Biology subjects

Llano, I.

Publications and source records attributed to Llano, I..

5 recordsLinked to original sources

bioRxiv2026

Streamlined Production of Recombinant Adeno-Associated Viruses Using Gateway Cloning Technology for Neural Circuit All-Optical Interrogation

The advent of optogenetics and the rapid development of genetically encoded actuators and reporters for calcium, voltage, neurotransmitters, and other molecules have revolutionized neuroscience research by enabling precise, non-invasive optical interrogation of neural circuits. Successful implementation, however, critically depends on efficient reporter expression in defined neuronal populations. We present a Gateway(R)-based cloning platform providing a robust pipeline for rapid, efficient construction of recombinant adeno-associated virus (rAAV) vectors optimized for neuroscience applications. This platform addresses promoter selection, indicator engineering, and capsid-type optimization, and enables systematic optimization of vector components across a range of optical tools. The benefit of this modular approach is illustrated in the case of ULoVE, a two-photon microscopy method based on acousto-optic deflectors (AODs). This method provides serial light-targeting with kHz sampling rates and high signal-to-noise ratio in vivo, which imposes stringent requirements on indicator expression -- including cell-type specificity, sparse labelling, and precise control of expression levels-- efficiently met through the combinatorial flexibility of the Gateway pipeline. As specific examples, Gateway-constructed Cre-driver viruses combined with Cre-dependent reporters enabled cell-type-specific labelling for two complementary applications. In cerebellar Purkinje cells, an L7::Cre driver virus paired with a Gateway-constructed voltage indicator (JEDI2P-Kv) and ULoVE two-photon excitation at ~5 kHz enables resolution of sub-millisecond dendritic voltage dynamics in awake, behaving mice, including optical detection of dendritic spikelets previously accessible only via intracellular electrophysiology. The same driver virus paired with calcium indicators (GCaMP6f, jRGECO1a) resolves climbing fiber-evoked calcium kinetics under the same conditions. In acute cerebellar slices, a kit::Cre driver virus enabled cell-type-specific expression of the optogenetic actuator ChR2(H134R) in molecular layer interneurons, where ULoVE doughnut-pattern photostimulation achieved single-cell, sub-millisecond optogenetic activation with micron-scale spatial resolution. Beyond ULoVE applications, the same sparse, strong labelling also proved suitable for anatomical tracing of axonal projections in cleared cerebellar tissue, resolving individual DCN axon terminals at the granule cell layer --molecular layer boundary. Taken together, our dual-front approach --combining a modular AAV expression pipeline with AOD-based optical acquisition --provides an integrated platform for developing sophisticated optical tools, including but not limited to AOD-based microscopy, for neural circuit interrogation.

neuroscience↗

bioRxiv2026

Synaptic vesicle undocking induces low frequency depression

Synaptic depression is often interpreted as reflecting depletion of the readily releasable pool (RRP) following exocytosis. Such a mechanism predicts little or no depression at low stimulation frequency, as RRP replenishment should then offset the loss of vesicles by exocytosis. Nevertheless, in several types of mammalian central synapses, repetitive presynaptic stimulation at low frequency (< 5 Hz) elicits synaptic depression (low frequency depression, or LFD). In the present work we count the number of synaptic vesicles released at individual active zones to study the RRP and its replenishment during LFD. Contrary to depletion models of synaptic depression, we find that LFD does not depend on previous SV consumption. We find that LFD displays a long recovery time course (tens of seconds) when challenged by isolated stimulations but is immediately reversed by a high frequency train. We suggest that LFD results from undocking, a shift between two classes of synaptic vesicles organized sequentially inside the RRP (replacement vs. docked vesicles) in favor of the upstream (replacement) state. While undocking is apparent hundreds of milliseconds after a stimulation, calcium dependent docking takes only a couple of milliseconds, explaining the fast LFD recovery when stimulating at high frequency. Consistent with the undocking model, we find that double presynaptic stimulations alleviate LFD as they favor vesicular docking and RRP replenishment. Finally, we expand our model to explain how stimulation frequency shapes short-term synaptic depression, changing from depression at low frequency to a facilitation-depression sequence at medium or high frequency trains.

neuroscience↗

bioRxiv2025

Early resident NK cell response to local HIV infection in lymphoid tissue

Natural killer (NK) cells are critical mediators of antiviral immunity, yet their role in lymphoid tissues--key reservoirs of HIV persistence--remains poorly defined. Here, we uncover a distinct cytotoxic signature of tonsil-resident NK cells essential for targeting HIV-infected CD4 T cells. Using a human tonsillar model of HIV infection and extracellular matrix-based functional assays, we identify a subset of NK cells co-expressing CD69, CD49a, CD103, and the adaptive marker NKG2C as potent effectors against autologous HIV-infected tissue CD4 T cells. Both CD16 and CD16- NK subsets exhibited cytotoxic antiviral activity. However, HIV infection induced profound functional alterations in these NK cells, including dysregulated expression of CD9, TRAF2, ITGA1, suggesting disrupted activation, signaling, and tissue residency. Functional assays corroborated a significant impairment in the cytotoxic capacity, indicating HIV-driven NK cell dysfunction. Intriguingly, a subset of immature CD16-CD69 NK cells underwent functional reprogramming, transitioning into a migration-competent, and metabolically primed state, driven by the upregulation of NUMAI, LCP1, SLC38A1, MT-ND2, NUMA1, MYH9, and CD44. Functional assays confirmed this transition, revealing changes in the expression of immune checkpoint receptors and a gain of cytotoxic function in these reprogrammed cells. These findings advance our understanding of NK cell biology in HIV infection and highlight novel avenues for NK-cell-based therapeutic strategies.

immunology↗

bioRxiv2025

Identification of the Inducible HIV reservoir in Tonsillar, Intestinal and Cervical Tissue Models of HIV Latency

HIV persists in diverse tissues, with distinct cellular reservoirs presenting a major barrier to a cure and requiring targeted therapeutic strategies to address this heterogeneity. Here, we developed tissue models of HIV latency using human tonsillar, intestinal and cervicovaginal tissues. These models revealed differential HIV infection across CD4+ T cell subpopulations, with ART partially restoring CD4+ T cells and reducing intact HIV DNA. T follicular helper cells (TFH CD69+ CCR7-) were the primary inducible reservoir in tonsils, while tissue-resident memory cells (TRM CD69+ CD49a+) dominated in the intestine. Identification of markers for inducible reservoirs revealed that CD69, CD45RO, and PD-1 were shared across tissues, while CXCR5 in the tonsils and CD49a in the intestine served as tissue-specific markers. Furthermore, using different latency reversal agents (LRAs) we found that Histone Deacetylase Inhibitors (HDACis) failed to induce HIV in any tissue, the SMAC mimetic AZD5582 was effective only in a resident-memory CD4+ T cell subpopulation in the intestine, and IL15 exhibited the broadest reactivation potential across tissues and CD4+ T subsets. These models recapitulate key aspects of HIV infection providing insights into the inducible reservoirs composition in different tissues and informing strategies for its elimination.

immunology↗

bioRxiv2023

A first morphological and electrophysiological characterization of FCs of the mouse cerebellum.

The "feathered" cells of Fananas (FCs) are cerebellar glia of unknown function. Initially described more than a century ago, they have been practically absent from the scientific literature. Here, we combined whole-cell patch-clamp recordings, dye loading and near UV-laser photolysis for a first characterization of FCs in terms of their morphology, electrophysiological properties and glutamate-evoked currents. FCs were identified in cerebellar slices by their small cell bodies located in the molecular layer and stubby processes. Despite a more compact shape compared to Bergmann glia (BGs) FCs had similar membrane resistances and basal currents, suggesting electrical coupling between neighboring glia. Dye filling and pharmacological experiments confirmed homo- and heterotypic gap junction coupling among FCs and BGs. Parallel-fiber stimulation evoked in FCs a slow inward current partially blocked by NBQX, along with superimposed fast (ms) transients. Repetitive stimulations resulted in a rapid desensitization of this AMPA-receptor mediated current, which recovered for stimulation intervals >0.5s. Laser photolysis of MNI-caged glutamate replicated fast inward currents with highest current densities in the distant process. We conclude that FCs respond with fast AMPA currents to local glutamate release, and that they integrate ambient glutamate to a slow inward current. Interestingly, FCs prevail throughout adulthood with markedly different densities among cerebellar lobes. Thus, FCs are not just displaced BGs as previously suggested, but they may have lobule specific functions, both locally and at the circuit level, yet to be uncovered.

neuroscience↗