Engineering, applications, and future perspectives of GPCR-based genetically encoded fluorescent indicators for neuromodulators.

This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity.

ΔFosB accumulation in hippocampal granule cells drives cFos pattern separation during spatial learning.

Mice display signs of fear when neurons that express cFos during fear conditioning are artificially reactivated. This finding gave rise to the notion that cFos marks neurons that encode specific memories. Here we show that cFos expression patterns in the mouse dentate gyrus (DG) change dramatically from day to day in a water maze spatial learning paradigm, regardless of training level. Optogenetic inhibition of neurons that expressed cFos on the first training day affected performance days later, suggesting that these neurons continue to be important for spatial memory recall. The mechanism preventing repeated cFos expression in DG granule cells involves accumulation of ΔFosB, a long-lived splice variant of FosB. CA1 neurons, in contrast, repeatedly expressed cFos. Thus, cFos-expressing granule cells may encode new features being added to the internal representation during the last training session. This form of timestamping is thought to be required for the formation of episodic memories.

Presynaptic cGMP sets synaptic strength in the striatum and is important for motor learning.

The striatum is a subcortical brain region responsible for the initiation and termination of voluntary movements. Striatal spiny projection neurons receive major excitatory synaptic input from neocortex and thalamus, and cyclic nucleotides have long been known to play important roles in striatal function. Yet, the precise mechanism of action is unclear. Here, we combine optogenetic stimulation, 2-photon imaging, and genetically encoded scavengers to dissect the regulation of striatal synapses in mice. Our data show that excitatory striatal inputs are tonically depressed by phosphodiesterases (PDEs), in particular PDE1. Blocking PDE activity boosts presynaptic calcium entry and glutamate release, leading to strongly increased synaptic transmission. Although PDE1 degrades both cAMP and cGMP, we uncover that the concentration of cGMP, not cAMP, controls the gain of striatal inputs. Disturbing this gain control mechanism in vivo impairs motor skill learning in mice. The tight dependence of striatal excitatory synapses on PDE1 and cGMP offers a new perspective on the molecular mechanisms regulating striatal activity.

Freeze-Frame Imaging of Dendritic Calcium Signals With TubuTag.

The extensive dendritic arbor of neurons is thought to be actively involved in the processing of information. Dendrites contain a rich diversity of ligand- and voltage-activated ion channels as well as metabotropic receptors. In addition, they are capable of releasing calcium from intracellular stores. Under specific conditions, large neurons produce calcium spikes that are locally restricted to a dendritic section. To investigate calcium signaling in dendrites, we introduce TubuTag, a genetically encoded ratiometric calcium sensor anchored to the cytoskeleton. TubuTag integrates cytoplasmic calcium signals by irreversible photoconversion from green to red fluorescence when illuminated with violet light. We used a custom two-photon microscope with a large field of view to image pyramidal neurons in CA1 at subcellular resolution. Photoconversion was strongest in the most distal parts of the apical dendrite, suggesting a gradient in the amplitude of dendritic calcium signals. As the read-out of fluorescence can be performed several hours after photoconversion, TubuTag will help investigating dendritic signal integration and calcium homeostasis in large populations of neurons.

Freeze-frame imaging of synaptic activity using SynTagMA.

Information within the brain travels from neuron to neuron across billions of synapses. At any given moment, only a small subset of neurons and synapses are active, but finding the active synapses in brain tissue has been a technical challenge. Here we introduce SynTagMA to tag active synapses in a user-defined time window. Upon 395-405 nm illumination, this genetically encoded marker of activity converts from green to red fluorescence if, and only if, it is bound to calcium. Targeted to presynaptic terminals, preSynTagMA allows discrimination between active and silent axons. Targeted to excitatory postsynapses, postSynTagMA creates a snapshot of synapses active just before photoconversion. To analyze large datasets, we show how to identify and track the fluorescence of thousands of individual synapses in an automated fashion. Together, these tools provide an efficient method for repeatedly mapping active neurons and synapses in cell culture, slice preparations, and in vivo during behavior.

Neuromorphometry of primary brain tumors by magnetic resonance imaging.

Magnetic resonance imaging is a technique for the diagnosis and classification of brain tumors. Discrete compactness is a morphological feature of two-dimensional and three-dimensional objects. This measure determines the compactness of a discretized object depending on the sum of the areas of the connected voxels and has been used for understanding the morphology of nonbrain tumors. We hypothesized that regarding brain tumors, we may improve the malignancy grade classification. We analyzed the values in 20 patients with different subtypes of primary brain tumors: astrocytoma, oligodendroglioma, and glioblastoma multiforme subdivided into the contrast-enhanced and the necrotic tumor regions. The preliminary results show an inverse relationship between the compactness value and the malignancy grade of gliomas. Astrocytomas exhibit a mean of [Formula: see text], whereas oligodendrogliomas exhibit a mean of [Formula: see text]. In contrast, the contrast-enhanced region of the glioblastoma presented a mean of [Formula: see text], and the necrotic region presented a mean of [Formula: see text]. However, the volume and area of the enclosing surface did not show a relationship with the malignancy grade of the gliomas. Discrete compactness appears to be a stable characteristic between primary brain tumors of different malignancy grades, because similar values were obtained from different patients with the same type of tumor.

[Beta-1 adrenoceptor blockade decreases the firing rate to painful stimuli in spinal wide-dynamic range neurons in rats]. / Los bloqueadores beta-1 adrenérgicos reducen la respuesta a estímulos dolorosos en neuronas de amplio rango dinámico de la médula espinal en ratas.

BACKGROUND: It is known that epinephrine/norepinephrine inhibit acute pain transmission. However, the role of ß-adrenoceptors is not clear. Thus, we analyzed if beta-1 and/or beta-2 adrenoceptors can modulate acute pain transmission by performing in vivo single unit recordings during painful and non-painful peripheral stimulation in rats. METHODS: Longitudinal study in which we analyzed seven groups of male rats Wistar: control group (n = 11): saline (0.9 %); EPI group (n = 8): epinephrine 100 mcg; beta-1 agonist group (n = 8): dobutamine 125 mcg; beta-1-antagonist group (n = 9): metoprolol 100 mcg; beta-2-agonist group (n = 7): clenbuterol 100 mcg; beta-2-antagonist group (n = 8): butoxamine 100 mcg; beta-1-antagonist + EPI group (n = 10): metoprolol 100 mcg + epinephrine 100 mcg. For the statistical analysis we used ANOVA. RESULTS: Epinephrine significantly reduced the basal firing rate (BFR) in 34.1 % (p < 0.05) and also the evoked response by painful stimulation in 56 % (p < 0.05). No change was observed in the evoked response by non-painful stimulation. ANTß1 was the only beta-adrenoceptor acting drug that significantly reduced the evoked response by painful stimulation in 41 % (p < 0.05). None of the other drugs alone affected either the BFR or the evoked response to non-painful or painful stimulation. CONCLUSIONS: It is the first time that a beta-1-adrenoceptor antagonist (metoprolol) probes to be effective in reducing the response to painful stimulation in WDR neurons.

Introducción: la epinefrina/norepinefrina inhibe la transmisión del dolor agudo; empero, no es claro el papel de los receptores beta-adrenérgicos. Por tanto, analizamos si los fármacos de estos receptores modulan la transmisión del dolor agudo mediante registro electrofisiológico unitario extracelular in vivo durante estimulación periférica dolorosa y no dolorosa en ratas. Métodos: estudio longitudinal en el que se cotejaron siete grupos de ratas: control (n = 11): solución salina (0,9 %); EPI (n = 8): 100 mcg epinefrina; agonista beta-1 (n = 8): 125 mcg dobutamina; antagonista beta-1 (n = 9): 100 mcg metoprolol; agonista beta-2 (n = 7): 100 mcg clembuterol; antagonista beta-2 (n = 8): butoxamina 100 mcg; antagonista beta-1 + EPI (n = 10): 100 mcg metoprolol + 100 mcg epinefrina. Se hizo análisis estadístico por medio de ANOVA. Resultados: La epinefrina redujo significativamente la tasa de disparo basal (RDB) en 34.1 % (p < 0.05) y la respuesta evocada por la estimulación dolorosa en 56 % (p < 0.05). No hubo cambios en la respuesta provocada por la falta de estimulación dolorosa. El antagonista beta-1 fue el único fármaco con acción beta-adrenérgica que redujo significativamente la respuesta evocada por la estimulación dolorosa en 41 % (p < 0.05). Conclusión: por primera vez un antagonista de los receptores beta-1-adrenérgicos (metoprolol) prueba ser eficaz en la reducción de la respuesta a la estimulación dolorosa en las neuronas ARD.