Computational Modeling of Orientation Tuning Dynamics in Monkey Primary Visual Cortex

In the primate visual pathway, orientation tuning of neurons is first observed in the primary visual cortex. The LGN cells that comprise the thalamic input to V1 are not orientation tuned, but some V1 neurons are quite selective. Two main classes of theoretical models have been offered to explain orientation selectivity: feedforward models, in which inputs from spatially aligned LGN cells are summed together by one cortical neuron; and feedback models, in which an initial weak orientation bias due to convergent LGN input is sharpened and amplified by intracortical feedback. Recent data on the dynamics of orientation tuning, obtained by a cross-correlation technique, may help to distinguish between these classes of models. To test this possibility, we simulated the measurement of orientation tuning dynamics on various receptive field models, including a simple Hubel-Wiesel type feedforward model: a linear spatiotemporal filter followed by an integrate-and-fire spike generator. The computational study reveals that simple feedforward models may account for some aspects of the experimental data but fail to explain many salient features of orientation tuning dynamics in V1 cells. A simple feedback model of interacting cells is also considered. This model is successful in explaining the appearance of Mexican-hat orientation profiles, but other features of the data continue to be unexplained.     

Age-related changes in nitric oxide synthase in the lateral geniculate nucleus of rats

Age-related changes in nitric oxide production in the visual system have not been well characterized. Therefore, we used staining and image-processing approaches to describe changes in levels of neuronal nitric oxide synthase (nNOS), the NADPH-diaphorase (NADPH-d) histochemical marker, and 3-nitrotyrosine in the lateral geniculate nucleus (LGN) of young and aged rats. The LGN plays an important role in the visual system, as it acts as a visual relay nucleus. Quantitative analysis of NADPH-d-positive and nNOS-immunoreactive neurons revealed significant optical density increases in the dorsal LGN and ventral LGN of aged rats; however, no significant changes were observed in the number of neurons with age. 3-Nitrotyrosine immunoreactivity was increased in the dorsal LGN and ventral LGN of aged rats. These results indicate that increased nitric oxide production and peroxynitrite may be associated with alterations in visual function during aging.     

Cortical Contributions to Saccadic Suppression

The stability of visual perception is partly maintained by saccadic suppression: the selective reduction of visual sensitivity that accompanies rapid eye movements. The neural mechanisms responsible for this reduced perisaccadic visibility remain unknown, but the Lateral Geniculate Nucleus (LGN) has been proposed as a likely site. Our data show, however, that the saccadic suppression of a target flashed in the right visual hemifield increased with an increase in background luminance in the left visual hemifield. Because each LGN only receives retinal input from a single hemifield, this hemifield interaction cannot be explained solely on the basis of neural mechanisms operating in the LGN. Instead, this suggests that saccadic suppression must involve processing in higher level cortical areas that have access to a considerable part of the ipsilateral hemifield.     

Deciphering the Leishmania exoproteome: what we know and what we can learn

Parasitic protozoa of the genus Leishmania are the causative agents of leishmaniasis. Survival and transmission of these parasites in their different hosts require membrane-bound or extracellular factors to interact with and modify their host environments. Over the last decade, several approaches have been applied to study all the extracellular proteins exported by an organism at a particular time or stage in its life cycle and under defined conditions, collectively termed the secretome or the exoproteome. In this review, we focus on emerging data shedding light on the secretion mechanisms involved in the production of the Leishmania exoproteome. We also describe other methodologies currently available that could be used to analyse the Leishmania exoproteome. Understanding the complexity of the Leishmania exoproteome is a key component to elucidating the mechanisms used by these parasites for exporting proteins to the extracellular space during its life cycle. Given the importance of extracellular factors, a detailed knowledge of the Leishmania exoproteome may provide novel targets for rational drug design and/or a source of antigens for vaccine development.     

Selection against the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major.

The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, and cultured mammalian cells. Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatment with gamma-irradiation, while the frequency of other events was less than 10(-6) and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the generation of both targeted and random mutations. We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by negative selection yielded parasites lacking DHFR-TS or foreign sequences. The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists. Such marker-free auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.     

Transcriptomics throughout the life cycle of Leishmania infantum: High down-regulation rate in the amastigote stage

Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in the Mediterranean Basin. The promastigote and amastigote stages alternate in the life cycle of the parasite, developing inside the sand-fly gut and inside mammalian phagocytic cells, respectively. High-throughput genomic and proteomic analyses have not focused their attention on promastigote development, although partial approaches have been made in Leishmania major and Leishmania braziliensis. For this reason we have studied the expression modulation of an etiological agent of visceral leishmaniasis throughout the life cycle, which has been performed by means of complete genomic microarrays. In the context of constitutive genome expression in Leishmania spp. described elsewhere and confirmed here (5.7%), we found a down-regulation rate of 68% in the amastigote stage, which has been contrasted by binomial tests and includes the down-regulation of genes involved in translation and ribosome biogenesis. These findings are consistent with the hypothesis of pre-adaptation of the parasite to intracellular survival at this stage.     

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.     

LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation

LGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), which is required for polarity establishment and asymmetric cell divisions during embryonic development. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis. LGN also regulates spindle organization, possibly through inhibition of NuMA function, but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, we now show that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. We therefore propose that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization.     

Mitochondrial damage contribute to epigallocatechin-3-gallate induced death in Leishmania amazonensis

Epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, has been reported to have antiproliferative effects on Trypanosoma cruzi however, the mechanism of protozoan action of EGCG has not been studied. In the present study, we demonstrate the mechanism for the antileishmanial activity of EGCG against Leishmania amazonensis promastigotes. Incubation with EGCG significantly inhibited L. amazonensis promastigote proliferation in a time- and dose-dependent manner. The IC(50) for EGCG at 120h was 0.063mM. Ultrastructural alterations of the mitochondria were observed in promastigote treated with EGCG, being the organelle injury reinforced by the decrease in rhodamine 123 fluorescence. The effects of several drugs that interfere directly with mitochondrial physiology in parasites such as Leishmania have been described. The unique mitochondrial features of Leishmania make this organelle an ideal drug target while minimizing toxicity. These data suggest mitochondrial collapse as a part of the EGCG mechanism of action and demonstrate the leishmanicidal effect of EGCG.     

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.     

Visual Receptive Field Properties of Neurons in the Mouse Lateral Geniculate Nucleus

The lateral geniculate nucleus (LGN) is increasingly regarded as a “smart-gating” operator for processing visual information. Therefore, characterizing the response properties of LGN neurons will enable us to better understand how neurons encode and transfer visual signals. Efforts have been devoted to study its anatomical and functional features, and recent advances have highlighted the existence in rodents of complex features such as direction/orientation selectivity. However, unlike well-researched higher-order mammals such as primates, the full array of response characteristics vis-à-vis its morphological features have remained relatively unexplored in the mouse LGN. To address the issue, we recorded from mouse LGN neurons using multisite-electrode-arrays (MEAs) and analysed their discharge patterns in relation to their location under a series of visual stimulation paradigms. Several response properties paralleled results from earlier studies in the field and these include centre-surround organization, size of receptive field, spontaneous firing rate and linearity of spatial summation. However, our results also revealed “high-pass” and “low-pass” features in the temporal frequency tuning of some cells, and greater average contrast gain than reported by earlier studies. In addition, a small proportion of cells had direction/orientation selectivity. Both “high-pass” and “low-pass” cells, as well as direction and orientation selective cells, were found only in small numbers, supporting the notion that these properties emerge in the cortex. ON- and OFF-cells showed distinct contrast sensitivity and temporal frequency tuning properties, suggesting parallel projections from the retina. Incorporating a novel histological technique, we created a 3-D LGN volume model explicitly capturing the morphological features of mouse LGN and localising individual cells into anterior/middle/posterior LGN. Based on this categorization, we show that the ON/OFF, DS/OS and linear response properties are not regionally restricted. Our study confirms earlier findings of spatial pattern selectivity in the LGN, and builds on it to demonstrate that relatively elaborate features are computed early in the visual pathway.     

Nutrient transport and pathogenesis in selected parasitic protozoa

Parasitic protozoa, such as malaria parasites, trypanosomes, and Leishmania, acquire a plethora of nutrients from their hosts, employing transport proteins located in the plasma membrane of the parasite. Application of molecular genetic approaches and the completion of genome projects have allowed the identification and functional characterization of a cohort of transporters and their genes in these parasites. This review focuses on a subset of these permeases that have been studied in some detail, that import critical nutrients, and that provide examples of approaches being undertaken broadly with these and other parasite transporters. Permeases reviewed include those for hexoses, purines, iron, polyamines, carboxylates, and amino acids. Topics of special emphasis include structure-function approaches, critical roles for transporters in parasite viability and physiology, regulation of transporter expression, and subcellular targeting. Investigations of parasite transporters impact a broad spectrum of basic biological problems in these protozoa.     

Monoclonal antibodies against defined Leishmania antigens protect mice against infection by different species of Leishmania

Mice monoclonal antibodies (IgG) have been raised against Leishmania infantum promastigotes by fusing SP 2/0 myeloma cells and immunized mice splenic cells. The monoclonal antibodies have been detected by indirect immunofluorescence. In vivo tests showed that some of them could inhibit the life cycle of several Leishmania species from the Old and the New World. Studies of these protective monoclonals by the western blot technique showed the presence of three constant antigens (40 kD, 70 kD and 113 kD) amongst the Leishmania species studied.     

Expression analysis and subcellular distribution of the two G-protein regulators AGS3 and LGN indicate distinct functionality. Localization of LGN to the midbody during cytokinesis

Activator of G-protein signaling 3 (AGS3) and LGN have a similar domain structure and contain four G-protein regulatory motifs that serve as anchors for the binding of the GDP-bound conformation of specific G-protein alpha subunits. As an initial approach to define further the different functional roles of AGS3 and LGN, we determined their expression profile and subcellular distribution. AGS3- and LGN-specific antisera indicated a widespread tissue distribution of LGN, whereas AGS3 is primarily enriched in brain. Brain punch biopsies of 13 discrete brain regions indicated that both AGS3 and LGN are expressed in all areas tested but are differentially regulated during development. LGN is expressed in neuronal, astroglial, and microglial cultures, whereas AGS3 expression is restricted to neurons. In primary neuronal cultures as well as in dividing cultures of PC12 cells, immunocytochemistry indicated distinct subcellular locations of AGS3 and LGN. The subcellular locations of the two proteins were differentially regulated by external stimuli and the cell cycle. In PC12 and COS7 cells, LGN moves from the nucleus to the midbody structure separating daughter cells during the later stages of mitosis, suggesting a role for G-proteins in cytokinesis. Thus, although AGS3 and LGN share a similar overall motif structure and both bind G-proteins, nature has endowed these proteins with different regulatory elements that allow functional diversity by virtue of tissue-specific expression and subcellular positioning.     

Leishmania (Viannia) lainsoni (Kinetoplastida: Trypanosomatidae), a divergent Leishmania of the Viannia subgenus--a mini review

Leishmania (Viannia) lainsoni is the Leishmania species that presents the most distinct biological (morphology, growth in axenic culture medium), biochemical (enzymatic electrophoresis profile), and molecular biology characteristics, when compared to other species of the Viannia subgenus. Development of promastigote forms of this parasite attached to the wall of the pyloric and hind gut regions of sand fly vectors is a solid characteristic that allows its positioning in the Viannia subgenus. However, taxonomic data from biochemical and molecular techniques on this Leishmania species are still not conclusive. It is evident the difficulty in taxonomically positioning this borderline Leishmania species. In this review we present the data accumulated since L. (Viannia) lainsoni has been described and we discuss its position in the Viannia subgenus.     

Predictive Feedback Can Account for Biphasic Responses in the Lateral Geniculate Nucleus

Biphasic neural response properties, where the optimal stimulus for driving a neural response changes from one stimulus pattern to the opposite stimulus pattern over short periods of time, have been described in several visual areas, including lateral geniculate nucleus (LGN), primary visual cortex (V1), and middle temporal area (MT). We describe a hierarchical model of predictive coding and simulations that capture these temporal variations in neuronal response properties. We focus on the LGN-V1 circuit and find that after training on natural images the model exhibits the brain's LGN-V1 connectivity structure, in which the structure of V1 receptive fields is linked to the spatial alignment and properties of center-surround cells in the LGN. In addition, the spatio-temporal response profile of LGN model neurons is biphasic in structure, resembling the biphasic response structure of neurons in cat LGN. Moreover, the model displays a specific pattern of influence of feedback, where LGN receptive fields that are aligned over a simple cell receptive field zone of the same polarity decrease their responses while neurons of opposite polarity increase their responses with feedback. This phase-reversed pattern of influence was recently observed in neurophysiology. These results corroborate the idea that predictive feedback is a general coding strategy in the brain.