Response: Visual number

While our brief Report [1] did not discuss previous work on texture density [2-7], we do acknowledge that these studies have some relevance to our own. However, we found some of Durgin's conclusions difficult to reconcile with our findings. From results obtained from two groups of subjects instructed to report either apparent numerosity or apparent density Durgin [6] claimed that "density is more influential [than numerosity] in the perception of high numerosities". He also suggests that numerosity may derive from what he claims is a correlate of it, kurtosis. We are unaware that kurtosis is a correlate of number, and in the case of spots uniformly either white or black the concept of kurtosis is inapplicable.     

A visual sense of number

Evidence exists for a nonverbal capacity for the apprehension of number, in humans [1] (including infants [2, 3]) and in other primates [4-6]. Here, we show that perceived numerosity is susceptible to adaptation, like primary visual properties of a scene, such as color, contrast, size, and speed. Apparent numerosity was decreased by adaptation to large numbers of dots and increased by adaptation to small numbers, the effect depending entirely on the numerosity of the adaptor, not on contrast, size, orientation, or pixel density, and occurring with very low adaptor contrasts. We suggest that the visual system has the capacity to estimate numerosity and that it is an independent primary visual property, not reducible to others like spatial frequency or density of texture [7].     

The role of visual information in numerosity estimation

Mainstream theory suggests that the approximate number system supports our non-symbolic number abilities (e.g. estimating or comparing different sets of items). It is argued that this system can extract number independently of the visual cues present in the stimulus (diameter, aggregate surface, etc.). However, in a recent report we argue that this might not be the case. We showed that participants combined information from different visual cues to derive their answers. While numerosity comparison requires a rough comparison of two sets of items (smaller versus larger), numerosity estimation requires a more precise mechanism. It could therefore be that numerosity estimation, in contrast to numerosity comparison, might rely on the approximate number system. To test this hypothesis, we conducted a numerosity estimation experiment. We controlled for the visual cues according to current standards: each single visual property was not informative about numerosity. Nevertheless, the results reveal that participants were influenced by the visual properties of the dot arrays. They gave a larger estimate when the dot arrays consisted of dots with, on average, a smaller diameter, aggregate surface or density but a larger convex hull. The reliance on visual cues to estimate numerosity suggests that the existence of an approximate number system that can extract numerosity independently of the visual cues is unlikely. Instead, we propose that humans estimate numerosity by weighing the different visual cues present in the stimuli.     

Ronald Ross and the malaria-mosquito cycle

This essay examines briefly Ronald Ross's research on malaria in India between 1895 and 1899, during which time he elucidated the role of the Anopheles mosquito in the transmission of the disease. During his crucial experimental work in 1898, he used birds, human malaria being relatively uncommon in Calcutta. This article is based largely on the correspondence between Ross and Patrick Manson, which has just been edited.     

Numerosity estimation in visual stimuli in the absence of luminance-based cues

Background

Numerosity estimation is a basic preverbal ability that humans share with many animal species and that is believed to be foundational of numeracy skills.It is notoriously difficult, however, to establish whether numerosity estimation is based on numerosity itself, or on one or more non-numerical cues like—in visual stimuli—spatial extent and density. Frequently, different non-numerical cues are held constant on different trials. This strategy, however, still allows numerosity estimation to be based on a combination of non-numerical cues rather than on any particular one by itself.

Methodology/Principal Findings

Here we introduce a novel method, based on second-order (contrast-based) visual motion, to create stimuli that exclude all first-order (luminance-based) cues to numerosity. We show that numerosities can be estimated almost as well in second-order motion as in first-order motion.

Conclusions/Significance

The results show that numerosity estimation need not be based on first-order spatial filtering, first-order density perception, or any other processing of luminance-based cues to numerosity. Our method can be used as an effective tool to control non-numerical variables in studies of numerosity estimation.     

A generalized sense of number

Much evidence has accumulated to suggest that many animals, including young human infants, possess an abstract sense of approximate quantity, a number sense. Most research has concentrated on apparent numerosity of spatial arrays of dots or other objects, but a truly abstract sense of number should be capable of encoding the numerosity of any set of discrete elements, however displayed and in whatever sensory modality. Here, we use the psychophysical technique of adaptation to study the sense of number for serially presented items. We show that numerosity of both auditory and visual sequences is greatly affected by prior adaptation to slow or rapid sequences of events. The adaptation to visual stimuli was spatially selective (in external, not retinal coordinates), pointing to a sensory rather than cognitive process. However, adaptation generalized across modalities, from auditory to visual and vice versa. Adaptation also generalized across formats: adapting to sequential streams of flashes affected the perceived numerosity of spatial arrays. All these results point to a perceptual system that transcends vision and audition to encode an abstract sense of number in space and in time.     

Radioautographic and quantitative study of insulin binding sites in the rat brain

In the present study, we describe the specificity and the autoradiographic distribution of insulin binding sites in the rat central nervous system (CNS) after in vitro incubation of brain sections with [125I]-14A insulin. Increasing concentrations of unlabeled insulin produced a dose-dependent inhibition of [125I]-insulin binding which represented 92 +/- 2% displacement with 3 X 10(-5) M, whatever the brain sections tested. Half-maximum inhibition with native insulin was obtained with 2.2 X 10(-9) M, with 10(-7) M proinsulin whereas glucagon had no effect. Under our experimental conditions, no degradation of [125I]-insulin was observed. Autoradiograms obtained by apposition of LKB 3H-Ultrofilm showed a widespread distribution of [125I]-insulin in rat CNS. However, quantitative analysis of the autoradiograms with 10(-10) M of labeled insulin, showed a high number of [125I]-insulin binding sites in the choroid plexus, olfactory areas, in both cerebral and cerebellar cortices, the amygdaloid complex and in the septum. In the hippocampal formation, the dorsal dentate gyrus and various subfields of CA1, CA2 and CA3 were labeled. Moreover, arcuate, dorso- and ventromedial nuclei of the hypothalamus contained high concentrations of [125I]-insulin whereas a low density was observed in the mesencephalon. The metabolic role of insulin in the CNS is supported by the large distribution of insulin binding sites in the rat brain. However, the presence of high affinity binding sites in selective areas involved in perception and integrative processes as well as in the regulation of both feeding behavior and neuroendocrine functions, suggests a neuromodulatory role of insulin in the brain.     

Internalization and degradation of receptor bound C-reactive protein by U-937 cells: induction of H2O2 production and tumoricidal activity

The presence of a membrane receptor for C-reactive protein (CRP-R) on the human monocytic cell line U-937 was the basis for determining the metabolic fate of the receptor-bound ligand and the functional response of the cells to CRP. Internalized [125I]CRP was measured by removing cell surface-bound [125I]CRP with pronase. Warming cells to 37 degrees C resulted in the internalization of approx. 50% of the receptor-bound [125I]CRP or receptor-bound [125I]CRP-PC-KLH complexes. U-937 cells degraded about 25% of the internalized [125I]CRP into TCA-soluble radiolabeled products. The lysosomotrophic agents (chloroquine, NH4Cl) greatly decreased the extent of CRP degradation without altering binding or internalization. In addition, a pH less than 4.0 resulted in dissociation of receptor-bound [125I]CRP. Treatment of U-937 cell with monensin, a carboxylic ionophore which prevents receptor recycling, resulted in accumulation of internalized [125I]CRP. Therefore, it appears that the CRP-R complex is internalized into an endosomal compartment where the CRP is uncoupled from its receptor and subsequently degraded. CRP initiated the differentiation of the U-937 cells so that they acquired the ability to produce H2O2 and also display in vitro tumoricidal activity. The results support the concept that internalization and degradation of CRP leads to the activation of monocytes during inflammation.     

Regulation of Rat Pineal α1-Adrenoceptors

Some aspects of the physiological regulation of the pineal alpha 1-adrenoceptor have been studied using the selective, high-affinity ligand [125I] iodo-2-[beta-(4-hydroxyphenyl)ethylaminomethyl]tetralone ([125I]HEAT). Pineal glands taken from rats housed in a diurnal lighting cycle showed no circadian rhythm in the number of specific [125I]HEAT binding sites, although a characteristic rhythm in pineal melatonin was seen. It was established that the pineal alpha 1-adrenoceptor is under neural control because interruption of neural stimulation of the pineal by bilateral superior cervical ganglionectomy (SCGX) or by exposing rats to constant light for 3 weeks doubled receptor density but did not change affinity for [125I]HEAT. Administration of various alpha 1-adrenoceptor agonists either acutely (i.p. injection) or chronically (s.c. infusion) did not alter the number of specific [125I]HEAT binding sites. Together these results indicate that the pineal alpha 1-adrenoceptor, like the pineal beta-adrenoceptor, is regulated by sympathetic nerve activity, probably through the physiological release of the neurotransmitter norepinephrine. However the absence of a circadian rhythm in alpha 1-adrenoceptor number and lack of down-regulation by adrenergic agonists imply different mechanisms of regulation.     

Ontogeny of endothelin and its receptors in rat brain.

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.     

A texture-processing model of the ‘visual sense of number’

It has been suggested that numerosity is an elementary quality of perception, similar to colour. If so (and despite considerable investigation), its mechanism remains unknown. Here, we show that observers require on average a massive difference of approximately 40% to detect a change in the number of objects that vary irrelevantly in blur, contrast and spatial separation, and that some naive observers require even more than this. We suggest that relative numerosity is a type of texture discrimination and that a simple model computing the contrast energy at fine spatial scales in the image can perform at least as well as human observers. Like some human observers, this mechanism finds it harder to discriminate relative numerosity in two patterns with different degrees of blur, but it still outpaces the human. We propose energy discrimination as a benchmark model against which more complex models and new data can be tested.     

Non-isopeptide-selective endothelin receptors in human Girardi heart cells.

We characterized the endothelin (ET) receptor in Girardi heart (GH) cells derived from human atrium. The ET isopeptides ET-1, ET-2 and ET-3 induced the monotonous and long-lasting rise in cytosolic free Ca2+ concentration [( Ca2+]i) with almost the same potency in GH cells. Scatchard analysis of [125I]ET-1 and [125I]ET-3 binding revealed that GH cells have almost the same number of binding sites for either labeled ligand. All ET isopeptides displaced either [125I]ET-1 or [125I]ET-3 binding in GH cells almost equipotently. These results reveal that the functional ET receptors in GH cells are of the ETB-type. GH cells are the first cell line to be found to express the functional ETB-receptor.     

Effects of ethanol on phosphoinositide hydrolysis and muscarinic acetylcholine receptor number in SH-SY5Y cells

Previous studies suggest that the effects of ethanol on carbachol-stimulated I(1,4,5)P3 formation and on the number of mAChRs may be independent of each other. The aim of this work was to further study this hypothesis. Human neuroblastoma SH-SY5Y cells were used as a model system. Acute exposure of the cells to 100 mM ethanol induced a decrease in [3H]N-methylscopolamine ([3H]NMS) binding at 30 seconds which was of lower magnitude and of shorter duration than the previously described ethanol-induced inhibition of the peak of carbachol-stimulated I(1,4,5)P3 formation. Long-term ethanol treatment of the cells induced a time- and concentration-dependent increase in [3H]NMS binding. Three hours of 100 mM ethanol treatment were sufficient to increase the number of mAChRs at the cell surface but these receptors were not immediately functionally active, suggesting that they may be newly synthesized. Furthermore, the ethanol-induced potentiation of carbachol-stimulated I(1,4,5)P3 formation, after two days, was, for all ethanol concentrations tested, of higher magnitude than the ethanol-induced increase in mAChR number. Together, these data indicate that both acute and chronic ethanol-induced changes in carbachol-stimulated I(1,4,5)P3 formation may not only be explained by changes in mAChR density at the cell surface but may rather be the consequence of actions of ethanol down-stream of the receptor.     

The pathways of endocytosed transferrin and secretory protein are connected in the trans-Golgi reticulum

We used a conjugate of transferrin and horseradish peroxidase (Tf/HRP) to label the intracellular transferrin receptor route in the human hepatoma cell line HepG2. The recycling kinetics of [125I]Tf/HRP were similar to those of unmodified [125I]Tf, implying identical routes for both ligands. 3,3'Diaminobenzidine (DAB)-cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were incubated with both Tf/HRP and [125I]Tf, and caused two different effects: (a) the equilibrium density of [125I]Tf containing microsomes in a Percoll density gradient was increased, and (b) the amount of immunoprecipitable [125I]Tf from density-shifted lysed microsomes was only 20% of that of nonDAB treated microsomes. The whole biosynthetic route of alpha 1-antitrypsin (AT), a typical secretory glycoprotein in HepG2 cells, was labeled during a 60-min incubation with [35S]methionine. DAB cytochemistry was performed on post-nuclear supernatants of homogenates of cells which were also incubated with Tf/HRP. DAB cytochemistry caused approximately 40% of microsome- associated "complex" glycosylated [35S]alpha 1-antitrypsin ([35S]c-AT) to shift in a Percoll density gradient. Only part of the density shifted [35S]c-AT could be recovered by immunoprecipitation. A maximum effect was measured already after 10 min of Tf/HRP uptake. The density distribution of the "high mannose" glycosylated form of 35S-alpha 1- anti-trypsin [( 35S]hm-AT) was not affected by Tf/HRP. If in addition to Tf/HRP also an excess of non-conjugated transferrin was present in the medium, [35S]c-AT was not accessible for Tf/HRP, showing the involvement of the transferrin receptor (TfR) in the process. Furthermore, we show that if Tf/HRP and [35S]c-AT were located in different vesicles, the density distribution of [35S]c-AT was not affected by DAB-cytochemistry. Pulse-labeling with [35S]methionine was used to show that [35S]c-AT became accessible to endocytosed Tf/HRP minutes after acquirement of the complex configuration. A common intracellular localization of endocytosed Tf/HRP and secretory protein could be confirmed by immuno-electron microscopy: cryosections labeled with anti-albumin (protein A-colloidal gold) as well as DAB reaction product showed double-labeling in the trans-Golgi reticulum.     

Absolute quantitative autoradiography of low concentrations of [125I]-labeled proteins in arterial tissue

We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of [125I]-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed [125I]-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural [125I]-low-density lipoprotein [( 125I]-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.     

Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.     

Insulin-like growth factor (IGF) binding to human fibroblast and glioblastoma cells: the modulating effect of cell released IGF binding proteins (IGFBPs)

The cell surface of human fibroblasts contains not only type I IGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF-I-cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125I]-IGF-I binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]-IGF-I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]-IGF-I added) since they have a higher affinity than type I IGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF-I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]-IGF-I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]-IGF-I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125I]-IGF-I that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]-IGF-I ([QAYL]-IGF-I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF-I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF-I and the [QAYL]-IGF-I compete equally with the [125I]-[QAYL]-IGF-I for binding to cell surfaces, whereas unlabeled [QAYL]-IGF-I is greater than 25-fold less potent compared to IGF-I in competing with [125I]-IGF-I for cell surface binding. Specific binding of [125I]-[QAYL]-IGF-I to GM10 and 1690 cell surfaces is less than 20% of [125I]-IGF-I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]-IGF-I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.     

Fate of asialofetuin endocytosed by rat liver

We have investigated the endocytosis by rat liver of asialofetuin coupled to [125I] tyramine cellobiose: [125I] TCASF. Subcellular distribution of radioactive compounds was established after differential and isopycnic centrifugation and by analysing the fractions by SDS electrophoresis. Labelling secondary lysosomes was performed by injecting rats with Triton WR 1339 four days before injecting the protein. Results show that after being associated with endosomes [125I] TCASF is recovered in organelles where they are subjected to a first degradation, the density of these organelles is practically not affected by Triton WR 1339 injection. Later the degradation products are associated with lysosomes whose density is markedly lowered by Triton WR 1339 treatment. These observations suggest that the first intracellular organelles where [125I] TCASF is subjected to digestion are distinct from the secondary lysosome population. This could be in agreement with the hypothesis that supposes that endosomes acquire enzymes from primary lysosomes before fusion with secondary lysosomes.     

The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I

The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[125I](trifluoromethyl)phenyldiazirinylacetogenin, [125I]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [125I]TDA, radioactivity was predominantly incorporated into an approximately 30 kDa band on a SDS gel. Blue native gel electrophoresis of the [125I]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at approximately 30 kDa. Immnoprecipitation of the [125I]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [125I]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of Deltalac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of Deltalac-acetogenins differs from that of traditional inhibitors.