Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Abstract

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

Figure 1. Photostimulation laser calibration.

(a) Photo-stimulation laser power output. Power at the sample plane (through a 60x/1.0 NA water-dipping objective) was measured for 405 nm and 473 nm photo-stimulation lasers at the indicated output setting. (b) Photo-stimulation laser calibration. Screen capture images show the spatial relationship between the intended photo-stimulation spot and the corresponding location where photo-stimulation occurred (burn-hole) before (left) and after (right) running calibration in MarkPoints. Please click here to view a larger version of this figure.

Figure 2. PA-Nic local application.

(a) Detection of PA-Nic from a local application pipette. 1 mM PA-Nic, photolysis by-product, or nicotine were dissolved in ACSF, loaded into a local application pipette, and dispensed onto brain tissue during 2PLSM (900 nm excitation) imaging using the same imaging settings for each drug. Laser scanning Dodt contrast transmission image shows the tissue/pipette whereas a GaAsP cathode PMT was used to capture fluorescence emission. (b) PA-Nic (1 mM) was perfused into brain tissue and imaged via 2PLSM as in (a) to show the lateral spread of PA-Nic using its intrinsic fluorescence. Please click here to view a larger version of this figure.

Figure 3. Acquisition of 2-photon laser scanning microscopy images.

(a) Optimal 2PLSM Z-stacks. Two examples of 2PLSM Z-stack maximum intensity projections are shown for MHb neurons with well-resolved dendrites and little to no interfering debris. (b) Sub-optimal 2PLSM Z-stacks. Two examples of 2PLSM Z-stack maximum intensity projections are shown for MHb neurons surrounded by debris (dye expelled from the pipette during cell approach). Such images are more difficult to interpret than images like those shown in (a). Please click here to view a larger version of this figure.

Figure 4. Laser flash photolysis of PA-Nic.

(a) MarkPoints reference images and inward currents evoked by PA-Nic photolysis. For one MHb neuron, raw reference images are shown for MarkPoints photo-stimulation trials at a single (indicated) cellular location. Note that for some photo-stimulation locations (the right-most image in this series), the dendritic structure is in focus but the soma and proximal dendrite are not. Below each reference image, the nicotine uncaging-evoked inward current is plotted. (b) Inter-stimulus intervals for PA-Nic photolysis. Exemplar recordings are shown for MHb neurons where nicotine was repeatedly uncaged at the same perisomatic location with an inter-stimulus interval of 1 s or 10 s. Please click here to view a larger version of this figure.