Relatively little is known about the properties of glutamate receptors within the synapse, as opposed to extrasynaptic receptors, because of their inaccessibility to conventional methods such as outside-out patch-clamp recording. Presynaptic nerve terminals, the conventional source of neurotransmitter for activating synaptic receptors, have variable release probabilities, making them a poor source of glutamate for many types of experiments. The properties of the synaptic receptors are important because they are thought to be the major determinant of the EPSC amplitude and time course (Jonas and Spruston, 1994), which determines the reliability and the precision of synaptic signaling (Galarreta and Hestrin, 2001; Cathala et al., 2003). Moreover, their properties are likely to be different from nonsynaptic receptors because other proteins to which they are bound can influence AMPA receptor (AMPAR) behavior and may be different at the two sites (Priel et al., 2005; Tomita et al., 2005). Silver, Thompson, Wang, and colleagues have designed systems for local photorelease of caged glutamate to examine the properties of glutamate receptor channels at single synapses and dendritic spines.
Silver's group has built, in collaboration with Prairie Technologies (Middleton, WI), a photolysis system that comprises a continuous-wave (CW) argon ion UV laser, an acousto-optical tunable filter (AOTF), and a single-mode fiber to deliver the light to the microscope. By filling the back aperture of the objective with the UV light, they can achieve a diffraction-limited illumination volume that has lateral dimensions approaching 220 nm (full-width at half-maximum), comparable with the size of a single postsynaptic density (PSD) and smaller than the lateral resolution of a two-photon microscope (Matsuzaki et al., 2001; Zipfel et al., 2003). The position of the uncaging spot is computer controlled with stepper motors and can be placed at the desired location with high precision using a CCD image of the preparation as a reference.
Thompson and colleagues have modified an upright microscope, equipped for whole-cell recording, to allow the UV light needed for photolysis to be combined with conventional wide-field illumination via a dichroic mirror in the excitation light path (Fig. 1A) (Wei et al., 2001). UV light from an argon ion or diode pumped solid-state laser is launched into a thin (10-25 µm) multimode quartz fiber and then focused in a plane conjugate to the preparation via a lens assembly above the microscope.
There are a number of advantages to these arrangements. First, the use of a CW or quasi-CW laser, particularly in combination with an AOTF, allows flexible uncaging protocols, including variable duration pulses, ramps, paired pulse, and trains. Second, UV laser excitation produces highly efficient uncaging of 4-methoxy-7-nitroindolinyl-caged L-glutamate (MNI-glutamate), allowing receptor activation with brief illumination pulses (20 µs) that mimic synaptic activation and produce minimal photodamage. In contrast, the two-photon cross-section of MNI-glutamate is low (Kiskin et al., 2002), so that it requires high light intensities (Matsuzaki et al., 2001; Kiskin et al., 2002) or extended illumination durations (Carter and Sabatini, 2004) to achieve millimolar concentrations of glutamate. The power levels necessary can induce photodamage (Kiskin et al., 2002), which increases nonlinearly with intensity for two-photon excitation (Koester et al., 1999; Hopt and Neher, 2001). Third, diffraction-limited illumination permits highly localized activation, allowing the investigation of spatial variations in receptor activation on the micrometer scale. It also permits paired-pulse activation protocols because the small photolysis volume can be replenished extremely rapidly by diffusion of unphotolysed caged molecules from outside the focal volume. Fourth, by having one fewer nonlinearity than two-photon excitation, calibration of the glutamate concentration within the UV illumination volume is potentially simpler. The limitations of using UV excitation include substantially larger scattering by tissue than with the long-wavelength light used for two-photon excitation and the fact that the defocused UV light can still cause photolysis above and below the focal illumination volume, albeit at a much lower rate. Unlike the case for two-photon uncaging, these properties will restrict the application of diffraction-limited UV uncaging to the surface of slices or to tissue culture preparations. For example, Thompson's group uses cultured hippocampal slices prepared with the roller tube method so that individual spines on cells loaded with fluorescent dye from the patch pipette may be targeted near the upper surface of the culture, thus minimizing the depth of tissue the light must traverse.
Synaptic AMPAR properties
Preliminary experiments from Silver's group show that a combination of diffraction-limited UV photolysis and whole-cell patch clamping can be used to study synaptic AMPA receptors in acute brain slices at room temperature (DiGregorio et al., 2004). The cerebellar granule cell is particularly suitable for studying synaptic receptors because their previous studies show that AMPA receptors are located exclusively in PSDs (DiGregorio et al., 2002). The initial findings suggest that they will be able to quantify the kinetic properties of synaptic channels by using various illumination protocols that can be generated with the AOTF. For example, Figure 2A shows AMPA receptor-mediated currents from a cerebellar granule cell under voltage clamp evoked with 20 µs UV pulses using MNI-glutamate. Application of a paired-pulse UV protocol, in which the illumination intensity of the first pulse is varied and that of the second is fixed, has allowed the investigation of the activation dependence of AMPAR desensitization (Fig. 2A). As the fraction of synaptic receptors activated increased, the size of their maximal response 10 ms later decreased attributable to an increased level of desensitization. Using their recently developed glutamate diffusion model of the synapse (Nielsen et al., 2004) and an experimental estimate of the uncaging efficiency, it is possible to predict both the concentration of the caged compound during the illumination pulse and the glutamate concentration generated (Fig. 2B). By combining these approaches, it should be possible to quantify the functional properties of receptors within the synaptic environment and build a kinetic model of the synaptic channels. This new approach should provide a better understanding of the role of receptor properties in determining the input-output function of single synaptic contacts.
NMDA receptor (NMDAR)-dependent long-term plasticity of excitatory synaptic transmission probably underlies learning and memory. Persistent uncertainty and controversy about the mechanisms of long-term potentiation (LTP) induction and expression remain, at least in part, because of the unreliability of transmitter release from presynaptic nerve terminals. For example, one of the stronger pieces of evidence for a presynaptic expression mechanism in LTP is the decrease in paired-pulse ratio that has been reported in some studies (Schulz et al., 1994). Matsuzaki et al. (2004) have presented preliminary evidence that potentiation of glutamate responses can be achieved with photorelease from caged glutamate targeted to dendritic spines, offering a powerful tool to answer fundamental questions about the kinetics and mechanisms of LTP expression.
Using microphotolysis of caged N-(6-nitro-7-coumarylmethyl)-L-glutamate (Ncm-Glu) (Cai et al., 2004) to activate receptors at single dendritic spines in CA1 cells in hippocampal slice cultures, Thompson's group can induce a long-lasting potentiation of the photolysis-induced AMPAR-mediated currents with a single pairing of photoreleased glutamate and brief postsynaptic depolarization (Bagal et al., 2005). The potentiation has a magnitude of 80% over the control amplitude, is stable for >30 min after induction (Fig. 1 B), and cannot be induced at extrasynaptic sites on the dendritic shaft. Like conventional synaptic LTP, this potentiation is blocked by NMDAR antagonists and is reversed with low-frequency photostimulation. Because they can induce the potentiation with a single pairing, they discovered that potentiation of photolytic responses develops at single spines in a rapid, stepwise manner after a 40 s delay. The potentiation is accompanied by a decrease in postsynaptic paired-pulse facilitation of the photolytic currents, indicating that a decrease in the proportion of glutamate receptor sub-type 2-lacking AMPARs at the spine head contributes to the potentiation. These results establish that activation of postsynaptic glutamate receptors by glutamate is not only necessary, but sufficient, for the induction of NMDA receptor-dependent LTP. Furthermore, the ability to stimulate single dendritic spines with photoreleased glutamate provides a powerful new means to study LTP expression.
Of course, learning does not involve single presynaptic and postsynaptic elements, however, but ensembles of cells and synapses acting in concert. Wang's laboratory is interested in synaptic learning rules and the cellular mechanisms that govern them. In brain slices, they are using photolysis of caged molecules as a means of rapidly and non-invasively manipulating biochemical signals with submicrometer spatial resolution. They have developed an optical system for rapid uncaging in arbitrary spatiotemporal patterns to emulate complex neural activity (Shoham et al., 2005) (Fig. 3A). They use patch-clamp recording, two-photon fluorescence microscopy and patterned single-photon uncaging of neurotransmitters and second messengers to probe network activity in functioning neural tissue. Uncaging is done using a frequency tripled Nd:YVO4 laser (DPSS, San Jose, CA) (pulse width, 50-60 nsec; = 355 nm; 100 kHz repetition rate) as the ultraviolet light source. The system uses TeO2 acousto-optical deflectors to steer the UV beam and can uncage at more than 20,000 locations per second. The uncaging beam is projected into the focal plane of a two-photon microscope, allowing patterned uncaging to be performed concurrently with imaging and electrophysiology. This technology has been used to generate precise, complex activity patterns of glutamate, other neurotransmitters, and second messengers. The spatial resolution of this system is well suited for studying integration in whole dendrites and neurons, in which signals are integrated on length scales of tens to hundreds of micrometers.
This technology promises to be a powerful technology for the study of dendritic integration or for applications that require the activation of many presynaptic neurons at once (Fig. 3B-D). In preliminary results, they have assayed local amplification by uncaging glutamate at multiple locations at once on a basal dendrite, each producing a response reminiscent of those measured with single-site stimulation (Polsky et al., 2004). They also find that uncaging sequences at dozens of locations distributed over a dendritic arbor give reproducible responses with multiple repetitions of the same sequence in either voltage clamp or current clamp. In current clamp, patterned activation evokes a sequence of action potentials with similar timing from trial to trial. These preliminary results demonstrate the utility of rapid patterned uncaging to act as a high-throughput assay for dendritic physiology and synaptic integration.