Photochemical and pharmacological evaluation of 7-nitroindolinyl-and 4-methoxy-7-nitroindolinyl-amino acids as novel, fast caged neurotransmitters
Introduction
Activation of synaptic transmission in the nervous system generally occurs on a sub-micron spatial scale with a sub-millisecond time scale and experimental approaches to synaptic function ideally require similar precision. A useful strategy for investigating the kinetics and distribution of synaptic processes is the photolytic release of neurotransmitters from caged precursors (Corrie and Trentham, 1993). This technique overcomes the problems of slow access and diffusional mixing, especially when working with brain slices or intact parts of the nervous system. It has been used in the nervous system to identify the neurotransmitter at the squid giant synapse (Corrie et al., 1993, Corrie and Trentham, 1993), to investigate the activation of post-synaptic receptors (Grewer, 1999, Grewer et al., 2000, Canepari et al., 2001b), and to map the connections between different regions in brain slices (Callaway et al., 1993, Kötter et al., 1998). However, despite the use of this technique in the last decade, development of stable caged neurotransmitters which are rapid and efficient in photorelease of neuroactive amino acids has posed difficulties, mainly in the development of stable precursors of amino acids.
A useful caged neurotransmitter must be thermally stable (i.e. it must not hydrolyse in aqueous solution), and fast and efficient in photorelease with respect to synaptic time scales and concentrations. Furthermore the caged precursor, the photolytic intermediates and by-products must be biologically inert and must have minimal interaction with the receptors, transporters and metabolism of the released neurotransmitter. Nor should they interfere with other components of synaptic transmission at the concentrations used. Many of these criteria have not been satisfied by the caged compounds used in the past. Among the best reagents the α-carboxy-2-nitrobenzyl-caged compounds have been found to be efficient (Qp≥0.1 at 308 nm) and fast (half time of the order of μs) in photorelease, but quite unstable in solution, with half time for hydrolysis (t1/2) of a few hours at pH 7 and room temperature (Wieboldt et al., 1994, Grewer et al., 2000). p-Hydroxyphenacyl esters of glutamate and GABA undergo photorelease on a sub-microsecond time scale (Givens et al., 1997) but are inefficient except under relatively short wavelength (308 nm) irradiation (Geibel et al., 2000). In contrast, the stable N-1-(2-nitrophenyl)ethoxycarbonyl-l-glutamate was slow in photorelease, with a half time ≥10 ms at pH 7 (Corrie et al., 1993, Corrie and Trentham, 1993).
Recently, 1-acyl-7-nitroindoline (NI) derivatives that release l-glutamate or other carboxylates have been described (Papageorgiou et al., 1999). These reagents are highly resistant to spontaneous hydrolysis, which is negligible at physiological pH and have t1/2≥6 h for hydrolysis at pH 12 and 30°C. The half time of the photorelease process was previously estimated as ≤0.26 ms (pH 7, 20°C) but more recent work shows that the true half-time for photorelease is in the sub-μs time domain (P. Wan, J. Morrison and J.E.T. Corrie, unpublished data). The NI-caged l-glutamate has been used to investigate the current generated by the activation of metabotropic glutamate receptors in cerebellar Purkinje neurones in slices (Canepari et al., 2001b). Subsequently, a study of substituent effects has shown that photorelease from derivatives based on 4-methoxy-7-nitroindoline (MNI) is 2–3-fold more efficient than from NI-caged compounds because of enhancements in both the absorption coefficient and the quantum yield (Papageorgiou et al., 2000, Canepari et al., 2001a). The MNI-caged compounds have the same resistance to hydrolysis and the same photochemistry, so are expected also to have rapid photorelease.
Characterisation of the photochemical and pharmacological properties of the NI-caged l-glutamate, GABA, glycine and an inert control compound, and of the MNI-caged l-glutamate are reported here. The structures of the NI-caged neurotransmitters and the MNI-caged l-glutamate are shown in Scheme 1. Methods to estimate the concentration of released products and to test the activation of ionotropic receptors by photorelease of neurotransmitters and the pharmacology of the caged precursors are described. The relative photorelease efficiencies of the NI-caged and MNI-caged neurotransmitters are also discussed. Part of this work has been presented in abstract form (Canepari et al., 2001a).
Section snippets
Cell preparation and external solutions
Patch clamp recordings were made from cerebellar granule cells, hippocampal pyramidal neurones or spinal cord neurones in culture, and from cerebellar Purkinje neurones in sagittal slices from 20 day rats. Cerebellar granule cell explant cultures were made from 250 to 500 μm diameter cerebellar fragments from 7-day-old male Wistar rats and cultured on coverslips previously coated with poly-d-lysine and laminin. Isolated hippocampal cells and spinal cord cells were prepared from papain-treated
Experimental calibration of photolytic conversion/flash in an epi-fluorescence microscope
With an upright compound microscope and water immersion optics it is not possible to make direct chemical calibration of photolysis in the focal region by removing samples for HPLC. Instead fluorescence changes induced by photolysis can be used to estimate the conversion per flash in a particular microscope by two methods, either from the fluorescence generated on photolysis of a ‘caged’ fluorophore, or by monitoring fluorescence changes induced by protons released stoichiometrically in the
Discussion
7-Nitroindolinyl and 4-methoxy-7-nitroindolinyl caged neurotransmitters are thermally stable, fast and efficient sources of free ligands. Photorelease from MNI-caged l-glutamate is about 2.5 times more efficient than NI-caged l-glutamate and is comparable to the release efficiency of NPE-ATP. NPE-caged phosphates, such as NPE-ATP, are among the most efficiently photolysed caged compounds in common use. Neurotransmitters are released from the nitroindolinyl cages with rates >3000 s−1, fast
Acknowledgements
Supported by the MRC and Marie Curie Fellowship of the European Community (to MC, HPMF-CT-1999-00349). We thank Chris Magnus for cell culture and Dr Ranjit Munasinghe for recording NMR spectra. We thank the MRC Biomedical NMR Centre for access to facilities.
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