholds on the molecular clock dynamics. We model the translational threshold by introducing a phenomenological Hill equation for the kinetics of PER translation and show how the parameters associated with translation kinetics affect the period, amplitude, and time delays between clock mRNA and clock protein expression. We show that our results are useful for analyzing experiments related to the translational regulation of negative elements of transcriptional-translational feedback loops. We also provide new elements for thinking about the translational threshold as a mechanism that favors the emergence of circadian rhythmicity, the tuning of the period-delay relationship and the cell capacity to control the protein oscillation amplitude with almost negligible changes in the mRNA amplitudes.

Circadian control of the pupillary light responses in an avian model of blindness, the GUCY1* chickens.

PURPOSE: The vertebrate inner retina has a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that express the nonvisual photopigment melanopsin. The intrinsically photosensitive retinal ganglion cells send light information from the environment to the brain to control, among other parameters, the amount of energy entering the eyes through the pupillary light reflex (PLR). A daily variation in the PLR in both mice and humans has recently been shown, indicating circadian control of this response. In a previous work involving the sensitivity spectra for the PLR, we showed that blind chickens (GUCY1*) display the highest sensitivity to light of 480 nm. The aim of the present study was to evaluate the potential circadian control of PLRs in blind birds under scotopic conditions. METHODS: Circadian PLR was performed on GUCY1* chickens with lights of different wavelengths (white or blue light of 475 nm) under scotopic conditions. RESULTS: We found a significant daily variation in the PLRs of chickens exposed to white or blue light of 475 nm, with increased sensitivity at circadian time 6 during the subjective day. CONCLUSIONS: Our observations clearly point to circadian control of PLRs even in blindness, strongly indicating that both the entry of light into the eyes and its quality are differentially regulated during the day in diurnal animals.

Effects of different per translational kinetics on the dynamics of a core circadian clock model.

Living beings display self-sustained daily rhythms in multiple biological processes, which persist in the absence of external cues since they are generated by endogenous circadian clocks. The period (per) gene is a central player within the core molecular mechanism for keeping circadian time in most animals. Recently, the modulation PER translation has been reported, both in mammals and flies, suggesting that translational regulation of clock components is important for the proper clock gene expression and molecular clock performance. Because translational regulation ultimately implies changes in the kinetics of translation and, therefore, in the circadian clock dynamics, we sought to study how and to what extent the molecular clock dynamics is affected by the kinetics of PER translation. With this objective, we used a minimal mathematical model of the molecular circadian clock to qualitatively characterize the dynamical changes derived from kinetically different PER translational mechanisms. We found that the emergence of self-sustained oscillations with characteristic period, amplitude, and phase lag (time delays) between per mRNA and protein expression depends on the kinetic parameters related to PER translation. Interestingly, under certain conditions, a PER translation mechanism with saturable kinetics introduces longer time delays than a mechanism ruled by a first-order kinetics. In addition, the kinetic laws of PER translation significantly changed the sensitivity of our model to parameters related to the synthesis and degradation of per mRNA and PER degradation. Lastly, we found a set of parameters, with realistic values, for which our model reproduces some experimental results reported recently for Drosophila melanogaster and we present some predictions derived from our analysis.

Differential regulation of feeding rhythms through a multiple-photoreceptor system in an avian model of blindness.

All organisms have evolved photodetection systems to synchronize their physiology and behavior with the external light-dark (LD) cycles. In nonmammalian vertebrates, the retina, the pineal organ, and the deep brain can be photoreceptive. Inner retinal photoreceptors transmit photic information to the brain and regulate diverse nonvisual tasks. We previously reported that even after preventing extraretinal photoreception, blind GUCY1* chickens lacking functional visual photoreceptors could perceive light that modulates physiology and behavior. Here we investigated the contribution of different photoreceptive system components (retinal/pineal and deep brain photoreceptors) to the photic entrainment of feeding rhythms. Wild-type (WT) and GUCY1* birds with head occlusion to avoid extraocular light detection synchronized their feeding rhythms to a LD cycle with light >12 lux, whereas at lower intensities blind birds free-ran with a period of >24 h. When released to constant light, both WT and blind chickens became arrhythmic; however, after head occlusion, GUCY1* birds free-ran with a 24.5-h period. In enucleated birds, brain illumination synchronized feeding rhythms, but in pinealectomized birds only responses to high-intensity light (≥800 lux) were observed, revealing functional deep brain photoreceptors. In chickens, a multiple photoreceptive system, including retinal and extraretinal photoreceptors, differentially contributes to the synchronization of circadian feeding behavior.

[Introduction of a rapid HIV test in Sexually Transmitted Infections Units]. / Implantación de una prueba rápida de VIH en Asturias (España).

INTRODUCTION: The aim of the study was to analyze the implementation of a rapid HIV test in Asturias (Spain). METHODS: The study was conducted in two STI Units using the Determine® HIV-1/2 test. RESULTS: A total of 1011 people were tested, of whom 65.3% had never been tested for HIV previously, and 71.4% were heterosexual men. Twenty-one tests were confirmed positive by Enzyme Immunoassay/Western Blot (EIA/WB) assay An increase was observed in the diagnosis of HIV. CONCLUSION: Awareness campaigns and rapid tests could be effective methods for the early diagnosis of HIV.

Expression of novel opsins and intrinsic light responses in the mammalian retinal ganglion cell line RGC-5. Presence of OPN5 in the rat retina.

The vertebrate retina is known to contain three classes of photoreceptor cells: cones and rods responsible for vision, and intrinsically photoresponsive retinal ganglion cells (RGCs) involved in diverse non-visual functions such as photic entrainment of daily rhythms and pupillary light responses. In this paper we investigated the potential intrinsic photoresponsiveness of the rat RGC line, RGC-5, by testing for the presence of visual and non-visual opsins and assessing expression of the immediate-early gene protein c-Fos and changes in intracellular Ca(2+) mobilization in response to brief light pulses. Cultured RGC-5 cells express a number of photopigment mRNAs such as retinal G protein coupled receptor (RGR), encephalopsin/panopsin (Opn3), neuropsin (Opn5) and cone opsin (Opn1mw) but not melanopsin (Opn4) or rhodopsin. Opn5 immunoreactivity was observed in RGC-5 cells and in the inner retina of rat, mainly localized in the ganglion cell layer (GCL). Furthermore, white light pulses of different intensities and durations elicited changes both in intracellular Ca(2+) levels and in the induction of c-Fos protein in RGC-5 cell cultures. The results demonstrate that RGC-5 cells expressing diverse putative functional photopigments display intrinsic photosensitivity which accounts for the photic induction of c-Fos protein and changes in intracellular Ca(2+) mobilization. The presence of Opn5 in the GCL of the rat retina suggests the existence of a novel type of photoreceptor cell.

Differential responses of the mammalian retinal ganglion cell line RGC-5 to physiological stimuli and trophic factors.

The rat retinal ganglion cell (RGC) line RGC-5 constitutes a widely used model for studying physiological processes in retinal cells. In this paper we investigated the expression of clock and immediately early genes, and calcium mediated responses to physiological stimuli in differentiated and mitotically active RGC-5 cells. To this end, we attempted to differentiate the RGC-5 cells with a variety of effectors classically used to induce morphological differentiation. No sign of morphological differentiation was observed after 24 h of treatment with BDNF (80 ng/mL), NGF (100 ng/mL) and retinoic acid (20 ng/mL), among others. Only staurosporine (SSP) was able to promote neurite outgrowth at concentrations ranging from 53.5 to 214 nM. However, apoptotic nuclei were seen at 24 h of treatment using DNA staining, and a few cells remained at 72 h post-treatment. Concentrations of SSP lower than 214 nM were partially effective in inducing cell differentiation. Dividing RGC-5 cells express the RGC marker Thy-1 and different clock genes such as Per1, Clock and Bmal1. When characterizing the responsiveness of proliferative RGC-5 cells we found that in most of them, brief pulses of 50% FBS induced c-Fos and PER1 expression. Subsets of RGC-5 cells displayed significant changes in intracellular Ca2+ levels by ATP (100 microM) but not by glutamate (100-200 microM) stimulation. On the basis of cell morphology, size and complexity and effector responsiveness it was possible to distinguish different subpopulations within the cell line. The results demonstrate that only SSP is effective in promoting RGC-5 morphological differentiation, though the treatment provoked cell death. Proliferative cells expressing the RGC marker Thy-1 and a number of clock genes, responded differentially to diverse physiological stimuli showing a rapid c-Fos and PER1 induction by FBS stimulation, and an increase in intracellular Ca2+ by ATP.

Inner retinal circadian clocks and non-visual photoreceptors: novel players in the circadian system.

Daily and annual changes in ambient illumination serve as specific stimuli that associate light with time and regulate the physiology of the organism through the eye. The eye acts as a dual sense organ linking light and vision, and detecting light that provides specific stimuli for non-classical photoreceptors located in the inner retina. These photoreceptors convey information to the master circadian pacemaker, the hypothalamic suprachiasmatic nuclei (SCN). Responsible for sensing the light that regulates several non-visual functions (i.e. behavior, pupil reflex, sleep, and pineal melatonin production), the retina plays a key role in the temporal symphony orchestra playing the musical score of life: it is intrinsically rhythmic in its physiological and metabolic activities. We discuss here recent evidence in support of the hypothesis that retinal oscillators distributed over different cell populations may act as clocks, inducing changes in the visual and circadian system according to the time of the day. Significant progress has recently been made in identifying photoreceptors/photopigments localized in retinal ganglion cells (RGCs) that set circadian rhythms and modulate non-visual functions. Autonomous retinal and brain oscillators could have a more complex organization than previously recognized, involving a network of "RGC clock/SCN clock cross-talk". The convergence of oscillatory and photoreceptive capacities of retinal cells could deeply impact on the circadian system, which in turn may be severely impaired in different retinal pathologies. The aim of this review is to discuss the state of the art on inner retinal cell involvement in the light and temporal regulation of health and disease.

Determinación de frecuencia cardiaca, frecuencia respiratoria, lactato deshidrogenasa, creatinkinasa y ácido láctico en caballos durante competencia de salto en la Sabana de Bogotá / Determination of cardiac and respiratory rate, Lactate dehydrogenase, creatin kinase and lactic acid during a horse jumping competition in Bogotá

El estudio presenta los primeros resultados de investigación en variables fisiológicas y sus cambios por el ejercicio en equinos en competencia de salto en el país. Se utilizarin 24 ejemplares de las razas Silla Argentina, PSI y mestizo de la Escuela de Equitación del Ejercito Nacional de Colombia, que participaron en pruebas de salto (1,10 – 1,20 m) en diferentes centros ecuestres de la Sabana de Bogotá. Se determinó frecuencia cardiaca, frecuencia respiratoria y enzimas musculares (creatinquinasa (CK), lactato deshidrogenasa (LDH) y el ácido láctico). Se tomaron tres muestras: reposo, inmediatamente después del ejercicio y a las 6 horas posejercicio. Para la frecuencia cardiaca se utilizó un monitor POLAR S625; la frecuencia respiratoria se determinó mediante fonendoscopio; y las enzimas y el ácido láctico se analizaron en laboratorio. Se utilizó el método estadístico ANAVA para enzimas musculares y acido láctico y estadística descriptiva para frecuencia cardiaca y respiratoria. En los resultados se encontró un comportamiento estadísticamente significativo (p < 0.05) del ácido láctico y la enzima creatinkinasa, a diferencia del comportamiento de la enzima lactato deshidrogenasa, que fue no significativo. Las frecuencias cardiaca y respiratoria se elevaron de manera significativa posejercicio y regresaron a lo normal alrededor de las 6 horas. El ácido láctico se incrementó posejercicio y disminuyó a las 6 horas, contrario a lo reportado por otros autores. Se concluye que una vez analizadas estas variables, hay diferencias con los valores obtenidos por otros autores, que pueden deberse a la altitud, al tipo y tiempo de ejercicio realizado. Por tanto es indispensable continuar haciendo estudios en este campo...

A nonmammalian vertebrate model of blindness reveals functional photoreceptors in the inner retina.

In mammals, photoreceptors located in the inner retina convey photic information to the brain, regulating diverse non-image-forming tasks such as pupillary light reflexes and photic synchronization (entrainment) of daily activity rhythms. In nonmammalian vertebrates, the retina, deep brain photoreceptors, and pineal organ may be photoreceptive. Here we investigated light perception in the absence of functional cone and rod photoreceptors using GUCY1* chickens, birds carrying a null mutation that causes blindness at hatch. They showed light responses in both the pupillary light reflex and the entrainment of feeding rhythms to a 12:12 h light-dark cycle. Light responses persisted even when the extraretinal photoperception was abolished, but they were lost after enucleation; this strongly indicates the essential role played by the inner retina. A sensitivity spectrum study for the pupillary reflex that combined pupil responses to different monochromatic lights of various intensities demonstrated that a single opsin/vitamin A-based photopigment peaking at 484 nm drives photic responses; the best fit (lowest sum of squares, R(2)=0.9622) was attained with an opsin:vitamin A2 template. The results are the first characterization of functional inner retinal photoreceptors participating in the regulation of non-image-forming activities in nonmammalian vertebrates.