Ca-dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca entry through Ca1.

Ca -dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses.

Bidirectional Neuronal Actuation by Uncaging with Violet and Green Light.

We have developed a photochemical protecting group that enables wavelength selective uncaging using green versus violet light. Change of the exocyclic oxygen of the laser dye coumarin-102 to sulfur, gave thio-coumarin-102, a new chromophore with an absorption ratio at 503/402 nm of 37. Photolysis of thio-coumarin-102 caged γ-aminobutyric acid was found to be highly wavelength selective on neurons, with normalized electrical responses >100-fold higher in the green versus violet channel.

Reverse Engineering Caged Compounds: Design Principles for their Application in Biology.

Light passes through biological tissue, and so it is used for imaging biological processes in situ. Such observation is part of the very essence of science, but mechanistic understanding requires intervention. For more than 50 years a "second function" for light has emerged; namely, that of photochemical control.

GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits.

GluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex.

The yin and yang of intracellular delivery of amphipathic optical probes using -butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible -butyl ester protecting group to deliver amphipathic cargoes to the cytosol.

Local recovery of cardiac calcium-induced calcium release interrogated by ultra-effective, two-photon uncaging of calcium.

Key Points: In cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two-photon photolysis of a new ultra-effective caged calcium compound.

Photochemical control of drug efficacy - a comparison of uncaging and photoswitching ifenprodil on NMDA receptors.

Ifenprodil is an important negative allosteric modulator of the N-methyl-D-aspartate (NMDA) receptors. We have synthesized caged and photoswitchable derivatives of this small molecule drug. Caged ifenprodil was biologically inert before photolysis, UV irradiation efficiently released the drug allowing selective inhibition of GluN2B-containing NMDA receptors.

Intracellular photoswitchable neuropharmacology driven by luminescence from upconverting nanoparticles.

Photoswitchable drugs are small-molecule optical probes that undergo chromatically selective control of drug efficacy using, most often, UV-visible light. Here we report that luminescence produced by near-infrared stimulation of NaYF4:TmYb nanoparticles can be used for "remote control" of an azobenzene-based photochromic ion channel blocker of neurons in living brain slices.

Useful Caged Compounds for Cell Physiology.

Light has been instrumental in the study of living cells since its use helped in their discovery in the late 17th century. Further, combining chemical technology with light microscopy was an essential part of the Nobel Prize for Physiology in 1906. Such landmark scientific findings involved passive observation of cells.

Optofluidic control of rodent learning using cloaked caged glutamate.

Glutamate is the major excitatory neurotransmitter in the brain, and photochemical release of glutamate (or uncaging) is a chemical technique widely used by biologists to interrogate its physiology. A basic prerequisite of these optical probes is bio-inertness before photolysis. However, all caged glutamates are known to have strong antagonism toward receptors of γ-aminobutyric acid, the major inhibitory transmitter.

Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmission.

We have developed photochromic probes for the nicotinic acetylcholine receptor that exploit the unique chemical properties of the tetrafluoroazobenzene (4FAB) scaffold. Ultraviolet light switching and rapid thermal relaxation of the metastable configuration are the main drawbacks associated with standard AB-based switches. We designed our photoprobes to take advantage of the excellent thermodynamic stability of the -4FAB configuration (thermal half-life > 12 days at 37 °C in physiological buffer) and photostationary states above 84%.

Chromatically independent, two-color uncaging of glutamate and GABA with one- or two-photon excitation.

Caged compounds enable fast, light-induced, and spatially-defined application of bioactive molecules to cells. Covalent attachment of a caging chromophore to a crucial functionality of a biomolecule renders it inert, while short pulses of light release the caged molecule in its active form. Caged neurotransmitters have been widely used to study diverse neurobiological processes such as receptor distribution, synaptogenesis, transport, and long-term potentiation.

Optical control of neuronal ion channels and receptors.

Light-controllable tools provide powerful means to manipulate and interrogate brain function with relatively low invasiveness and high spatiotemporal precision. Although optogenetic approaches permit neuronal excitation or inhibition at the network level, other technologies, such as optopharmacology (also known as photopharmacology) have emerged that provide molecular-level control by endowing light sensitivity to endogenous biomolecules. In this Review, we discuss the challenges and opportunities of photocontrolling native neuronal signalling pathways, focusing on ion channels and neurotransmitter receptors.

Dendrimer Conjugation Enables Multiphoton Chemical Neurophysiology Studies with an Extended π-Electron Caging Chromophore.

We have developed a caged neurotransmitter using an extended π-electron chromophore for efficient multiphoton uncaging on living neurons. Widely studied in a chemical context, such chromophores are inherently bioincompatible due to their highly lipophilic character. Attachment of two polycarboxylate dendrimers, a method we call "cloaking", to a bisstyrylthiophene (or BIST) core effectively transformed the chromophore into a water-soluble optical probe, whilst maintaining the high two-photon absorption of over 500 GM.

Locked-Azobenzene: Testing the Scope of a Unique Photoswitchable Scaffold for Cell Physiology.

Azobenzenes are the most widely studied photoswitches, and have become popular optical probes for biological systems. The cis configuration is normally metastable, meaning the trans configuration is always thermodynamically favored. The unique exception to this rule is an azobenzene having a two-carbon bridge between ortho positions, substitutions that lock the photoswitch in its cis configuration.

Two-Photon Uncaging of Glutamate.

Two-photon microscopy produces the excited singlet state of a chromophore with wavelengths approximately double that used for normal excitation. Two photons are absorbed almost simultaneously, via a virtual state, and this makes the excitation technique inherently non-linear. It requires ultra-fast lasers to deliver the high flux density needed to access intrinsically very short lived intermediates, and in combination with lenses of high numerical aperture, this confines axial excitation highly.

Optical probing of acetylcholine receptors on neurons in the medial habenula with a novel caged nicotine drug analogue.

Key Points: A new caged nicotinic acetylcholine receptor (nAChR) agonist was developed, ABT594, which is photolysed by one- and two-photon excitation. The caged compound is photolysed with a quantum yield of 0.20.

Thermodynamically Stable, Photoreversible Pharmacology in Neurons with One- and Two-Photon Excitation.

Photoswitchable bioprobes enable bidirectional control of cell physiology with different wavelengths of light. Many current photoswitches use cytotoxic UV light and are limited by the need for constant illumination owing to thermal relaxation in the dark. Now a photoswitchable tetrafluoroazobenzene(4FAB)-based ion channel antagonist has been developed that can be efficiently isomerized in two separate optical channels with visible light.

Coumarin-diene photoswitches for rapid and efficient isomerization with visible light.

We have developed 7-diethylaminocoumarin-based chromophores that photoisomerize with visible light. These photoswitches possess many desirable attributes, including large extinction coefficients (18 600-59 100 M-1 cm-1), high quantum yields (0.45-0.

Two-color, one-photon uncaging of glutamate and GABA.

Neuronal cells receive a variety of excitatory and inhibitory signals which they process to generate an output signal. In order to study the interaction between excitatory and inhibitory receptors with exogenously applied transmitters in the same preparation, two caging chromophores attached to glutamate and GABA were developed that were selectively photolyzed by different wavelengths of light. This technique has the advantage that the biologically inactive caged compound can be applied at equilibrium prior to the near instantaneous release of the transmitters.

Comparative one- and two-photon uncaging of MNI-glutamate and MNI-kainate on hippocampal CA1 neurons.

Background: The light-induced release of neurotransmitters from caging chromophores provides a powerful means to study the underlying receptors in a physiologically relevant context. Surprisingly, most caged neurotransmitters, including the widely used 4-methoxy-7-nitroindolinyl (MNI)-glutamate, show strong antagonism against GABA-A receptors. Kainate has been shown to exhibit a higher efficacy at glutamate receptors compared to glutamate itself.

Intracellular Uncaging of cGMP with Blue Light.

We have made a new caged cGMP that is photolyzed with blue light. Using our recently developed derivative of 7-diethylaminocourmarin (DEAC) called DEAC450, we synthesized coumarin phosphoester derivatives of cGMP with two negative charges appended to the DEAC450 moiety. DEAC450-cGMP is freely soluble in physiological buffer without the need for any organic cosolvents.

A Critical Role for Astrocytes in Hypercapnic Vasodilation in Brain.

Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO, arterial O, and brain activity and is largely constant in the awake state. Although small changes in arterial CO are particularly potent to change CBF (1 mmHg variation in arterial CO changes CBF by 3%-4%), the coupling mechanism is incompletely understood. We tested the hypothesis that astrocytic prostaglandin E (PgE) plays a key role for cerebrovascular CO reactivity, and that preserved synthesis of glutathione is essential for the full development of this response.

Cloaked Caged Compounds: Chemical Probes for Two-Photon Optoneurobiology.

Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a "cloaking" technology that effectively reduces such antagonism to very low levels.