Search bioRxiv⌕ Search

Biology subjects

Moreno, A.

Publications and source records attributed to Moreno, A..

At least 37 records · Page 2Linked to original sources
bioRxiv2024

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination ([~]1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

immunology↗
bioRxiv2024

Identification of the main barriers to Ku accumulation in chromatin

Repair of DNA double strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Given its high affinity for ends, multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to [~]1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unknown. Here we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process which actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism which operates in S-phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together our data shed light on the multiple layers of coordinated mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions. HighlightsO_LIDNA-PKcs structurally blocks Ku sliding into chromatin in human & Xenopus C_LIO_LIA neddylation/FBXL12-dependent mechanism limits Ku accumulation on chromatin C_LIO_LIIn S-phase, ATM/CtIP overcomes Ku accumulation C_LIO_LIIn absence of DNA-PKcs, transcription at the DNA end vicinity is inhibited C_LI eTOC blurbThe DNA end binding protein Ku can slide onto naked DNA but this is limited in cells. Using human cells and Xenopus egg extracts, DNA-PKcs is identified as the main structural barrier to Ku entry into chromatin, along with two active mechanisms which limit Ku accumulation in absence of DNA-PKcs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/574002v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@4ebc45org.highwire.dtl.DTLVardef@12a7805org.highwire.dtl.DTLVardef@12db0a8org.highwire.dtl.DTLVardef@a925cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗
bioRxiv2023

PERK modulation, with GSK2606414, Sephin1 or salubrinal, failed to produce therapeutic benefits in the SOD1G93A mouse model of ALS

Amyotrophic lateral sclerosis (ALS) has been linked to overactivity of the protein kinase RNA-like ER kinase (PERK) branch of the unfolded protein response (UPR) pathway, both in ALS patients and mouse models. However, attempts to pharmacologically modulate PERK for therapeutic benefit have yielded inconsistent and often conflicting results. This study sought to address these discrepancies by comprehensively evaluating three commonly used PERK modulators (GSK2606414, salubrinal, and Sephin1) in the same experimental models, with the goal of assessing the viability of targeting the PERK pathway as a therapeutic strategy for ALS. To achieve this goal, a tunicamycin-challenge assay was developed using wild-type mice to monitor changes in liver UPR gene expression in response to PERK pathway modulation. Subsequently, multiple dosing regimens of each PERK modulator were tested in standardized, well-powered, gender-matched, and litter-matched survival efficacy studies using the SOD1G93A mouse model of ALS. The alpha-2-adrenergic receptor agonist clonidine was also tested to elucidate the results obtained from the Sephin1, and of the previously reported guanabenz studies, by comparing the effects of presence or absence of -2 agonism. The results revealed that targeting PERK may not be an ideal approach for ALS treatment. Inhibiting PERK with GSK2606414 or activating it with salubrinal did not confer therapeutic benefits. While Sephin1 showed some promising therapeutic effects, it appears that these outcomes were mediated through PERK-independent mechanisms. Clonidine also produced some favorable therapeutic effects, which were unexpected and not linked to the UPR. In conclusion, this study highlights the challenges of pharmacologically targeting PERK for therapeutic purposes in the SOD1G93A mouse model and suggests that exploring other targets within, and outside, the UPR may be more promising avenues for ALS treatment.

neuroscience↗
bioRxiv2023

Non-Hebbian plasticity produces long-lasting associative memories

The dominant models of learning and memory, such as Hebbian plasticity, propose that experiences are transformed into memories through input-specific synaptic plasticity at the time of learning. However, synaptic plasticity is neither strictly input specific nor restricted to the time of its induction. The impact of such forms of non-Hebbian plasticity on memory has been difficult to test, hence poorly understood. Here, we demonstrate that synaptic manipulations can deviate from the Hebbian model of learning, yet produce a lasting memory. First, we established a weak associative conditioning protocol in mice, where optogenetic stimulation of sensory thalamic input to the amygdala was paired with a footshock, but no detectable memory was formed. However, when the same input was potentiated minutes before or after, or even 24 hours later, the associative experience was converted to a lasting memory. Importantly, potentiating an independent input to the amygdala minutes but not 24 hours after the pairing produced a lasting memory. Thus, our findings suggest that the process of transformation of a transient experience into a memory is neither restricted to the time of the experience nor to the synapses triggered by it; instead, it can be influenced by past and future events.

neuroscience↗
bioRxiv2023

Extremely potent pan-sarbecovirus neutralizing antibodies generated by immunization of macaques with an AS03-adjuvanted monovalent subunit vaccine against SARS-CoV-2

The rapid emergence of SARS-CoV-2 variants that evade immunity to vaccination has placed a global health imperative on the development of therapeutic countermeasures that provide broad protection against SARS-CoV-2 and related sarbecoviruses. Here, we identified extremely potent pan-sarbecovirus antibodies from non-human primates vaccinated with an AS03 adjuvanted subunit vaccine against SARS-CoV-2 that recognize conserved epitopes in the receptor binding domain (RBD) with femtomolar affinities. Longitudinal analysis revealed progressive accumulation of somatic mutation in the immunoglobulin genes of antigen-specific memory B cells for at least one year following primary vaccination. 514 monoclonal antibodies (mAbs) were generated from antigen-specific memory B cells. Antibodies isolated at 5 to 12 months following vaccination displayed greater potency and breadth, relative to those identified at 1.4 months. Notably, 15 out of 338 ([~]4.4%) antibodies isolated at 1.4[~]6 months after the primary vaccination showed extraordinary neutralization potency against SARS-CoV-2 omicron BA.1, despite the absence of BA.1 neutralization in serum. Two of them, 25F9 and 20A7, neutralized authentic clade Ia sarbecoviruses (SARS-CoV, WIV-1, SHC014) and clade Ib sarbecoviruses (SARS-CoV-2 D614G, SARS-CoV-2 BA.1, Pangolin-GD) with half-maximal inhibition concentrations of (0.85 ng/ml, 3 ng/ml, 6 ng/ml, 6 ng/ml, 42 ng/ml, 6 ng/ml) and (13 ng/ml, 2 ng/ml, 18 ng/ml, 9 ng/ml, 6 ng/ml, 345 ng/ml), respectively. Furthermore, 20A7 and 27A12 showed potent neutralization against all SARS-CoV-2 variants of concern and multiple Omicron sublineages, including BA.1, BA.2, BA.3, BA.4/5, BQ.1, BQ.1.1 and XBB variants. X-ray crystallography studies revealed the molecular basis of broad and potent neutralization through targeting conserved RBD sites. In vivo prophylactic protection of 25F9, 20A7 and 27A12 was confirmed in aged Balb/c mice. Notably, administration of 25F9 provided complete protection against SARS-CoV-2, SARS-CoV-2 BA.1, SARS-CoV, and SHC014 challenge, underscoring that these mAbs are promising pan-sarbecovirus therapeutic antibodies. One Sentence SummaryExtremely potent pan-sarbecovirus neutralizing antibodies

immunology↗
bioRxiv2022

Degree-day-based model to predict egg hatching of Philaenus spumarius (Hemiptera: Aphrophoridae), the main vector of Xylella fastidiosa in Europe

Philaenus spumarius L., the main vector of Xylella fastidiosa (Wells) in Europe, is a univoltine species that overwinters in the egg stage, and its nymphs emerge in late winter or spring. Predicting the time of egg hatching is essential for determining the precise times for deploying control strategies against insect pests. Here, we monitored P. spumarius eggs from oviposition to egg hatching together with the daily temperatures and relative humidities at four field locations that were located at different altitudes in central Spain. The collected data were used to build a growing degree day (GDD) model to forecast egg hatching in the Iberian Peninsula. Furthermore, the model was validated with field observations that were conducted in Spain. The model was then used as a decision-support tool to calculate the optimum timing for applying control actions against P. spumarius. Our results suggest that controlling nymphs at two different dates would target the highest percentages of nymphal populations present in the field. Our model represents a first step for predicting the emergence of nymphs and adopting timely control actions against P. spumarius. These actions could limit disease spread in areas where X. fastidiosa is present.

animal behavior and cognition↗
bioRxiv2022

mRNA bivalent booster enhances neutralization against BA.2.75.2 and BQ.1.1

The emergence of the highly divergent SARS-CoV-2 Omicron variant has jeopardized the efficacy of vaccines based on the ancestral spike. The bivalent COVID-19 mRNA booster vaccine within the United States is comprised of the ancestral and the Omicron BA.5 spike. Since its approval and distribution, additional Omicron subvariants have been identified with key mutations within the spike protein receptor binding domain that are predicted to escape vaccine sera. Of particular concern is the R346T mutation which has arisen in multiple subvariants, including BA.2.75.2 and BQ.1.1. Using a live virus neutralization assay, we evaluated serum samples from individuals who had received either one or two monovalent boosters or the bivalent booster to determine neutralizing activity against wild-type (WA1/2020) virus and Omicron subvariants BA.1, BA.5, BA.2.75.2, and BQ.1.1. In the one monovalent booster cohort, relative to WA1/2020, we observed a reduction in neutralization titers of 9-15-fold against BA.1 and BA.5 and 28-39-fold against BA.2.75.2 and BQ.1.1. In the BA.5-containing bivalent booster cohort, the neutralizing activity improved against all the Omicron subvariants. Relative to WA1/2020, we observed a reduction in neutralization titers of 3.7- and 4-fold against BA.1 and BA.5, respectively, and 11.5- and 21-fold against BA.2.75.2 and BQ.1.1, respectively. These data suggest that the bivalent mRNA booster vaccine broadens humoral immunity against the Omicron subvariants.

immunology↗
bioRxiv2022

From individual behaviors to collective outcomes: fruiting body formation in Dictyostelium as a group-level phenotype

Collective phenotypes, which arise from the interactions among individuals, can be important for the evolution of higher levels of biological organization. However, how a groups composition determines its collective phenotype remains poorly understood. When starved, cells of the social amoeba Dictyostelium discoideum cooperate to build a multicellular fruiting body, and the morphology of the fruiting body is likely advantageous to the surviving spores. We assessed how the number of strains, as well as their genetic and geographic relationships to one another, impact the groups morphology and productivity. We find that some strains consistently enhance or detract from the productivity of their groups, regardless of the identity of the other group members. We also detect extensive pairwise and higher-order genotype interactions, which collectively have a large influence on the group phenotype. Whereas previous work in Dictyostelium has focused almost exclusively on whether spore production is equitable when strains cooperate to form multicellular fruiting bodies, our results suggest a previously unrecognized impact of chimeric co-development on the group phenotype. Our results demonstrate how interactions among members of a group influence collective phenotypes and how group phenotypes might in turn impact selection on the individual.

evolutionary biology↗
bioRxiv2022

A Novel Caenorhabditis elegans Gene Network Uncovers Mechanisms of Mitochondrial Maintenance

Mitochondria play key roles in cellular health and metabolism and are a critical determinant of the activation of multiple cell death processes. Although several pathways for regulating and re-establishing mitochondrial homeostasis have been identified within the past twenty years, large gaps remain in our understanding of how cells keep mitochondria healthy. To address this limitation, have developed a network of genes that underlie mitochondrial health. We began by compiling a list of frequently mutated genes using publicly available data from multiple human cancer cell lines. RNAi was used to disrupt orthologous genes in the model organism Caenorhabditis elegans in a series of assays to evaluate these genes ability to support mitochondrial health, as evidenced by precocious activation of mitochondrial autophagy and sensitivity to acute mitochondrial damage. Iterative screening of ~1000 genes yielded a network of 139 genes showing significant connectivity. Functional validation of a panel of genes from the network indicated that disruption of each gene triggered at least one phenotype consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of ATP, NADH, or ROS, and altered oxygen consumption. Importantly, RNAi-mediated knockdown of these genes often exacerbated -synuclein aggregation in a C. elegans model of Parkinsons disease, indicating significant changes to cellular health. Additionally, human orthologs of the final mitochondrial health gene network showed enrichment for roles in a number of human disorders identified in the OMIM database. This gene network provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis.

genetics↗
bioRxiv2022

Spontaneous suppressors against debilitating transmembrane mutants of CaMdr1 disclose novel interdomain communication via Signature motifs of the Major Facilitator Superfamily

The Major Facilitator Superfamily (MFS) includes multiple families of proteins operating as uniporters, symporters and antiporters for a wide spectrum of substrates. Among them, the multidrug resistance-1 drug:H+ antiporter CaMdr1 from Candida albicans is responsible for the efflux of structurally-diverse antifungals. MFS share a common fold of 12-14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS show a high primary structure diversity and few conserved Signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously rescuing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1->G140DTMH1) or 3-TMHs repeat (F216ATMH4->G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3->D235HTMH4, L480ATMH10->A435TTMH9), and finally between the N- and C-domains (G230ATMH4->P528HTMH12). Remarkably, most of these mutants belongs to the different Signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS. Results point also to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.

biochemistry↗
bioRxiv2022

The maternal X chromosome impairs cognition and accelerates brain aging through epigenetic modulation in female mice

Female mammalian cells harbor two X chromosomes, one of maternal and one of paternal origin. During development, one X randomly inactivates1-4. This renders either the maternal or paternal X active, causing X mosaicism that varies among individual females, with some showing considerable or complete skew in the general female population5-7. Parent-of-X-origin can modify epigenetics via DNA methylation8, 9 and possibly gene expression; thus, mosaicism could buffer dysregulated processes in aging and disease. However, whether X skewing - or its mosaicism - alters functions in females is largely unknown. Here we tested whether skew toward the maternal X (Xm) influences key functions of the body. Among cardiac, bone, metabolic, and brain functions, Xm selectively impaired cognition in female mice throughout the lifespan. Cognitive deficits were accompanied by Xm-mediated acceleration of biologic or epigenetic aging of the female hippocampus, a key center for learning and memory. Xm showed epigenetic imprinting of several genes within hippocampal neurons, suggesting silenced cognitive loci. Thus, the maternal X chromosome impaired cognition, accelerated brain aging, and silenced genes. Understanding how the maternal X impairs brain function could lead to new understanding of female heterogeneity in cognitive heath and to X chromosome-derived pathways against cognitive deficits and brain aging.

neuroscience↗
bioRxiv2021

Optimizing large organ scale micro computed tomography imaging in pig and human hearts using a novel air-drying technique

Underlying electrical propagation in the heart and potentially fatal arrhythmia is the cardiac microstructure. Despite the critical role of muscle architecture, a non-destructive approach to examine not only myocyte orientation, but cellular arrangement in to laminar organization is lacking in hearts from translational animal models and humans. X-ray micro computed tomography using contrast enhancing agents achieves three-dimensional images at near-histological resolutions. However, imaging large mammalian hearts presents challenges including X-ray over-attenuation and loss of image contrast. The goal of this study was to rethink tissue pre-treatment to optimize, and benefit from micro computed tomography imaging resolution in large tissues. Whole pig and human hearts were dehydrated and perfused with a tissue reinforcing agent, hexamethyldisilazane, and slowly air-dried. Heart morphology was conserved and temporally stable. This enabled direct air-mounting for micro computed tomography imaging. Moreover, the desiccated tissue density was significantly reduced compared to the initial hydrated state (P=0.04). Three-dimensional image reconstructions of air-dried hearts segmented using a single intensity threshold revealed detailed microstructural architecture of myolaminae. Conversely, one-step segmentation of hearts loaded with contrast agents poorly estimated the gross anatomical morphology of the heart and lacked identification of tissue microarchitecture. Air-drying large mammalian hearts optimizes X-ray imaging of cardiac microstructure.

physiology↗
bioRxiv2021

Clinical recovery of Macaca fascicularis infected with Plasmodium knowlesi

BackgroundKra monkeys (Macaca fascicularis), a natural host of Plasmodium knowlesi, control parasitaemia caused by this parasite species and escape death without treatment. Knowledge of the disease progression and resilience in kra monkeys will aid the effective use of this species to study mechanisms of resilience to malaria. This longitudinal study aimed to define clinical, physiological and pathological changes in kra monkeys infected with P. knowlesi, which could explain their resilient phenotype. MethodsKra monkeys (n = 15, male, young adults) were infected intravenously with cryopreserved P. knowlesi sporozoites and the resulting parasitaemias were monitored daily. Complete blood counts, reticulocyte counts, blood chemistry and physiological telemetry data (n = 7) were acquired as described prior to infection to establish baseline values and then daily after inoculation for up to 50 days. Bone marrow aspirates, plasma samples, and 22 tissue samples were collected at specific time points to evaluate longitudinal clinical, physiological and pathological effects of P. knowlesi infections. ResultsAs expected, the kra monkeys controlled parasitaemia and remained with low-level, persistent parasitaemias without antimalarial intervention. Unexpectedly, early in the infection, fevers developed, which ultimately returned to baseline, as well as mild to moderate thrombocytopaenia, and moderate to severe anaemia. Mathematical modeling and the reticulocyte production index indicated that the anaemia was largely due to the removal of uninfected erythrocytes and not impaired production of erythrocytes. Mild tissue damage was observed, and tissue parasite load was associated with tissue damage even though parasite accumulation in the tissues was generally low. ConclusionsKra monkeys experimentally infected with P. knowlesi sporozoites presented with multiple clinical signs of malaria that varied in severity among individuals. Overall, the animals shared common mechanisms of resilience characterized by controlling parasitaemia 3-5 days after patency, and controlling fever, coupled with physiological and bone marrow responses to compensate for anaemia. Together, these responses likely minimized tissue damage while supporting the establishment of chronic infections, which may be important for transmission in natural endemic settings. These results provide new foundational insights into malaria pathogenesis and resilience in kra monkeys, which may improve understanding of human infections.

pathology↗
bioRxiv2021

Dual-color optical activation and suppression of neurons with high temporal precision

A well-known phenomenon in the optogenetic toolbox is that all light-gated ion channels, including red-shifted channelrhodopsins (ChRs), are activated by blue light, whereas blue-shifted ChRs are minimally responsive to longer wave-lengths. Here, we took advantage of this feature to create a system which allows high-frequency activation of neurons with pulses of red light, while permitting the suppression of action potentials with millisecond precision by blue light. We achieved this by pairing an ultrafast red-shifted ChR with a blue light-sensitive anion channel of appropriately matching kinetics. This required screening several anion-selective ChRs, followed by a model-based mutagenesis strategy to optimize their kinetics and light spectra. Slice electrophysiology in the hippocampus as well as behavioral inspection of vibrissa movement demonstrate a minimal excitation from blue light. Of significant potential value, in contrast to existing tools, the system we introduce here allows high frequency optogenetic excitation of neurons with red light, while blue light suppression of action potentials is confined within the duration of the light pulse.

neuroscience↗
bioRxiv2020

Dispersal ability of Neophilaenus campestris, a vector of Xylella fastidiosa, from olive groves to over-summering hosts

Neophilaenus campestris is one of the recently identified spittlebugs (Hemiptera: Cercopoidea) able to transmit Xylella fastidiosa to olive trees. Considering its vector ability and the wide distribution of this species in Spain, N. campestris should be considered a serious threat to key crops that are vital for Spanish agriculture such as olive, almonds and grapevines. Migration and dispersal abilities of insect vectors have profound implications in the spread of vector-borne diseases. Thus, knowledge on the dispersal ability of N. campestris is essential to model, predict and limit the spread of the diseases caused by X. fastidiosa. A mark-release-recapture technique was developed to track between-field movements of N. campestris during its late spring migration from the ground cover grasses within olive groves to sheltered areas dominated by pine trees. An indoor assay showed that the fluorescent dust used for marking did not affect the survival nor the flying ability of N. campestris. Spittlebug adults captured in olive groves at Los Santos de la Humosa (Madrid, Spain) during late spring, 2019 were dusted with four fluorescent colours and released in four different locations. Six recapture samplings were performed 23 to 42 days after release in 12 different sites located within a maximum distance of 2.8 km from the release point. Results indicated that N. campestris was able to disperse a maximum distance of 2473 m in 35 days from the olive groves to areas dominated by pine trees. Furthermore, our flight mill studies also showed that N. campestris was able to fly long distances, reaching almost 1.4 km in an 82 minutes single flight. Moreover, we carried out a survey of directional movement of potential vectors of X. fastidiosa in an olive grove located in Villa del Prado (Madrid). We used yellow sticky bands, a Malaise trap and a vertical yellow sticky net to assess the directional movement from olive groves to surrounding managed and unmanaged areas. The captures obtained in the yellow sticky bands showed that spittlebugs dispersal from the olive grove to surrounding vegetation matched with the time when the ground cover dried out. The highest number of spittlebugs was captured in the border between the olive grove and a vineyard close by. Altogether, our findings suggest that eradication measures by rooting-up X. fastidiosa-infected and non-infected trees in a radius of 100 m are of limited value because vectors are able to disperse rapidly over distances much longer than expected.

ecology↗
bioRxiv2018

Chemogenetic ligands for translational neurotheranostics

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a popular chemogenetic technology for manipulation of neuronal activity in uninstrumented awake animals with potential for precision medicine-based clinical theranostics. DREADD ligands developed to date are not appropriate for such translational applications. The prototypical DREADD agonist clozapine N-oxide (CNO) lacks brain entry and converts to clozapine. The second-generation DREADD agonist, Compound 21 (C21), was developed to overcome these limitations. We found that C21 has low brain penetrance, weak affinity, and low in vivo DREADD occupancy. To address these drawbacks, we developed two new DREADD agonists, JHU37152 and JHU37160, and the first dedicated positron emission tomography (PET) DREADD radiotracer, [18F]JHU37107. JHU37152 and JHU37160 exhibit high in vivo DREADD potency. [18F]JHU37107 combined with PET allows for DREADD detection in locally-targeted neurons and at their long-range projections, enabling for the first time, noninvasive and longitudinal neuronal projection mapping and potential for neurotheranostic applications.

neuroscience↗
bioRxiv2018

Sudden heart rate reduction upon optogenetic release of acetylcholine from cardiac parasympathetic neurons in perfused hearts

The balance of sympathetic and parasympathetic tone provides exquisite control of heart rate and contractility and has also been shown to modulate coronary flow and inflammation. Understanding how autonomic balance is altered by cardiac disease is an active area of research and developing new ways to control this balance is providing insights into disease therapies. However, achieving acute neuron-specific stimulation of autonomic neurons can be difficult in experiments that measure the acute effects of nerve stimulation on the heart. Conventional electrical and pharmacological approaches can be spatially and temporally non-selective. Cell-specific expression of light-activated channels (channelrhodopsin, ChR2) is a powerful approach that enables control of the timing and distribution of cellular stimulation using light. We present such an optogenetic approach where parasympathetic cardiac neurons are selectively photoactivated at high temporal precision to initiate cholinergic-mediated slowing of heart rate.\n\nMice were crossbred to express ChR2 in peripheral cholinergic neurons using Cre-Lox recombination driven by a choline acetyltransferase (ChAT) promoter. Hearts from adult mice were excised, perfused, and the epicardium was illuminated (peak 460-465nm) to photoactivate ChR2. In one set of studies, hearts were illuminated using a large-field LED light source. In other studies, a micro LED was placed on the right atrium to selectively illuminate the junction of the superior vena cava and right atrium. The ECG was acquired before, during, and after tissue illumination to measure changes in heart rate. Upon illumination, hearts exhibited sudden and dramatic reductions in heart rate with restoration of normal heart rate after cessation of illumination. Delays in atrioventricular conduction were also observed. Heart rate reductions at the highest irradiance levels were similar to heart rate reductions caused by application of bethanechol (10{micro}M) or acetylcholine (800{micro}M). Atropine (50nM) completely blocked the effect of ChR2 photoactivation, confirming cholinergic mediation.\n\nOptogenetic activation of intrinsic parasympathetic neurons reduced heart rate in an immediate, dose-dependent fashion, resembling the slowing of sinus rate in response to acetylcholine. Our results demonstrate a new approach for controlling parasympathetic modulation of cardiac function by selectively activating the endogenous release of acetylcholine from intrinsic cardiac cholinergic neurons.\n\nKey MessageOptogenetic photoactivation of intrinsic cardiac neurons provides immediate, tissuespecific stimulation with minimal cross-reactivity. Our results demonstrate that selective expression of channelrhodopsin within cardiac cholinergic neurons enables photoactivated release of acetylcholine, thereby instantaneously slowing sinus rate and altering atrioventricular conduction. This provides for indepth examination of the endogenous interplay between cardiac autonomic neurons and the functional outcomes of downstream post-synaptic receptor activation.

physiology↗