Repeat controlled human Plasmodium falciparum infections delay bloodstream patency and reduce symptoms.

Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity.

Juxtacellular recordings from identified neurons in the mouse locus coeruleus.

The locus coeruleus (LC) is the primary source of noradrenergic transmission in the mammalian central nervous system. This small pontine nucleus consists of a densely packed nuclear core-which contains the highest density of noradrenergic neurons-embedded within a heterogeneous surround of non-noradrenergic cells. This local heterogeneity, together with the small size of the LC, has made it particularly difficult to infer noradrenergic cell identity based on extracellular sampling of in vivo spiking activity. Moreover, the relatively high cell density, background activity and synchronicity of LC neurons have made spike identification and unit isolation notoriously challenging. In this study, we aimed at bridging these gaps by performing juxtacellular recordings from single identified neurons within the mouse LC complex. We found that noradrenergic neurons (identified by tyrosine hydroxylase, TH, expression; TH-positive) and intermingled putatively non-noradrenergic (TH-negative) cells displayed similar morphologies and responded to foot shock stimuli with excitatory responses; however, on average, TH-positive neurons exhibited more prominent foot shock responses and post-activation firing suppression. The two cell classes also displayed different spontaneous firing rates, spike waveforms and temporal spiking properties. A logistic regression classifier trained on spontaneous electrophysiological features could separate the two cell classes with 76% accuracy. Altogether, our results reveal in vivo electrophysiological correlates of TH-positive neurons, which can be useful for refining current approaches for the classification of LC unit activity.

Sensory and behavioral modulation of thalamic head-direction cells.

Head-direction (HD) neurons are thought to exclusively encode directional heading. In awake mice, we found that sensory stimuli evoked robust short-latency responses in thalamic HD cells, but not in non-HD neurons. The activity of HD cells, but not that of non-HD neurons, was tightly correlated to brain-state fluctuations and dynamically modulated during social interactions. These data point to a new role for the thalamic compass in relaying sensory and behavioral-state information.

Opto-juxtacellular interrogation of neural circuits in freely moving mice.

Neural circuits are assembled from an enormous variety of neuronal cell types. Although significant advances have been made in classifying neurons on the basis of morphological, molecular and electrophysiological properties, understanding how this diversity contributes to brain function during behavior has remained a major experimental challenge. Here, we present an extension to our previous protocol, in which we describe the technical procedures for performing juxtacellular opto-tagging of single neurons in freely moving mice by using Channelrhodopsin-2-expressing viral vectors. This method allows one to selectively target molecularly defined cell classes for in vivo single-cell recordings. The targeted cells can be labeled via juxtacellular procedures and further characterized via post-hoc morphological and molecular analysis. In its current form, the protocol allows multiple recording and labeling attempts to be performed within individual animals, by means of a mechanical pipette micropositioning system. We provide proof-of-principle validation of this technique by recording from Calbindin-positive pyramidal neurons in the mouse hippocampus during spatial exploration; however, this approach can easily be extended to other behaviors and cortical or subcortical areas. The procedures described here, from the viral injection to the histological processing of brain sections, can be completed in ~4-5 weeks.This protocol is an extension to: Nat. Protoc. 9, 2369-2381 (2014): https://doi.org/10.1038/nprot.2014.161.

Extending the range of Plasmodium falciparum transmission blocking antibodies.

Recent work demonstrating that asymptomatic carriers of P. falciparum parasites make up a large part of the infectious reservoir highlights the need for an effective malaria vaccine. Given the historical challenges of vaccine development, multiple parasite stages have been targeted, including the sexual stages required for transmission. Using flow cytometry to efficiently screen for P. falciparum gamete/zygote surface reactivity, we identified 82 antibodies that bound live P. falciparum gametes/zygotes. Ten antibodies had significant transmission-reducing activity (TRA) in a standard membrane feeding assay and were subcloned along with 9 nonTRA antibodies as comparators. After subcloning, only eight of the monoclonals obtained have significant TRA. These eight TRA mAbs do not recognize epitopes present in any of the current recombinant transmission-blocking vaccine candidates, Pfs230D1M, Pfs48/45.6C, Pf47 D2 and rPfs25. One TRA mAb immunoprecipitates two surface antigens, Pfs47 and Pfs230, that are expressed by both gametocytes and gametes/zygotes. These two proteins have not previously been reported to associate and the recognition of both by a single TRA mAb suggests the Pfs47/Pfs230 complex is a new vaccine target. In total, Pfs230 was the dominant target antigen, with five of the eight TRA mAbs and 8 of 11 nonTRA gamete/zygote surface reactive mAbs interacting with Pfs230. Of the three remaining TRA mAbs, two recognized non-reduced, parasite-produced Pfs25 and one bound non-reduced, parasite-produced Pfs48/45. None of the TRA mAbs bound protein on an immunoblot of reduced gamete/zygote extract and two TRA mAbs were immunoblot negative, indicating none of the new TRA epitopes are linear. The identification of eight new TRA mAbs that bind epitopes not included in any of the constructs currently under advancement as transmission-blocking vaccine candidates may provide new targets worthy of further study.

Modular microcircuit organization of the presubicular head-direction map.

Our internal sense of direction is thought to rely on the activity of head-direction (HD) neurons. We find that the mouse dorsal presubiculum (PreS), a key structure in the cortical representation of HD, displays a modular "patch-matrix" organization, which is conserved across species (including human). Calbindin-positive layer 2 neurons within the "matrix" form modular recurrent microcircuits, while inputs from the anterodorsal and laterodorsal thalamic nuclei are non-overlapping and target the "patch" and "matrix" compartments, respectively. The apical dendrites of identified HD cells are largely restricted within the "matrix," pointing to a non-random sampling of patterned inputs and to a precise structure-function architecture. Optogenetic perturbation of modular recurrent microcircuits results in a drastic tonic suppression of firing only in a subpopulation of HD neurons. Altogether, our data reveal a modular microcircuit organization of the PreS HD map and point to the existence of cell-type-specific microcircuits that support the cortical HD representation.

Juxtacellular opto-tagging of hippocampal CA1 neurons in freely moving mice.

Neural circuits are made of a vast diversity of neuronal cell types. While immense progress has been made in classifying neurons based on morphological, molecular, and functional properties, understanding how this heterogeneity contributes to brain function during natural behavior has remained largely unresolved. In the present study, we combined the juxtacellular recording and labeling technique with optogenetics in freely moving mice. This allowed us to selectively target molecularly defined cell classes for in vivo single-cell recordings and morphological analysis. We validated this strategy in the CA1 region of the mouse hippocampus by restricting Channelrhodopsin expression to Calbindin-positive neurons. Directly versus indirectly light-activated neurons could be readily distinguished based on the latencies of light-evoked spikes, with juxtacellular labeling and post hoc histological analysis providing 'ground-truth' validation. Using these opto-juxtacellular procedures in freely moving mice, we found that Calbindin-positive CA1 pyramidal cells were weakly spatially modulated and conveyed less spatial information than Calbindin-negative neurons - pointing to pyramidal cell identity as a key determinant for neuronal recruitment into the hippocampal spatial map. Thus, our method complements current in vivo techniques by enabling optogenetic-assisted structure-function analysis of single neurons recorded during natural, unrestrained behavior.

Output-Specific Adaptation of Habenula-Midbrain Excitatory Synapses During Cocaine Withdrawal.

Projections from the lateral habenula (LHb) control ventral tegmental area (VTA) neuronal populations' activity and both nuclei shape the pathological behaviors emerging during cocaine withdrawal. However, it is unknown whether cocaine withdrawal modulates LHb neurotransmission onto subsets of VTA neurons that are part of distinct neuronal circuits. Here we show that, in mice, cocaine withdrawal, drives discrete and opposing synaptic adaptations at LHb inputs onto VTA neurons defined by their output synaptic connectivity. LHb axons innervate the medial aspect of VTA, release glutamate and synapse on to dopamine and non-dopamine neuronal populations. VTA neurons receiving LHb inputs project their axons to medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and lateral hypothalamus (LH). While cocaine withdrawal increases glutamate release from LHb onto VTA-mPFC projectors, it reduces presynaptic release onto VTA-NAc projectors, leaving LHb synapses onto VTA-to-LH unaffected. Altogether, cocaine withdrawal promotes distinct adaptations at identified LHb-to-VTA circuits, which provide a framework for understanding the circuit basis of the negative states emerging during abstinence of drug intake.

Structural Correlates of CA2 and CA3 Pyramidal Cell Activity in Freely-Moving Mice.

Plasticity within hippocampal circuits is essential for memory functions. The hippocampal CA2/CA3 region is thought to be able to rapidly store incoming information by plastic modifications of synaptic weights within its recurrent network. High-frequency spike-bursts are believed to be essential for this process, by serving as triggers for synaptic plasticity. Given the diversity of CA2/CA3 pyramidal neurons, it is currently unknown whether and how burst activity, assessed in vivo during natural behavior, relates to principal cell heterogeneity. To explore this issue, we juxtacellularly recorded the activity of single CA2/CA3 neurons from freely-moving male mice, exploring a familiar environment. In line with previous work, we found that spatial and temporal activity patterns of pyramidal neurons correlated with their topographical position. Morphometric analysis revealed that neurons with a higher proportion of distal dendritic length displayed a higher tendency to fire spike-bursts. We propose that the dendritic architecture of pyramidal neurons might determine burst-firing by setting the relative amount of distal excitatory inputs from the entorhinal cortex.SIGNIFICANCE STATEMENT High-frequency spike-bursts are thought to serve fundamental computational roles within neural circuits. Within hippocampal circuits, spike-bursts are believed to serve as potent instructive signals, which increase the efficiency of information transfer and induce rapid modifications of synaptic efficacies. In the present study, by juxtacellularly recording and labeling single CA2/CA3 neurons in freely-moving mice, we explored whether and how burst propensity relates to pyramidal cell heterogeneity. We provide evidence that, within the CA2/CA3 region, neurons with higher proportion of distal dendritic length display a higher tendency to fire spike-bursts. Thus, the relative amount of entorhinal inputs, arriving onto the distal dendrites, might determine the burst propensity of individual CA2/CA3 neurons in vivo during natural behavior.

Interspike interval analysis and spikelets in presubicular head-direction cells.

Head-direction (HD) neurons are thought to provide the mammalian brain with an internal sense of direction. These cells, which selectively increase their firing when the animal's head points in a specific direction, use the spike rate to encode HD with a high signal-to-noise ratio. In the present work, we analyzed spike train features of presubicular HD cells recorded juxtacellularly in passively rotated rats. We found that HD neurons could be classified into two groups on the basis of their propensity to fire spikes at short interspike intervals. "Bursty" neurons displayed distinct spike waveforms and were weakly but significantly more modulated by HD compared with "nonbursty" cells. In a subset of HD neurons, we observed the occurrence of spikelets, small-amplitude "spike-like" events, whose HD tuning was highly correlated to that of the co-recorded juxtacellular spikes. Bursty and nonbursty HD cells, as well as spikelets, were also observed in freely moving animals during natural behavior. We speculate that spike bursts and spikelets might contribute to presubicular HD coding by enhancing its accuracy and transmission reliability to downstream targets. NEW & NOTEWORTHY We provide evidence that presubicular head-direction (HD) cells can be classified into two classes (bursty and nonbursty) on the basis of their propensity to fire spikes at short interspike intervals. Bursty cells displayed distinct electrophysiological properties and stronger directional tuning compared with nonbursty neurons. We also provide evidence for the occurrence of spikelets in a subset of HD cells. These electrophysiological features (spike bursts and spikelets) might contribute to the precision and robustness of the presubicular HD code.

Manipulating Hippocampal Place Cell Activity by Single-Cell Stimulation in Freely Moving Mice.

Learning critically depends on the ability to rapidly form and store non-overlapping representations of the external world. In line with their postulated role in episodic memory, hippocampal place cells can undergo a rapid reorganization of their firing fields upon contextual manipulations. To explore the mechanisms underlying such global remapping, we juxtacellularly stimulated 42 hippocampal neurons in freely moving mice during spatial exploration. We found that evoking spike trains in silent neurons was sufficient for creating place fields, while in place cells, juxtacellular stimulation induced a rapid remapping of their place fields to the stimulus location. The occurrence of complex spikes was most predictive of place field plasticity. Our data thus indicate that plasticity-inducing stimuli are able to rapidly bias place cell activity, simultaneously suppressing existing place fields. We propose that such competitive place field dynamics could support the orthogonalization of the hippocampal map during global remapping.

Structural modularity and grid activity in the medial entorhinal cortex.

Following the groundbreaking discovery of grid cells, the medial entorhinal cortex (MEC) has become the focus of intense anatomical, physiological, and computational investigations. Whether and how grid activity maps onto cell types and cortical architecture is still an open question. Fundamental similarities in microcircuits, function, and connectivity suggest a homology between rodent MEC and human posteromedial entorhinal cortex. Both are specialized for spatial processing and display similar cellular organization, consisting of layer 2 pyramidal/calbindin cell patches superimposed on scattered stellate neurons. Recent data indicate the existence of a further nonoverlapping modular system (zinc patches) within the superficial MEC layers. Zinc and calbindin patches have been shown to receive largely segregated inputs from the presubiculum and parasubiculum. Grid cells are also clustered in the MEC, and we discuss possible structure-function schemes on how grid activity could map onto cortical patch systems. We hypothesize that in the superficial layers of the MEC, anatomical location can be predictive of function; thus relating functional properties and neuronal morphologies to the cortical modules will be necessary for resolving how grid activity maps onto cortical architecture. Imaging or cell identification approaches in freely moving animals will be required for testing this hypothesis.

Testing the Efficacy of Single-Cell Stimulation in Biasing Presubicular Head Direction Activity.

To support navigation, the firing of head direction (HD) neurons must be tightly anchored to the external space. Indeed, inputs from external landmarks can rapidly reset the preferred direction of HD cells. Landmark stimuli have often been simulated as excitatory inputs from "visual cells" (encoding landmark information) to the HD attractor network; when excitatory visual inputs are sufficiently strong, preferred directions switch abruptly to the landmark location. In the present work, we tested whether mimicking such inputs via juxtacellular stimulation would be sufficient for shifting the tuning of individual presubicular HD cells recorded in passively rotated male rats. We recorded 81 HD cells in a cue-rich environment, and evoked spikes trains outside of their preferred direction (distance range, 11-178°). We found that HD tuning was remarkably resistant to activity manipulations. Even strong stimulations, which induced seconds-long spike trains, failed to induce a detectable shift in directional tuning. HD tuning curves before and after stimulation remained highly correlated, indicating that postsynaptic activation alone is insufficient for modifying HD output. Our data are thus consistent with the predicted stability of an HD attractor network when anchored to external landmarks. A small spiking bias at the stimulus direction could only be observed in a visually deprived environment in which both average firing rates and directional tuning were markedly reduced. Based on this evidence, we speculate that, when attractor dynamics become unstable (e.g., under disorientation), the output of HD neurons could be more efficiently controlled by strong biasing stimuli.SIGNIFICANCE STATEMENT The activity of head direction (HD) cells is thought to provide the mammalian brain with an internal sense of direction. To support navigation, the firing of HD neurons must be anchored to external landmarks, a process thought to be supported by associative plasticity within the HD system. Here, we investigated these plasticity mechanisms by juxtacellular stimulation of single HD neurons in vivo in awake rats. We found that HD coding is strongly resistant to external manipulations of spiking activity. Only in a visually deprived environment was juxtacellular stimulation able to induce a small activity bias in single presubicular neurons. We propose that juxtacellular stimulation can bias HD tuning only when competing anchoring inputs are reduced or not available.

Cutis Laxa Acquisita After Urticarial Vasculitis in SLE Patients.

Cutis laxa is a rare connective tissue disease involving damage to dermal elastic fibers creating a clinical appearance of loose, sagging skin. The condition can be either acquired or genetic. Autoimmune diseases, neoplasms, infections, and medications have been proposed as the cause of, or in association with, the acquired form. In nearly 50% of cases, erythematous plaques present before the onset of cutis laxa. Separately, urticarial vasculitis and systemic lupus erythematosus have been linked to cutis laxa acquisita. Our case is the first in the literature documenting a coexistence of cutis laxa acquisita, hypocomplementemic urticarial vasculitis, and systemic lupus erythematosus.

Linking neuronal structure to function in rodent hippocampus: a methodological prospective.

Since the discovery of place cells, hippocampus-dependent spatial navigation has proven to be an ideal model system for resolving the relationship between neural coding and behavior. Electrical recordings from the hippocampal formation in freely moving animals have revealed a rich repertoire of spatial firing patterns and have enormously advanced our understanding of the neural principles of spatial representation. However, limited progress has been achieved in resolving the underlying cellular mechanisms. This is partially attributable to the inability of standard recording techniques to link neuronal structure to function directly. In this review, we summarize recent efforts aimed at filling this gap. We also highlight the development of methodologies that allow functional measurements from identified neuronal elements in behaving rodents. Recent progress in the dentate gyrus serves as a showcase to reveal the potential of such methodologies and the necessity of resolving structure-function relationships in order to access the cellular mechanisms of hippocampal circuit computations.

Cuantificación de trazas de soja con kits de ELISA en galletitas y productos extrudidos / Soya traces quantification in cookies and extruded products with ELISA kits / Quantificação de traços de soja com kits de ELISA em biscoitos e produtos extrusados

Se evaluó la detección de trazas de soja en sistemas modelo (SM) de galletitas y extrudidos que contenían 5000, 250, 100, 50, 25, 10, 5 y 0 ppm de concentrado de proteína de soja (CPS). Se analizaron, además, dos lotes diferentes de ocho clases de galletitas comerciales y dos tipos de extrudidos que declaraban la presencia de soja a través de la frase "Contiene ..." o a través de frases de advertencia. La determinación de proteínas de soja se realizó utilizando tres kits ELISA (R-Biopharm, Neogen-Veratox y Romer). Solo el kit de R-Biopharm permitió la cuantificación de soja a partir de 25 ppm de CPS en galletitas y a partir de 5 ppm de CPS en productos extrudidos. Los otros dos kits resultaron menos sensibles. De acuerdo con los resultados obtenidos en el análisis de los SM de galletitas y de productos extrudidos con diferentes kits comerciales se concluye que debe estudiarse el desempeño de cada kit para cada alergeno y para cada matriz en particular. Esto permitiría establecer cuál es el método más adecuado para el control de los productos comerciales a los fines de evaluar la correcta declaración de alergenos en los respectivos rótulos.

Detection of traces of soy was evaluated in cookies and extruded product model systems (MS) containing 5000, 250, 100, 50, 25, 10, 5 and 0 ppm of soy protein concentrate (SPC). Besides, two different batches of eight different types of commercial cookies and two types of extruded products declaring soy presence through either the statement: "Contains…" or precautionary phrases were analyzed. Soy protein determination was carried out using three different commercial ELISA kits (R-Biopharm, Neogen-Veratox and Romer). Only the R-Biopharm kit allowed quantification of soy from 25 ppm of SPC in cookies and from 5 ppm of SPC in the extruded products. The other two kits had lower sensitivity. According to the results obtained in the analysis of cookies and extruded product MS with different commercial kits, it can be concluded that the performance of every kit should be studied for all allergens and each particular food matrix. This would enable establishing the most suitable method for commercial products control, in order to assess the correct allergen labeling.

Avaliou-se a detecção de traços de soja em sistemas modelo (SM) de biscoitos e extrusados que continham 5000, 250, 100, 50, 25, 10, 5 e 0 ppm de concentrado de proteína de soja (CPS). Analisaram-se, também dois lotes diferentes de oito tipos de biscoitos comerciais e dois tipos de extrusados que declaravam a presença de soja através da frase "Contém.... ou através de frases de advertência. A determinação de proteínas de soja foi realizada utilizando três kits ELISA (R-Biopharm, Neogen-Veratox e Romer). Apenas o kit de R-Biopharm permitiu a quantificação de soja a partir de 25 ppm de CPS em biscoitos e de a partir de 5 ppm de CPS em produtos extrusados. Os outros dois kits resultaram menos sensíveis. De acordo com os resultados obtidos na análise dos SM de biscoitos e de produtos extrusados com diferentes kits comerciais conclui-se que é preciso estudar o desempenho de cada kit para cada alergeno e para cada matriz em particular. Isto permitiria estabelecer qual é o método mais adequado para o controle dos produtos comerciais viando avaliar a correta declaração de alergenos nas respectivas etiquetas.

Declaración de alérgenos y detección de trazas de leche, soja y huevo en alimentos de consumo frecuente por niños / ALLERGENS DECLARATION AND DETECTION OF MILK, SOY AND EGG TRACES IN FOODS OF FREQUENT CONSUMPTION BY CHILDREN

Introducción: las alergias alimentarias constituyen un problema creciente tanto en los países desarrollados como en los emergentes. En las últimas décadas la prevalencia de las alergias a alimentos ha aumentado considerablemente y este tema constituye un desafío tanto desde el punto de vista clínico como para la industria de los alimentos. Objetivos: evaluar la declaración de alérgenos y la presencia de frases de advertencia en los rótulos de alimentos que habitualmente consumen los niños y realizar la cuanti cación de proteínas de soja, leche y huevo con kits de ELISA en todas las muestras analizadas. Materiales y métodos: se analizaron dos lotes diferentes de 11 productos comerciales que se adquirieron de manera aleatoria en supermercados de la Ciudad Autónoma de Buenos Aires, Argentina. A partir de la observación de lo declarado en cada rótulo, se determinó cuál sería la declaración correcta de alérgenos cuando el Art. 235 del Código Alimentario Argentino esté vigente. Se realizó la determinación de alérgenos utilizando dos kits comerciales de ELISA diferentes para cada uno de ellos de las marcas R-Biopharm, Neogen y Romer. En el caso de los alérgenos leche y huevo, ambos kits tuvieron el mismo comportamiento en todos los alimentos estudiados y en general coincidieron con lo declarado en los rótulos. Resultados: los resultados obtenidos para el alérgeno soja fueron muy dispares tanto al comparar los dos kits empleados para el análisis como entre los diferentes lotes de cada muestra, sin existir correlación entre los resultados obtenidos y lo declarado en los rótulos. Conclusiones: resultaría muy importante que en el rótulo de los alimentos complementarios se utilizaran frases de advertencia reales y con ables para incrementar el acceso de estos alimentos para los niños con alergias alimentarias.

Supported and Unsupported Chiral Squaramides as Organocatalysts for Stereoselective Michael Additions: Synthesis of Enantiopure Chromenes and Spirochromanes.

Novel supported chiral bifunctional squaramides have been easily prepared starting from diamines derived from natural amino acids and commercially available aminoalkyl polystyrene resins. These squaramides behave as excellent stereoselective recoverable organocatalysts in different Michael additions, in neat conditions at room temperature. The reaction on 2-(2-nitrovinyl) phenol as electrophile lead, in excellent yields and enantioselectivities, to intermediates that can be easily transformed into 4H-chromenes, and spirochromanones.

Sparse activity of identified dentate granule cells during spatial exploration.

In the dentate gyrus - a key component of spatial memory circuits - granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure-function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population.