Spatial distributions of GABA receptors and local inhibition of Ca2+ transients studied with GABA uncaging in the dendrites of CA1 pyramidal neurons.

GABA (γ-amino-butylic acid)-mediated inhibition in the dendrites of CA1 pyramidal neurons was characterized by two-photon uncaging of a caged-GABA compound, BCMACM-GABA, and one-photon uncaging of RuBi-GABA in rat hippocampal slice preparations. Although we found that GABA(A)-mediated currents were diffusely distributed along the dendrites, currents elicited at the branch points of the apical dendritic trunk were approximately two times larger than those elsewhere in the dendrite. We examined the inhibitory action of the GABA-induced currents on Ca(2+) transients evoked with a single back-propagating action potential (bAP) in oblique dendrites. We found that GABA uncaging selectively inhibited the Ca(2+) transients in the region adjacent (<20 µm) to the uncaging site, and that GABA uncaging was effective only within a short period after bAP (<20 ms). The strength of inhibition was linearly related to the amplitudes of the GABA currents, suggesting that the currents inhibited a sustained, subthreshold after-depolarization without preventing propagation of bAP. GABA uncaging at the dendritic branch points inhibited Ca(2+) transients farther into dendritic branches (>20 µm). Our data indicate that GABA inhibition results in spatially confined inhibition of Ca(2+) transients shortly after bAP, and suggest that this effect is particularly potent at the dendritic branch points where GABA receptors cluster.

Simultaneous two-photon activation of presynaptic cells and calcium imaging in postsynaptic dendritic spines.

BACKGROUND: Dendritic spines of pyramidal neurons are distributed along the complicated structure of the dendritic branches and possess a variety of morphologies associated with synaptic strength. The location and structure of dendritic spines determine the extent of synaptic input integration in the postsynaptic neuron. However, how spine location or size relates to the position of innervating presynaptic cells is not yet known. This report describes a new method that represents a first step toward addressing this issue. RESULTS: The technique combines two-photon uncaging of glutamate over a broad area (~500 × 250 × 100 µm) with two-photon calcium imaging in a narrow region (~50 × 10 × 1 µm). The former was used for systematic activation of layer 2/3 pyramidal cells in the rat motor cortex, while the latter was used to detect the dendritic spines of layer 5 pyramidal cells that were innervated by some of the photoactivated cells. This technique allowed identification of various sizes of innervated spine located <140 µm laterally from the postsynaptic soma. Spines distal to their parent soma were preferentially innervated by cells on the ipsilateral side. No cluster of neurons innervating the same dendritic branch was detected. CONCLUSIONS: This new method will be a powerful tool for clarifying the microarchitecture of synaptic connections, including the positional and structural characteristics of dendritic spines along the dendrites.

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

We developed a caged GABA (gamma-aminobutyric acid), which, when combined with an appropriate caged glutamate, allows bimodal control of neuronal membrane potential with subcellular resolution using optically independent two-photon uncaging of each neurotransmitter. We used two-color, two-photon uncaging to fire and block action potentials from rat hippocampal CA1 neurons in brain slices with 720-nm and 830-nm light, respectively. Our method should be generalizable to other chemical messenger pairs.