Summary Unmodified neurons can be directly stimulated with light to produce action potentials, but such techniques have lacked localization of the delivered light energy. Here we show that gold nanoparticles can be conjugated to high-avidity ligands for a variety of cellular targets. Once bound to a neuron, these particles transduce millisecond pulses of light into heat which changes membrane capacitance, depolarizing the cell and eliciting action potentials. Compared to non-functionalized nanoparticles, ligand-conjugated nanoparticles highly resist convective washout, and enable photothermal stimulation with lower delivered energy and resulting temperature increase. Ligands targeting three different membrane proteins were tested; all showed similar activity and washout resistance. This suggests that many types of ligands can be bound to nanoparticles, preserving ligand and nanoparticle function, and that many different cell phenotypes can be targeted by appropriate choice of ligand. The findings have applications as an alternative to optogenetics, and potentially for therapies involving neuronal photostimulation.

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Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander (Ambystoma tigrinum). The delay period (7" c ) that preceded recovery of the dark current by a criterion amount (3 pA) was analyzed in relation to the flash intensity (If), and to the corresponding fractional bleach (R* 0 /R lol ) of the visual pigment; Rl/R lol was compared with R*/R lol , the fractional bleach at which the peak level of activated transducin approaches saturation. Over an approximately 8 In unit range of If that included the predicted value of R*/R lol , T c increased linearly with In If. Within the linear range, the slope of the function yielded an apparent exponential time constant (T C ) of 1.7 ± 0.2 s (mean ± S.D.). Background light reduced the value of T c measured at a given flash intensity but preserved a range over which T c increased linearly with In If-, the linear-range slope was similar to that measured in the absence of background light. The intensity dependence of T c resembles that of a delay (T d ) seen in light-scattering experiments on bovine retinas, which describes the period of essentially complete activation of transducin following a bright flash; the slope of the function relating T d and In flash intensity is thought to reflect the lifetime of photoactivated visual pigment (/?*) (Pepperberg et al., 1988; Kahlert et al., 1990). The present data suggest that the electrophysiological delay has a similar basis in the deactivation kinetics of / ? ' , and that T C represents T R >, the lifetime of R* in the phototransduction process. The results furthermore suggest a preservation of the "dark-adapted" value of T R * within the investigated range of background intensity.

Electroretinograms (ERGs) were recorded corneally from C57BL/6J mice using a paired‐flash procedure in which a brief test flash at time zero was followed at time tprobe by a bright probe flash of fixed strength, and in which the probe response amplitude was determined at time t=tprobe+ 6 ms. Probe responses obtained in a series of paired‐flash trials were analysed to derive A(t), a family of amplitudes that putatively represents the massed response of the rod photoreceptors to the test flash. A central aim was to obtain a mathematical description of the normalized derived response A(t)/Amo as a function of Itest, the test flash strength. With fixed tprobe (80 ≤tprobe≤ 1200 ms), A(t)/Amo was described by the saturating exponential function [1 ‐ exp(‐ktItest)], where kt is a time‐dependent sensitivity parameter. For t= 86 ms, a time near the peak of A(t), k86 was 7·0 ± 1·2 (scotopic cd s m−2)−1 (mean ± s.d.; n= 4). A(t)/Amo data were analysed in relation to the equation below, a time‐generalized form of the above exponential function in which (k86Itest) is replaced by the product [k86Itestu(t)], and where u(t) is independent of the test flash strength. The function u(t) was modelled as the product of a scaling factor γ, an activation term 1 ‐ exp[‐α(t ‐ td)2]}, and a decay term exp(‐t/τω): where td is a brief delay, τω is an exponential time constant, and α characterizes the acceleration of the activation term. For Itest up to ∼2·57 scotopic cd s m−2, the overall time course of A(t) was well described by the above equation with γ= 2·21, td= 3·1 ms, τω= 132 ms and α= 2·32 × 10−4 ms−2. An approximate halving of α improved the fit of the above equation to ERG a‐wave and A(t)/Amo data obtained at t about 0‐20 ms. Kinetic and sensitivity properties of A(t) suggest that it approximates the in vivo massed photocurrent response of the rods to a test flash, and imply that u(t) in the above equation is the approximate kinetic description of a unit, i.e. single photon, response.

In the human eye, domination of the electroretinogram (ERG) by the b−wave and other postreceptor components ordinarily obscures all but the first few milliseconds of the rod photoreceptor response to a stimulating flash. However, recovery of the rod response after a bright test flash can be analyzed using a paired-flash paradigm in which the test flash, presented at time zero, is followed at time t by a bright probe flash that rapidly saturates the rods (Birch et al., 1995). In ERG experiments on normal subjects, the hypothesis that a similar method can be used to obtain the full time course of the rod response to test flashes of subsaturating intensity was tested. Rod-only responses to probe flashes presented at varying times t after the test flash were used to derive a family of amplitudes A(t) that represented the putative rod response to the test flash. These rod-only responses to the probe flash were obtained by computational subtraction of the cone-mediated component of each probe flash response. With relatively weak test flashes (11–15 scot-td-s), the time course of the rod response to the test flash derived in this manner was consistent with a four-stage impulse response function of time-to-peak ≃170 ms. A(170), the amplitude of the derived response at 170 ms, increased with test flash intensity (Itest) to a maximum value Amo and exhibited a dependence on Itest given approximately by the relation, A(170)/Amo = 1 - exp(-kItest), where k = 0.092 (scot-td-s)−1. In steady background light, the falling (i.e. recovery) phase of the derived response began earlier, and the sensitivity parameter k was reduced several-fold from its dark-adapted value. As the sensitivity, kinetics, and light-adaptation properties of the derived response correspond closely with those of photocurrent flash responses previously obtained from isolated rods in vitro, it was concluded that the response derived here from the human ERG approximates the course of the massed in vivo rod response to a test flash.

Millisecond pulses of laser light delivered to gold nanoparticles residing in close proximity to the surface membrane of neurons can induce membrane depolarization and initiate an action potential. An optocapacitance mechanism proposed as the basis of this effect posits that the membrane-interfaced particle photothermally induces a cell-depolarizing capacitive current, and predicts that delivering a given laser pulse energy within a shorter period should increase the pulse's action-potential-generating effectiveness by increasing the magnitude of this capacitive current. Experiments on dorsal root ganglion cells show that, for each of a group of interfaced gold nanoparticles and microscale carbon particles, reducing pulse duration from milliseconds to microseconds markedly decreases the minimal pulse energy required for AP generation, providing strong support for the optocapacitance mechanism hypothesis.

Functionalization of highly fluorescent CdSe/ZnS core-shell nanocrystals (quantum dots, qdots) is an emerging technology for labeling cell surface proteins. We have synthesized a conjugate consisting of ~150-200 muscimols (a GABA receptor agonist) covalently joined to the qdot via a poly(ethylene glycol) (PEG) linker (~78 ethylene glycol units), and investigated the binding of this muscimol-PEG-qdot conjugate to homomeric ρ1 GABA C receptors expressed in Xenopus oocytes. GABA C receptors mediate inhibitory synaptic signaling at multiple locations in the CNS. Binding of the conjugate was analyzed quantitatively by determining the fluorescence intensity of the oocyte surface membrane in relation to that of the surrounding incubation medium. Upon 5-to 10-min incubation with muscimol-PEG-qdots (34 nM in qdot concentration), GABA C -expressing oocytes exhibited a fluorescent halo at the surface membrane that significantly exceeded the fluorescence of the incubation medium. This halo was absent following muscimol-PEG-qdot treatment of oocytes lacking GABA C receptors. Incubation of the oocyte with free muscimol (100 μM -5 mM), PEGmuscimol (500 μM) or GABA (100 μM -5 mM) substantially reduced or eliminated the fluorescence halo produced by muscimol-PEG-qdots, and the removal of GABA or free muscimol led to a recovery of muscimol-PEG-qdot binding. Unconjugated qdots and PEG-qdots that lacked conjugated muscimol neither exhibited significant binding activity nor diminished the subsequent binding of muscimol-PEG-qdots. The results indicate that muscimol joined to qdots via a long-chain PEG linker exhibits specific binding activity at the ligand-binding pocket of expressed GABA C receptors, despite the presence of both the long PEG linker and the sterically bulky qdot.

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