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. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two-photon uncaging microscopy for the first time.

Age-related changes to layer 3 pyramidal cells in the rhesus monkey visual cortex.

The effects of normal aging on morphologic and electrophysiologic properties of layer 3 pyramidal neurons in rhesus monkey primary visual cortex (V1) were assessed with whole-cell, patch-clamp recordings in in vitro slices. In another cohort of monkeys, the ultrastructure of synapses in the layers 2-3 neuropil of V1 was assessed using electron microscopy. Distal apical dendritic branching complexity was reduced in aged neurons, as was the total spine density, due to specific loss of mushroom spines from the apical tree and of thin spines from the basal tree. There was also an age-related decrease in the numerical density of symmetric and asymmetric synapses. In contrast to these structural changes, intrinsic membrane, action potential (AP), and excitatory and inhibitory synaptic current properties were the same in aged and young neurons. Computational modeling using morphologic reconstructions predicts that reduced dendritic complexity leads to lower attenuation of voltage outward from the soma (e.g., backpropagating APs) in aged neurons. Importantly, none of the variables that changed with age differed in neurons from cognitively impaired versus unimpaired aged monkeys. In summary, there are age-related alterations to the structural properties of V1 neurons, but these are not associated with significant electrophysiologic changes or with cognitive decline.

Caged compounds for multichromic optical interrogation of neural systems.

Caged compounds are widely used by neurophysiologists to study many aspects of cellular signaling in glia and neurons. Biologically inert before irradiation, they can be loaded into cells via patch pipette or topically applied in situ to a defined concentration; photolysis releases the caged compound in a very rapid and spatially defined way. As caged compounds are exogenous optical probes, they include not only natural products such neurotransmitters, calcium and IP3 but non-natural products such as fluorophores, drugs and antibodies. In this Technical Spotlight we provide a short introduction to the uncaging technique by discussing the nitroaromatic caging chromophores most widely used in such experiments [e.g. α-carboxy-ortho-nitrobenyl (CNB), dimethoxynitrobenzyl (DMNB), 4-methoxy-7-nitroindolinyl (MNI) and 4-carboxymethoxy-7-nitroindolinyl (CDNI)]. We show that recently developed caging chromophores [rutheniumbipyridial (RuBi) and 7-diethylaminocoumarin (DEAC)450] that are photolyzed with blue light (~ 430-480 nm range) can be combined with traditional nitroaromatic caged compounds to enable two-color optical probing of neuronal function. For example, one-photon uncaging of either RuBi-GABA or DEAC450-GABA with a 473-nm laser is facile, and can block nonlinear currents (dendritic spikes or action potentials) evoked by two-photon uncaging of CDNI-Glu at 720 nm. We also show that two-photon uncaging of DEAC450-Glu and CDNI-GABA at 900 and 720 nm, respectively, can be used to fire and block action potentials. Our experiments illustrate that recently developed chromophores have taken uncaging out of the 'monochrome era', in which it has existed since 1978, so as to enable multichromic interrogation of neuronal function with single-synapse precision.

Influence of highly distinctive structural properties on the excitability of pyramidal neurons in monkey visual and prefrontal cortices.

Whole-cell patch-clamp recordings and high-resolution 3D morphometric analyses of layer 3 pyramidal neurons in in vitro slices of monkey primary visual cortex (V1) and dorsolateral granular prefrontal cortex (dlPFC) revealed that neurons in these two brain areas possess highly distinctive structural and functional properties. Area V1 pyramidal neurons are much smaller than dlPFC neurons, with significantly less extensive dendritic arbors and far fewer dendritic spines. Relative to dlPFC neurons, V1 neurons have a significantly higher input resistance, depolarized resting membrane potential, and higher action potential (AP) firing rates. Most V1 neurons exhibit both phasic and regular-spiking tonic AP firing patterns, while dlPFC neurons exhibit only tonic firing. Spontaneous postsynaptic currents are lower in amplitude and have faster kinetics in V1 than in dlPFC neurons, but are no different in frequency. Three-dimensional reconstructions of V1 and dlPFC neurons were incorporated into computational models containing Hodgkin-Huxley and AMPA receptor and GABA(A) receptor gated channels. Morphology alone largely accounted for observed passive physiological properties, but led to AP firing rates that differed more than observed empirically, and to synaptic responses that opposed empirical results. Accordingly, modeling predicts that active channel conductances differ between V1 and dlPFC neurons. The unique features of V1 and dlPFC neurons are likely fundamental determinants of area-specific network behavior. The compact electrotonic arbor and increased excitability of V1 neurons support the rapid signal integration required for early processing of visual information. The greater connectivity and dendritic complexity of dlPFC neurons likely support higher level cognitive functions including working memory and planning.

Wavelength-selective one- and two-photon uncaging of GABA.

We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of γ-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named "DEAC450". DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological buffer. We found that DEAC450-caged GABA is relatively inactive toward two-photon excitation at 720 nm, so when paired with a nitroaromatic caged glutamate that is efficiently excited at such wavelengths, we could photorelease glutamate and GABA around single spine heads on neurons in brain slices with excellent wavelength selectivity using two- and one-photon photolysis, respectively. Furthermore, we found that DEAC450-caged GABA could be effectively released using two-photon excitation at 900 nm with spatial resolution of about 3 µm. Taken together, our experiments show that the DEAC450 caging chromophore holds great promise for the development of new caged compounds that will enable wavelength-selective, two-color interrogation of neuronal signaling with excellent subcellular resolution.