Biology subjects

Patriarchi, T.

Publications and source records attributed to Patriarchi, T..

At least 19 recordsLinked to original sources

bioRxiv2026

A chemigenetic fluorescence lifetime-based biosensor for imaging of ERK activity

Extracellular signal-regulated kinase (ERK) governs many cellular processes, and its spatiotemporal activity dynamics are important for downstream signaling specificity. Existing fluorescent ERK activity biosensors, however, fall short in enabling spectrally multiplexed, spatially resolved, or deep tissue measurements. Here, we introduce a chemigenetic, single-fluorophore biosensor, WHaloEKAR, that reports ERK activities through changes in fluorescence lifetime. WHaloEKAR combines large lifetime changes (-0.81 ns) with the spectral versatility of the self-labeling protein HaloTag giving access to orange, red, and far-red ERK biosensors. The latter enabled the investigation of heterogeneous ERK activity at distinct subcellular locations in cultured cell lines and primary cortical rat neurons via fluorescence lifetime imaging microscopy (FLIM). Moreover, multiplexed FLIM measurements allowed to study ERK activity in parallel to the second messengers calcium and cyclic adenosine monophosphate. Finally, WHaloEKAR enabled the investigation of ERK activity in 3D samples as well as in live animals.

cell biology↗

bioRxiv2026

An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function

Opioid receptors (ORs) orchestrate pain relief, reward, and dependence, yet their signaling arises from diverse cell types and subcellular compartments that cannot be selectively interrogated with existing pharmacological or genetic approaches. Single-domain antibodies, or nanobodies (Nbs), can probe receptor states, but their potential as tools for controlling native receptor signaling at the system level has remained unexplored. Here, we engineer a suite of high-affinity intracellular Nbs that bind active ORs through structure-guided evolution and in silico design. Iterative optimization yields Nb64, a potent inhibitor that rapidly suppresses transducer engagement, receptor internalization, and downstream signaling, including endogenous pathways in neuronal cells. Organelle targeting highlights Nb64s capacity to control OR activity with subcellular precision, while bio-reversible cell-penetrating peptide (CPP) conjugation enables non-genetic cytosolic delivery. Cell-type-specific expression of Nb64 in VTA interneurons attenuates fentanyl-evoked dopamine release and behavioral responses in mice, demonstrating targeted control of opioid actions in vivo. Nb64 provides a versatile strategy for dissecting OR biology and establishes a generalizable framework for precision inhibition of native GPCR signaling in vivo.

neuroscience↗

bioRxiv2025

Temporal dynamics of noradrenaline release at fine spatial scales during motor learning

Noradrenaline (NA) released from the locus coeruleus (LC) has been known to play pivotal roles in arousal, sensory processing, decision-making, and learning through global release across the entire neocortex. Recent studies have demonstrated heterogeneous and modular NA release in distinct brain regions and highlighted its spatiotemporal dynamics across the neocortex. However, the spatiotemporal specificity of NA release at fine scales within a single brain region remains unclear, and it has not been reported whether the release patterns evolve functionally throughout prolonged learning processes such as motor skill acquisition. Here, by employing in vivo two-photon imaging with various genetically encoded NA sensors, we reveal that behavior-induced NA release in the primary motor cortex (M1) is spatially heterogeneous at the scale of local microcircuitry. Over the course of learning, the release pattern is locally refined, achieving consistent spatial precision within M1. Intriguingly, pharmacological manipulations that disrupt the spatial specificity also alter local neurons activity and representations. Furthermore, LC-NA axonal calcium imaging uncovered two distinct temporal activity patterns, in which non-behavior-related rapid axonal activity (sub-second duration events) profoundly affect the temporally precise behavior-induced persistent axonal activity (seconds duration events). Closed-loop optogenetic manipulations that bi-directionally modulate non-behavior-related rapid events directly impacted the learning process. Together, our results provide novel insights into the temporal dynamics of NA release at fine spatial scales within one brain region and underscore the critical role of local NA specificity in regulating circuit plasticity during motor skill acquisition.

neuroscience↗

bioRxiv2025

Experience-dependent sharp-wave ripple deficits in an Alzheimer's disease mouse model

Amyloid pathology is a hallmark of Alzheimers disease (AD). Hippocampal sharp-wave ripples (SWRs) play a role in memory consolidation and are impaired in various AD mouse models. However, it remains unclear how experience affects SWRs and how extrinsic signals contribute to SWR generation in AD. Here, by combining behavioral, in vivo electrophysiological and fiber photometry approaches, we show that an experience-dependent increase in hippocampal SWRs is disrupted in male and female 5xFAD mice. In wild-type mice, SWRs during non-rapid eye movement sleep (NREMS) increased after exploring a novel environment and the SWR rate gradually decreased with NREMS episodes and multiple behavioral sessions. However, 5xFAD mice did not show such experience-dependent SWR rate changes. A similar deficit was observed after a novel object recognition test. On the other hand, sleep spindles were intact in 5xFAD mice under all conditions. Since deficits in basal forebrain cholinergic neurons have been implicated in 5xFAD mice and SWRs are regulated by hippocampal cholinergic tone, we examined if hippocampal cholinergic signals could explain experience-dependent SWR deficits. Using fiber photometry and expressing a genetically encoded acetylcholine (ACh) sensor in the hippocampus, we found that ACh dynamics in the hippocampus were intact in 5xFAD mice across sleep-wake cycles, including NREMS, while we also found a negative correlation of infraslow cortical signal power dynamics with hippocampal ACh signals during NREMS regardless of genotypes. These results suggest that experience-dependent SWR deficits stem from non-cholinergic pathological changes.

neuroscience↗

bioRxiv2025

Slow-Timescale Regulation of Dopamine Release and Mating Drive over Days

The rise and fall of motivational states may take place over timescales as long as many days. We used mouse mating behavior to model how the brain orchestrates slow-timescale changes in motivation. Male mice become sexually satiated after successful matings, and their motivation to mate gradually recovers over a week. Using deep-brain fluorescence-lifetime imaging in the medial preoptic area (MPOA), we found that tonic dopamine transmission--which regulates mating drive--also declined after mating and re-emerged over a week. Two mechanisms regulated dopamine transmission. First, successful mating transiently reduced tonic firing of hypothalamic dopamine-releasing neurons, thereby inhibiting dopamine release and mating behavior. Second, mating reduced the ability of these neurons to produce and release dopamine, and this ability gradually returned over the week-long recovery time course. Therefore, fast and slow mechanisms of neuronal plasticity cooperate to control the early and late phases of motivational dynamics, respectively.

neuroscience↗

bioRxiv2025

Effects of altered gravity on adrenergic-mediated cAMP signalling in intact cells

Spaceflight-induced cardiac atrophy and rhythm disorders are linked to dysregulation of the adrenergic-cAMP-PKA pathway. Gravity-dependent alterations in adrenergic signaling, particularly cAMP dynamics, remain poorly understood. Using fluorescence biosensors, we studied intact cells under simulated microgravity and hypergravity. We observed shifts in the EC50 of cAMP production: leftward under hypergravity and rightward in microgravity, with faster cAMP accumulation kinetics in hypergravity. Cytoskeletal remodelling, hypothesized to be a determinant of such chances, was negligible, suggesting alternative mechanisms. These findings highlight significant gravity-induced offsets in the pharmacology of a prototypical GPCR, with implications not only for adrenergic signalling but also for other pathways of pharmacological interest, potentially informing countermeasures for astronaut health and pharmacology in altered gravity settings.

cell biology↗

bioRxiv2025

Neurometabolic signaling and control of policy complexity

Cognition and adaptive behavior emerge from neural information processing. This must operate within finite metabolic constraints, since neural information processing is metabolically expensive. While neural implementations of action selection and learning are well-studied, systems allocating the informational capacity required to encode complex behavioral policies remain unknown. We hypothesized that hypothalamic hypocretin/orexin neurons (HONs) are uniquely positioned to signal and control policy complexity, given that they are activated by metabolic depletion and influence decision-making systems. To explore this, we employed a set of cell/neurotransmitter-specific imaging and causal manipulations during a multi-armed bandit task where freely behaving mice learned probabilistic state-action-reward relationships (together [~]100,000 decisions from >100 mice). Miniscope recordings of HON activity revealed that pre-choice, but not post-choice activity correlates with decision policy, dissociating decisions from feedback. Furthermore, manipulating HON signals with optogenetics and pharmacology confirmed that they causally regulate the development of complex policies. Finally, neurotransmitter-specific sensors revealed that hypocretin/orexin receptors modulate decision policy-related dopamine and noradrenaline dynamics in the nucleus accumbens and medial prefrontal cortex. These findings identify HONs as subcortical regulators of policy complexity, encoding critical signals for decision-making adjustments. This opens a new window for the development of comprehensive mechanistic models of strategic learning which account for interplay between decision-making policies and metabolic/informational constraints, and may guide the development of potential treatments for disorders with policy deficits such as autism and schizophrenia.

neuroscience↗

bioRxiv2024

PinkyCaMP a mScarlet-based calcium sensor with exceptional brightness, photostability, and multiplexing capabilities

Genetically encoded calcium (Ca2+) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches. Here, we present the development of PinkyCaMP, the first mScarlet-based Ca2+ sensor that outperforms current red fluorescent sensors in brightness, photostability, signal-to-noise ratio, and compatibility with optogenetics and neurotransmitter imaging. PinkyCaMP is well-tolerated by neurons, showing no toxicity or aggregation, both in vitro and in vivo. All imaging approaches, including single-photon excitation methods such as fiber photometry, widefield imaging, miniscope imaging, as well as two-photon imaging in awake mice, are fully compatible with PinkyCaMP.

neuroscience↗

bioRxiv2024

Innervation density governs crosstalk of GPCR-based norepinephrine and dopamine sensors

GPCR-based fluorescent sensors are widely used to correlate neuromodulatory signaling with brain function. While experiments in transfected cells often reveal selectivity for individual neurotransmitters, sensor specificity in the brain frequently remains uncertain. Pursuing experiments in brain slices and in vivo, we find that norepinephrine and dopamine cross-activate the respective sensors. Non-specific activation occurred when innervation of the cross-reacting transmitter was high, and silencing of specific innervation was indispensable for interpreting sensor fluorescence.

neuroscience↗

bioRxiv2024

Distinct bandwidths of orexin neuron activity independently encode body movement and metabolic state

Tracking net body movement in real time may enable the brain to estimate ongoing demands and thus better orchestrate muscle tone, energy balance, and arousal. To identify neural populations specializing in tracking net body movement, here we compared self-initiated movement-related activity across genetically-defined subcortical neurons in the mouse brain, including dopaminergic, glutamatergic, noradrenergic, and key peptidergic neurons. We show that hypothalamic orexin/hypocretin-producing neurons (HONs) are exceptionally precise movement-trackers, encoding net body movement across multiple classified behaviors with a high degree of precision, independent of head acceleration. This tracking was so precise, that video analysis of the mouse body movement reliably served as a low-cost biometric for HON population activity. The movement tracking was independent of internal nutritional states, and occurred in a communication bandwidth distinct from HON encoding of blood glucose. At key projection targets, orexin/hypocretin peptide outputs correlated with self-initiated movement in a projection-specific manner, indicating functional heterogeneity in HON outputs. Finally, we found that body movement was not encoded to the same extent in other key neural populations related to arousal or energy. These findings indicate that subcortical orchestrators of arousal and metabolism are finely tuned to encode net body movement, constituting a bridge multiplexing ongoing motor activity with internal energy resources.

neuroscience↗

bioRxiv2024

Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model for Parkinson's disease

Parkinsons disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, several follow-up studies have questioned the validity of this astrocyte to DAN conversion model. In this study, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, we observed that PTBP1 downregulation in neurons of the substantia nigra pars compacta and striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of the TH-positive cells in the striatum co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where neuronal, but not astrocytic, downregulation of PTBP1 can mitigate symptoms in PD mice.

neuroscience↗

bioRxiv2024

Multimodal intrinsic activation of GPCRs in ultrastable plasma membrane nanodomains

G protein-coupled receptors (GPCRs) mediate many physiological functions and are key targets in drug development1-3. A long-held tenet of molecular pharmacology is that GPCRs can spontaneously sample preexisting active conformations. This concept is pivotal to our understanding of ligand pharmacology4, however, direct evidence supporting it has only been obtained with reconstituted receptors5-12. Here, we introduce a method for quantitatively imaging the intrinsic activation probability of GPCRs directly at the plasma membrane of live cells, utilizing fluorescent conformational biosensors13,14. Our findings unveil a remarkable spatial multimodality in intrinsic activation probability, with a significant majority (up to 99%) of plasma membrane-expressed receptors showing negligible spontaneous activation. In contrast, the remaining minority of receptors exhibits spontaneous activation up to 22-fold higher than previously estimated. Experiments and theoretical calculations revealed that receptors diffuse into and out of ultralong-lived ([~]5 minutes) nanodomains where the local membrane curvature allosterically enhances activation in the absence and presence of ligands. Extensive testing across five prototypic GPCRs indicates spatial nanoscale multimodality is ubiquitous, but varying in magnitude depending on the receptor and cell type. Upending conventional wisdom, this study reveals that drug efficacy is not a constant number but a spatiotemporal function {varepsilon} (x, y, z, t) whose properties define and multiplex the signaling potency and efficacy of ternary complexes of GPCRs and likely other plasma membrane-receptors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/582451v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e4680borg.highwire.dtl.DTLVardef@16aaebcorg.highwire.dtl.DTLVardef@f5df52org.highwire.dtl.DTLVardef@18a5ea_HPS_FORMAT_FIGEXP M_FIG C_FIG GPCR spontaneous activation and intrinsic efficacy are not uniform across the plasma membrane but exhibit ultralong-lived spatial multimodality. Spatial variations in the curvature and composition of the plasma membrane, lead to the emergence of ultralong-lived nanodomains with contrasting physicochemical properties that allosterically regulate GPCR conformations. This results in a multimodal landscape of intrinsic efficacy{epsilon} (x, y, z, t) that ultimately governs cell signaling. XY scalebar: 500 nm. Z-range: 100 nm.

biophysics↗

bioRxiv2024

Probing PAC1 receptor activation across species with an engineered sensor

Class-B1 G protein-coupled receptors (GPCRs) are an important family of clinically relevant drug targets that remain difficult to investigate via high-throughput screening and in animal models. Here, we engineered PAClight1P78A, a novel genetically-encoded sensor based on a class-B1 GPCR (the human PAC1 receptor, hmPAC1R) endowed with high dynamic range ({Delta}F/F0 = 1100%), excellent ligand selectivity and rapid activation kinetics ({tau}ON = 1.15 sec). To showcase the utility of this tool for in vitro applications, we thoroughly characterized and compared its expression, brightness and performance between PAClight1P78A transfected and stably-expressing cells. Demonstrating its use in animal models, we show robust expression and fluorescence responses upon exogenous ligand application ex vivo and in vivo in mice, as well as in living zebrafish larvae. Thus, the new GPCR-based sensor can be used for a wide range of applications across the life sciences empowering both basic research and drug development efforts.

neuroscience↗

bioRxiv2023

Stimulation of VTA dopamine inputs to LH upregulates orexin neuronal activity in a DRD2-dependent manner

Dopamine and orexins (hypocretins) play important roles in regulating reward-seeking behaviors. It is known that hypothalamic orexinergic neurons project to dopamine neurons in the ventral tegmental area (VTA), where they can stimulate dopaminergic neuronal activity. Although there are reciprocal connections between dopaminergic and orexinergic systems, whether and how dopamine regulates the activity of orexin neurons is currently not known. Here we implemented an opto-Pavlovian task in which mice learn to associate a sensory cue with optogenetic dopamine neuron stimulation to investigate the relationship between dopamine release and orexin neuron activity in the LH. We found that dopamine release can be evoked in LH upon optogenetic stimulation of VTA dopamine neurons, and is also naturally evoked by cue presentation after opto-Pavlovian learning. Furthermore, orexin neuron activity could also be upregulated by local stimulation of dopaminergic terminals in the LH in a way that is partially dependent on dopamine D2 receptors (DRD2). Our results reveal previously unknown orexinergic coding of reward expectation and unveil an orexin-regulatory axis mediated by local dopamine inputs in the LH. HighlightsO_LIOptical VTA DA neuron stimulation is sufficient to elicit a Pavlovian-like dopamine transient in the NAc C_LIO_LIDopamine in the LH encodes both negative and positive reward prediction errors C_LIO_LIDopamine in the LH positively modulates orexin neuronal activity locally in a D2R dependent way C_LI

neuroscience↗

bioRxiv2023

Development of a genetically-encoded sensor for probing endogenous nociceptin opioid peptide release

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

neuroscience↗

bioRxiv2023

Prime editing of the beta-1 adrenoceptor in the brain reprograms mouse behavior

Prime editing is a highly versatile genome editing technology that holds great potential for treating genetic diseases1, 2. While in vivo prime editing has recently been conducted in the brain, liver, heart, and retina3-6, application of this technology to modulate neural circuits in the brain has not been reported yet. Here, we employ adeno-associated viral vectors to deliver optimized intein-split prime editors into the brain of mice. Delivery into newborn pups via intracerebroventricular injection resulted in up to 44.0% editing at the Dnmt1 locus in the cortex (on average 34.8{+/-}9.8% after 6 months). In addition, we obtained up to 28.1% editing at the Adrb1 locus in the cortex (on average 14.7{+/-}11.6% after 6 months). The introduced Adrb1A187V mutation is a naturally occurring variant of the {beta}1-adrenergic receptor, which has previously been linked to increased activity and natural short sleep7. Similarly, we observed an increase in the activity and exploratory behavior of treated animals. This study demonstrates the potential of prime editing for treating genetic diseases in the central nervous system and for reprogramming molecular pathways that modulate animal behavior.

genetics↗

bioRxiv2023

Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution

The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here we introduce a novel genetically-encoded sensor based on the engineering of a circularly-permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max {Delta}F/F0 = 528%) and temporal resolution ({tau}ON = 4.7 sec). We further demonstrated that GLPLight1 shows comparable responses to GLP-1 derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.

biochemistry↗

bioRxiv2022

Locus Coeruleus firing patterns selectively modulate brain activity and dynamics

Noradrenaline (NA) release from the brainstem nucleus locus coeruleus (LC) changes activity and connectivity in neuronal networks across the brain, thus modulating multiple behavioural states. NA release is mediated by both tonic and burst-like neuronal LC activity. However, it remains unknown whether the functional changes in downstream projection areas depend on these firing patterns. Using optogenetics, pupillometry, photometry, and functional MRI in mice, we show that tonic and burst LC firing patterns elicit brain responses that are specific to the activation frequency and temporal pattern. Tonic activation of the LC evokes nonlinear responses in prefrontal, limbic, and cerebellar regions, in line with the proposed inverted-U relationship between LC activity and behaviour. We further demonstrate that LC activity enhances network integration and acts as a facilitator of brain state transitions, hence increasing brain flexibility. Together, these findings reveal how the LC-NA system achieves a nuanced regulation of global circuit operations.

neuroscience↗