Linking neural microcircuit function to emergent properties of the mammalian brain requires fine-scale manipulation and measurement of neural activity during behavior, where each neuron’s coding and dynamics can be characterized. We developed an optical method for simultaneous cellular-resolution stimulation and large-scale recording of neuronal activity in behaving mice. Dual-wavelength two-photon excitation allowed largely independent functional imaging with a green fluorescent calcium sensor (GCaMP3, λ = 920 ± 6 nm) and single-neuron photostimulation with a red-shifted optogenetic probe (C1V1, λ = 1,064 ± 6 nm) in neurons coexpressing the two proteins. We manipulated task-modulated activity in individual hippocampal CA1 place cells during spatial navigation in a virtual reality environment, mimicking natural place-field activity, or ‘biasing’, to reveal subthreshold dynamics. Notably, manipulating single place-cell activity also affected activity in small groups of other place cells that were active around the same time in the task, suggesting a functional role for local place cell interactions in shaping firing fields.

We demonstrate that channelrhodopsin-2 (CR), a light-gated ion channel that is conventionally activated by using visible-light excitation, can also be activated by using IR two-photon excitation (TPE). An empirical estimate of CR's two-photon absorption crosssection at λ = 920 nm is presented, with a value (260 ± 20 GM) indicating that TPE stimulation of CR photocurrents is not typically limited by intrinsic molecular excitability [1 GM = 10 −50 (cm 4 s)/ photon]. By using direct physiological measurements of CR photocurrents and a model of ground-state depletion, we evaluate how saturation of CR's current-conducting state influences the spatial resolution of focused TPE photostimulation, and how photocurrents stimulated by using low-power scanning TPE temporally summate. We show that TPE, like visible-light excitation, can be used to stimulate action potentials in cultured CR-expressing neurons.optogenetics | photostimulation | laser-scanning microscopy C hannelrhodopsin-2 (CR) is a microbially derived cation channel that transiently opens in response to blue-light illumination (1). Several modified forms of the channel, each encoded by a single gene, have been expressed heterologously in excitable mammalian neurons to recapitulate this light-gated conductance. It has been shown that in CR-expressing neurons, action potentials can be triggered by single-photon excitation, using blue-light illumination, with millisecond temporal resolution (2-7). Where multiple cells are photosensitized, the optical resolution with which each cell can be independently excited determines the precision of optical neuronal interrogation.Spatially localized excitation of individual cells is, however, difficult to achieve in thick biological tissue by using single-photon excitation. Brain tissue, for example, is an optically scattering medium that defocuses light, reducing the spatial definition and intensity of a focused beam or projected spatial pattern with increasing tissue depth (8). Additionally, excitation cannot be confined to a single z-focal plane of interest perpendicular to the axis of illumination (but see ref. 9), a geometry typically favored for in vivo studies of neural circuits.Two-photon laser-scanning microscopy (TPLSM) using a focused IR laser beam is the method of choice by which to achieve spatially localized fluorescence excitation deep in scattering tissue, providing an intrinsic optical section around the plane of focus (10, 11). We hypothesized that an analogous method to activate CR photocurrents by using two-photon excitation (TPE) would confer a much higher degree of spatial precision for targeted neuronal photostimulation than is currently possible by using single-photon (blue-light) illumination.Here we use whole-cell recordings of photocurrents in cultured cells to provide an initial biophysical characterization of TPE of CR. At typical light intensities used for TPLSM, we find that brief (<25 ms) CR photocurrents are described by a singlephotocycle model incorporating depletion of the CR ground state....

Molecular motors drive genome packaging into preformed procapsids in many dsDNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage λ. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage ϕ29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ~600 bp/s, which is 3.5-fold higher than ϕ29, and the motor processivity was also 3-fold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging in many cases, and greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid.

In many viruses, DNA is confined at such high density that its bending rigidity and electrostatic self-repulsion present a strong energy barrier in viral assembly. Therefore, a powerful molecular motor is needed to package the DNA into the viral capsid. Here, we investigate the role of electrostatic repulsion on single DNA packaging dynamics in bacteriophage 29 via optical tweezers measurements. We show that ionic screening strongly affects the packing forces, confirming the importance of electrostatic repulsion. Separately, we find that ions affect the motor function. We separate these effects through constant force measurements and velocity versus load measurements at both low and high capsid filling. Regarding motor function, we find that eliminating free Mg 2؉ blocks initiation of packaging. In contrast, Na ؉ is not required, but it increases the motor velocity by up to 50% at low load. Regarding internal resistance, we find that the internal force was lowest when Mg 2؉ was the dominant ion or with the addition of 1 mM Co 3؉ . Forces resisting DNA confinement were up to Ϸ80% higher with Na ؉ as the dominant counterion, and only Ϸ90% of the genome length could be packaged in this condition. The observed trend of the packing forces is in accord with that predicted by DNA charge-screening theory. However, the forces are up to six times higher than predicted by models that assume coaxial spooling of the DNA and interaction potentials derived from DNA condensation experiments. The forces are also severalfold higher than ejection forces measured with bacteriophage .optical tweezers ͉ single molecule D uring the assembly of many dsDNA viruses, the genome is compacted to near-crystalline density (1). Because the size of viral capsids is on the order of the persistence length of the DNA (Ϸ50 nm), significant DNA bending must occur during packaging (2-7). Moreover, due to the negatively charged phosphate backbone of DNA, a large repulsive electrostatic barrier must be overcome during DNA confinement (2-7). In some cases, more than half of the physically available space inside the capsid is taken up by the viral genome (1, 7).In the case of bacteriophage 29, the 19.3-kbp genome (Ϸ6.5 m in length) is packed inside a prolate icosahedral capsid Ϸ45-nm wide and 54-nm long (8). As with many other dsDNA viruses, DNA is translocated into the preformed precursor capsid (prohead) by an ATP-powered molecular motor (9-11). The 29 motor is situated at a unique vertex of the prohead and consists of a ring of RNA molecules (pRNA) sandwiched between two protein rings: the head-tail connector (gene product 10, gp10) and the packaging ATPase (gp16) (12).Previously, we developed an optical tweezers assay that allowed us to measure the packaging of a single DNA molecule into a single 29 prohead (9). We found that the rate of packaging decreased during capsid filling or when an external force was applied to the DNA substrate. From these measurements, we showed that a large internal force builds during packaging because of DNA confinement ...

During the assembly of many viruses, a powerful molecular motor compacts the genome into a preassembled capsid. Here, we present measurements of viral DNA packaging in bacteriophage phi29 using an improved optical tweezers method that allows DNA translocation to be measured from initiation to completion. This method allowed us to study the previously uncharacterized early stages of packaging and facilitated more accurate measurement of the length of DNA packaged. We measured the motor velocity versus load at near-zero filling and developed a ramped DNA stretching technique that allowed us to measure the velocity versus capsid filling at near-zero load. These measurements reveal that the motor can generate significantly higher velocities and forces than detected previously. Toward the end of packaging, the internal force resisting DNA confinement rises steeply, consistent with the trend predicted by many theoretical models. However, the force rises to a higher magnitude, particularly during the early stages of packaging, than predicted by models that assume coaxial inverse spooling of the DNA. This finding suggests that the DNA is not arranged in that conformation during the early stages of packaging and indicates that internal force is available to drive complete genome ejection in vitro. The maximum force exceeds 100 pN, which is about one-half that predicted to rupture the capsid shell.