Biology subjects

Cragg, S. J.

Publications and source records attributed to Cragg, S. J..

12 recordsLinked to original sources

bioRxiv2026

Calcium-independent biosensors for multiplexed in vivo imaging of extracellular lactate dynamics

Extracellular L-lactate reports cellular metabolism, but resolving its dynamics alongside intracellular metabolism and neuronal activity remains challenging. Here we develop eLACCO3 and R-eLACCO3, green and red cell-surface lactate biosensors with Ca2+-independent responses and faster response and recovery kinetics in cultured cells. Selected variants provide larger fluorescence responses than their predecessors in brain cells, slices, and living mice and improve detection of glucose-evoked extracellular lactate oscillations. Combining R-eLACCO3 with GCaMP6f enabled imaging of extracellular L-lactate simultaneously with odor-evoked activity in anesthetized mice and cortical activity in awake mice. These biosensors enable multiplexed measurements of extracellular lactate dynamics in relation to intracellular metabolism and neuronal activity.

molecular biology↗

bioRxiv2026

Selective GRAB sensors reveal distinct endocannabinoid dynamics in vivo

The endocannabinoid system modulates diverse physiological processes via two endogenous lipid ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); however, their specific spatiotemporal dynamics remain poorly understood owing to the lack of selective probes. Here, we developed GRAB2-AG2.0 and GRABAEA2.0, two genetically encoded fluorescent sensors that selectively detect 2-AG and AEA, respectively. Both sensors exhibited high apparent affinity and molecular specificity for their respective ligands, enabling the real-time detection of 2-AG and AEA release evoked by electrical stimulation in cultured neurons and acute brain slices. In freely behaving mice, these sensors revealed ligand- and context-specific eCB dynamics: aversive stimulation preferentially evoked 2-AG, whereas psychoactive drugs produced distinct 2-AG and AEA responses. Notably, {Delta}9-THC elicited a sustained 2-AG signal in the nucleus accumbens shell, and local deletion of Dagla markedly attenuated both this signal and {Delta}9-THC-induced hypolocomotion. These sensors therefore enable detecting 2-AG and AEA signaling seperately and reveal an endogenous 2-AG component of the behavioral response to {Delta}9-THC.

neuroscience↗

bioRxiv2026

5-HT4 receptor ligand RS67333 modulates striatal acetylcholine and dopamine release via inhibition of acetylcholinesterase

Serotonin 5-HT4 receptors (5-HT4Rs) have emerged as potential therapeutic targets in neuropsychiatric and neurodegenerative disorders by modulating circuits that shape mood, cognition, and motor function. Ligands for 5-HT4Rs can modify dopamine (DA) and acetylcholine (ACh) transmission but mechanisms and circuits have not been fully resolved. Some 5-HT4R agonists have been suggested to have effects that include inhibition of acetylcholinesterase (AChE), raising speculation that 5-HT4R ligands might modulate ACh and/or DA through this action. Here, we investigated the impact of RS67333, a partial 5-HT4R agonist, on DA and ACh release dynamics in the striatum detected ex vivo in mouse brain slices using fast-scan cyclic voltammetry and genetically encoded ACh sensor GRABACh3.0 respectively. We found that RS67333 significantly modulated electrically evoked DA release in dorsolateral striatum and nucleus accumbens core, effects that were abolished by a nicotinic receptor (nAChR) antagonist. In parallel, RS67333 altered evoked ACh signals by extending extracellular ACh lifetime, and correspondingly, RS67333 was found to inhibit striatal AChE enzymatic activity. By contrast, BIMU8, an alternative 5-HT4R ligand that did not inhibit striatal AChE, had no effect on evoked striatal ACh or DA release. These findings indicate that RS67333 modulates striatal ACh transmission, which shapes downstream regulation of DA release by nAChRs, not through 5-HT4Rs but through AChE inhibition. These findings emphasize the caution due in attributing functions to 5-HT4Rs, but also highlight an alternative pharmacological profile of some purported 5-HT4R ligands as AChE inhibitors of potential utility for treating ACh/DA disorders.

neuroscience↗

bioRxiv2026

Dopamine release from Parkinson's patient-derived neurons is disrupted due to impaired synaptic vesicle loading

Striatal dopamine release defects are an early pathological feature observed in diverse models of Parkinsons disease. However, the underlying molecular mechanisms responsible, and potential links to disease aetiology in humans, have been elusive. Here, we tested the hypothesis that dopamine release deficits are a characteristic feature of disease-relevant human neurons, using human Parkinsons patient iPSC-derived dopamine neurons carrying the SNCA-triplication mutation. We reveal deficits in dopamine release from SNCA-triplication patient-derived neurons, and identify that this is due to reduced dopamine content arising from a lower capacity to store dopamine through reduced expression and function of vesicular monoamine transporter 2 (VMAT2) compared to healthy controls. In turn, by imaging VMAT substrate FFN206, and reporters for synaptic vesicular dynamics, SynaptopHluorin and CypHer5E, we reveal corresponding deficits in the size of either VMAT-containing, presynaptic releasing or recycling vesicle pools. Consistent with diminished synaptic vesicle loading and recycling, the cytosolic turnover of dopamine indicated by the ratio of concentrations of dopamine metabolite DOPAC to dopamine was elevated. By contrast, glutamate release events and VGLUT2 levels in neurons in the same preparations were not disturbed, demonstrating that vesicular dysfunction is limited to vesicles for dopamine. These findings therefore reveal dopamine loading into vesicles as a locus of dysfunction in human Parkinsons-derived neurons. These disturbances will not only drive deficits in dopamine release but could potentially also be detrimental to dopamine neuron viability through an increased burden of oxidative stress associated with elevated cytosolic dopamine, thus contributing to both symptoms and aetiology of Parkinsons pathology and offering a strategic target for improved therapies.

neuroscience↗

bioRxiv2025

Gabapentinoids promote striatal dopamine release and rescue multiple deficits of a mouse model of early Parkinson's

Neuronal entry and handling of intracellular calcium have long-been hypothesised to burden vulnerable dopamine neurons in Parkinsons disease. However, no treatments for Parkinsons target calcium biology. Gabapentinoid drugs bind to 2{delta} subunits of voltage-gated calcium channels (VGCCs) and are licensed for neurological disorders including dopamine-dysregulated restless leg syndrome, suggesting their potential utility to modify both calcium biology and dopamine signalling. We therefore tested whether gabapentinoids modulate dopamine signalling, underlying VGCC-dependence, and potential for treating Parkinsons. In mouse striatum, we reveal that gabapentinoids ex vivo promote dopamine release, via sex-specific dependence on 2{delta}1/2 subunits and alterations to the calcium- and VGCC-subtype-dependence of dopamine release or its tonic inhibition by striatal GABA. In vivo administration of gabapentinoids to a mouse model of early Parkinsons rescued deficits in dopamine release, dysregulation of GABAergic inhibition and dopamine content, and abolished parkinsonian deficits in movement transitions. Thus, gabapentinoids urgently deserve attention for repurposing for Parkinsons disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/675586v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@16cd7e1org.highwire.dtl.DTLVardef@d104e5org.highwire.dtl.DTLVardef@98683eorg.highwire.dtl.DTLVardef@1e3e3ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

The superior colliculus gates dopamine responses to conditioned stimuli in visual classical conditioning

In classical Pavlovian conditioning, it is well-established that midbrain dopamine neurons respond to conditioned stimuli (CS) that predict a reward. However, how the dopamine neurons associate a neutral CS to a reward remains unknown. Here, we show that the superior colliculus (SC) develops neuronal responses to a visual CS during conditioning, which in turn drive the responses of dopamine neurons. Visual responses in the SC were only potentiated when a behaviorally meaningful time interval separated the visual stimulus and reward. Potentiation also required the convergence of visual, dopamine and serotonin inputs to the SC. Importantly, blocking potentiation of the visual response was sufficient to suppress the dopamine responses following a CS. These results reveal a mechanism for how the brain forms associations between unconditioned stimuli and behaviorally meaningful visual information during classical conditioning.

neuroscience↗

bioRxiv2024

Rapid modulation of striatal cholinergic interneurons and dopamine release by satellite astrocytes

Astrocytes are increasingly thought to have underestimated and important roles in modulating neuronal circuits. Astrocytes in striatum can regulate dopamine transmission by governing the extracellular tone of axonal neuromodulators, including GABA and adenosine. However, here we reveal that striatal astrocytes occupy a cell type-specific anatomical and functional relationship with cholinergic interneurons (ChIs), through which they rapidly excite ChIs and govern dopamine release via nicotinic acetylcholine receptors on subsecond timescales. We identify that ChI somata are in unexpectedly close proximity to astrocyte somata, in mouse and human, forming a "soma-to-soma" satellite-like configuration not typically observed for other striatal neurons. Transient depolarization of astrocytes in mouse striatum reversibly regulated ChI excitability by decreasing extracellular calcium. These findings reveal a privileged satellite astrocyte-interneuron interaction for striatal ChIs operating on subsecond timescales via regulation of extracellular calcium dynamics to shape downstream striatal circuit activity and dopamine signaling.

neuroscience↗

bioRxiv2023

Dopamine D2 receptor upregulation in dorsal striatum in the LRRK2-R1441C rat model of early Parkinson's disease revealed by in vivo PET imaging

LRRK2 mutations are the most common cause of dominantly inherited Parkinsons disease (PD). Here, we conducted PET imaging in aged transgenic rats carrying human pathogenic LRRK2 R1441C or G2019S mutations with [18F]FDOPA and dopamine D2/3 receptor ligand [18F]fallypride. We interrogate presynaptic integrity and postsynaptic dopamine receptor availability, and compared these to non-transgenic rats. LRRK2 mutant rats displayed similar [18F]FDOPA uptake to non-transgenic animals, consistent with intact dopamine synthesis in striatal axons. However, LRRK2-R1441C rats demonstrated greater binding of [18F]fallypride than LRRK2-G2019S or non-transgenic controls, exhibiting regionally selective binding increase in the dorsal striatum. Immunocytochemical labelling post-mortem confirmed a greater density of D2 receptors in LRRK2-R1441C than other genotypes restricted to the dorsal striatum, consistent with upregulation of D2-receptors as a compensatory response to the greater dopamine release deficit observed in this genotype. These results show that [18F]fallypride PET imaging is sensitive to dysregulation of dopamine signalling in the LRRK2-R1441C rat, detecting upregulation of D2 receptors that parallels observations in early human sporadic PD. Future studies of candidate therapies could exploit this non-invasive approach to assess treatment efficacy.

neuroscience↗

bioRxiv2021

Dopamine neuron morphology and output are differentially controlled by mTORC1 and mTORC2

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties and whether they act together or independently is unknown. Here we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. As mTOR is involved in several brain disorders caused by dopaminergic dysregulation including Parkinsons disease and addiction, our results have implications for understanding the pathophysiology and potential therapeutic strategies for these diseases.

neuroscience↗

bioRxiv2020

L-type calcium channel contribution to striatal dopamine release is governed by calbindin-D28K, the dopamine transporter, D2-receptors, α2δ-subunits and sex differences

Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes respectively to pacemaker activity and DA release, and has long been thought to contribute to vulnerability to degeneration in Parkinsons disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC-control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, -synuclein, DA transporters (DATs), and calbindin-D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast-scan cyclic voltammetry, and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, -synuclein, DAT, and a dorsolateral co-ordinate, but limited by factors associated with protection i.e. female sex, glucocerebrosidase activity, Calb1, and ventromedial co-ordinate. Together, these data show that LTCC function in DA axons, and isradipine effect, are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/186411v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1ccc783org.highwire.dtl.DTLVardef@13e7296org.highwire.dtl.DTLVardef@d4e3d3org.highwire.dtl.DTLVardef@b9427b_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

Astrocytic striatal GABA transporter activity governs dopamine release and shows maladaptive downregulation in early parkinsonism

Striatal dopamine (DA) is critical for action and learning. Recent data show DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be governed by plasma membrane GABA uptake transporters (GATs) on astrocytes. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs were downregulated and tonic GABAergic inhibition of DA release augmented, with corresponding attenuation of GABA co-release from dopaminergic axons. These data define previously unappreciated and important roles for GATs and astrocytes in determining DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions. HighlightsO_LIGABA transporters set the level of GABA inhibition of DA output in dorsal striatum C_LIO_LIAstrocytes facilitate DA release levels by limiting tonic GABA inhibition C_LIO_LITonic inhibition of DA release is augmented in a mouse model of early parkinsonism C_LIO_LIDA and GABA co-release are reduced in a mouse model of early parkinsonism C_LI

neuroscience↗

bioRxiv2018

Plasticity in striatal dopamine release is governed by release-independent depression and the dopamine transporter

Mesostriatal DA neurons possess extensively branched axonal arbours. Whether action potentials are converted to DA output in striatum will be influenced dynamically and critically by axonal properties and mechanisms that are poorly understood. We addressed the roles for mechanisms governing release probability and axonal activity in determining short-term plasticity of DA release, using fast-scan cyclic voltammetry in ex vivo mouse striatum. Brief short-term facilitation (STF) and longer short-term depression (STD) were only weakly dependent on the level of initial release, i.e. were release-insensitive. Rather, short-term plasticity was strongly determined by mechanisms which governed axonal activation, including K+-gated excitability and the dopamine transporter (DAT), particularly in dorsal striatum. We identify the DAT as a master regulator of DA short-term plasticity, governing the balance between release-dependent and independent mechanisms that also show region-specific gating.\n\nKey FindingsO_LIShort-term plasticity in dopamine release is only weakly governed by initial release\nC_LIO_LIShort-term depression is strongly dependent on axonal excitability and activation\nC_LIO_LIThe dopamine transporter controls short-term plasticity and drives short-term depression\nC_LIO_LIDopamine transporters govern the balance between release-dependent and -independent mechanisms\nC_LI

neuroscience↗