Synthesis of photolabile precursors of amino acid neurotransmitters

Wilcox, M. E.; Viola, Randall W.; Johnson, Katherine Waltman; Billington, Andrew P.; Carpenter, Barry K.; McCray, James A.; Guzikowski, Anthony P.; Hess, George P.

doi:10.1021/jo00292a038

Cited by 126 publications

(75 citation statements)

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“…Photostimulation. A particularly attractive way of delivering pulses of agonist is to photorelease the active compound from an inactive, photolabile precursor (24)(25)(26)(27)(28). One such precursor, the DMNPE ester of ATP (18,24), was available from a commercial source; another was synthesized by reacting capsaicin with DMNB chloroformate to attach a DMNB blocking group in carbonate ester linkage to the phenolic hydroxyl function of capsaicin (Fig.…”

Section: Resultsmentioning

confidence: 99%

Photochemical gating of heterologous ion channels: Remote control over genetically designated populations of neurons

Zemelman

Nesnas

Lee

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

227

164

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Biochemistry has led the way in reducing what once were considered irreducible biological phenomena to fundamental physico-chemical principles. Keys to this accomplishment were the ability to trace the transformations of defined chemical substrates in cellular extracts or intact cells and the reconstitution of biochemical processes with pure components. For neuroscience to follow this reductionist lead, analogous experimental strategies need to be developed: an ability to trace the transformations of defined neuronal activity patterns in explanted neural tissues or intact nervous systems and a capacity to reconstitute neurally encoded information and the behaviors it guides with synthetic neural signals.The initial hurdle to both of these strategies lies in the difficulty of feeding artificial neural signals into functionally circumscribed but anatomically dispersed populations of neurons. An organic solution to this problem is to harness proteins mediating neuronal excitation as conduits for artificial stimuli and to restrict the expression of these transducers genetically to a predetermined group of target neurons (1, 2). If, for example, a set of generalist neurons could be programmed to express signal transduction machinery that normally is present only in specialized sensory cells, these neurons also might acquire the capacity to respond selectively to the adequate physical or chemical triggers. Not only could all members of a population of neurons then be addressed simultaneously, susceptibility to stimulation rather than the stimulus itself would be localized, and even diffusely broadcast stimuli could elicit precise, patterned responses (1, 2).Neurons offer two principal routes for the transduction of excitatory signals (3). Metabotropic signaling systems consist of heptahelical receptors that communicate with their effectors through heterotrimeric G proteins. The activation of some of these effectors is coupled to changes in membrane potential (3-7). Ionotropic signaling systems effect changes in membrane potential directly, via chemically or physically gated conductances (3). Previously, we have used components of a metabotropic system, the phototransduction cascade of the fruit fly (7), to sensitize vertebrate neurons to light (2). Expression of what was termed ''chARGe'' to commemorate the essential elements (arrestin-2, rhodopsin, and a G protein ␣-subunit) created a light-controlled source of depolarizing current whose activation sparked action potentials (2). We now introduce the use of ionotropic mechanisms for the selective chemical and optical stimulation of genetically designated populations of neurons. In addition to multiple trigger modes, these ionotropic systems possess numerous practical advantages over chARGe, including simplicity, fast kinetics, and broad tunability. Materials and MethodsHeterologous Expression of Ion Channels. Candidate ion channels were expressed under the control of the cytomegalovirus promoter in pCI-fluor, a derivative of the mammalian expression vector pCI-neo (Pr...

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Section: Resultsmentioning

confidence: 99%

Photochemical gating of heterologous ion channels: Remote control over genetically designated populations of neurons

Zemelman

Nesnas

Lee

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

227

164

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“…o-NB groups are widely used as photocages in the presence of live cells, [8][9][10][11][12] and also as degradable linkages in polymer materials 13 . Several different o-NB groups have been reported [14][15][16] , and small modifications to the structure of the o-NB linker have a significant impact on the photolysis rate.…”

Section: Introductionmentioning

confidence: 99%

Photodegradable Macromers and Hydrogels for Live Cell Encapsulation and Release

Griffin¹,

Kasko²

2012

J. Am. Chem. Soc.

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Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extra-cellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (k app ) of each via photorheology at 370 nm, 10 mW/cm 2 . Decreasing the number of aryl ethers on the o-NB group increases k app , and changing the functionality from primary to seconday at the benzylic site dramatically increases k app . Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability post-encapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs).

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“…Furthermore, the performance of their photostimulation probes varied between cell types and was occasionally species-specific. Here we report development of a photostimulation method, based on the use of caged glutamate (8) adult, from the visual cortex of ferrets aged 4-5 weeks postnatal, or from the visual cortex of adult cats. Slices (400 ,um thick) were cut using a specialized slicer and maintained at 33-34°C in an interface holding chamber as described (1).…”

mentioning

confidence: 99%

“…At least 1 h after cutting, individual slices were moved to a recording chamber on the fixed stage of an inverted microscope and submerged in ACSF containing 1 mM L-glutamic acid a-(4,5-dimethoxy-2-nitrobenzyl) ester [caged glutamate (8), obtained from Molecular Probes] at room temperature. In some control experiments, the ACSF contained no caged glutamate.…”

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confidence: 99%

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Photostimulation using caged glutamate reveals functional circuitry in living brain slices.

Callaway

Katz

1993

Proc. Natl. Acad. Sci. U.S.A.

370

282

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An approach for high-spatial-resolution mapping of functional circuitry in living mammalian brain slices has been developed. The locations of neurons making functional synaptic connections to a single neuron are revealed by photostimulation of highly restricted areas of the slice (50-100 ,um in diameter) while maintaining a whole-cell recording of the neuron of interest. Photostimulation is achieved by bathing brain slices in a molecularly caged form ofthe neurotransmitter glutamate [L-glutamic acid a-(4,5-dimethoxy-2-nitrobenzyl) ester], which is then converted to the active form by brief pulses (<1 ms in duration) of ultraviolet irradiation. Direct activation ofreceptors on recorded neurons in rat hippocampus and ferret visual cortex demonstrates that photostimulation is reliable and reproducible and can be repeated at the same site at least 30 times without obvious decrement in neuronal responsiveness. Photostimulation of presynaptic neurons at sites distant to the recorded neuron evoked synaptic responses in hippocampal and cortical cells at distances of up to several millmeters from the recorded neuron. Stimulation of 25-100 distinct presynaptic sites while recording from a single postsynaptic neuron was easily achieved. Caged glutamate-based photostimulation eliminates artifacts and limitations inherent in conventional stimulation methods, including stimulation of axons of passage, desensitization, and poor temporal resolution of "puffer" pipettes, and current artifacts of iontophoretic application. This approach allows detailed physiological investigation and manipulation of the complex intrinsic circuitry of the mammalian brain.Understanding the detailed organization of neuronal networks is important for elucidating computations responsible for brain function. The limitations of presently available methods for revealing such circuits are especially apparent in the study of detailed local circuits, as in the mammalian cerebral cortex, in which very closely spaced neurons often make up distinctly different components of the network (1). Conventional physiological and anatomical methods, such as current source density analysis (2, 3), single-unit recordings, cross-correlation analyses, intracellular recording, and dye filling (4-6), allow only limited insight into the connectivity of neuronal assemblies. To overcome these limitations, Farber and Grinvald (7) developed an approach based on lightbased neuronal stimulation ("photostimulation") for dissecting neuronal networks. They designed photostimulation molecules (analogs of voltage-sensitive dyes) that allowed stained neuronal elements to be activated by small spots of laser light and were able to sequentially stimulate numerous neurons in invertebrate ganglionic preparations to identify those that were presynaptic to an intracellularly impaled neuron. Since single neurons could easily be targeted for stimulation in the isolated ganglia, the fact that their methodThe publication costs of this article were defrayed in part by page charge payment. T...

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Synthesis of photolabile precursors of amino acid neurotransmitters

Cited by 126 publications

References 0 publications

Photochemical gating of heterologous ion channels: Remote control over genetically designated populations of neurons

Photochemical gating of heterologous ion channels: Remote control over genetically designated populations of neurons

Photodegradable Macromers and Hydrogels for Live Cell Encapsulation and Release

Photostimulation using caged glutamate reveals functional circuitry in living brain slices.

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