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Alipasha Vaziri

Our goal is to understand how stochasticity, non-linearity, correlations, and coupled excited state dynamics of biological systems and networks contribute to their function. We investigate these questions in different systems and at different levels, from the fundamental principles of protein and bio-molecular dynamics on the nanoscale to dynamic interactions in cellular networks, giving rise to physiological function. To address these questions, we adopt a multidisciplinary approach and develop new methods and technologies, such as advanced imaging and spectroscopy techniques based on ultrafast and quantum optics, and combine these with molecular biology, optogenetics, and electrophysiology.

Dynamics of coupled biological systems – methods and phenomena

Optogenetics and dynamics of neuronal circuits

The recently discovered class of genetically expressible photoactivatable ion-channels, such as Channelrhodopsin, has enabled the optical control of neural activity. The most widely used approach has been the optical activation of the genetically expressed light-gated ion-channel, Channelrhodopsin-2 (ChR2), to initiate population activity in neuronal circuits. However, given the low channel conductance, the initiation of action potentials is only possible when a sufficiently large number of channels are activated at the same time, which has made single cell resolution of optogenetic activation a major challenge.

To overcome these limitations, we have recently developed a scheme for fast, selective and targeted control of neuronal activity with single cell resolution in mouse and rat hippocampal slices. Using the scanningless technique of temporal focusing, for which the axial beam profile can be controlled independently of its lateral distribution, a large number of channels on individual neurons can be excited simultaneously leading to strong depolarizations. At the same time, the application of this method on the neuronal network level allows for delivering defined neuronal input patterns onto neuronal populations.

This can be used to interfere with and manipulate the spatio-temporal structure of the pre-synaptic inputs that a local neuronal population is receiving. We have demonstrated the power of this approach by mimicking the perisomatic inhibition patterns that hippocampal CA1 pyramidal neurons receive during place cell activity. We are interested in using the speed and flexibility that this technique provides to explore the role of stochastically induced sub-threshold spontaneous activity pattern of individuals, and how they are integrated into the network dynamics by combing it with Ca+ imaging and electrophysiology.

High-speed 3D calcium imaging of neuronal activity

Figure 1: Experimental setup of volumetric fluorescence imaging using wide-field two-photon light sculpting. Schematic depicting the light-sculpting microscope and microfluidic sample holder. The pulses at the bottom sketch the geometric dispersion in temporal focusing as a function of axial position. The inset at top right is an artistic rendering of a C. elegans head, indicating axially scanned light discs and the imaged region. Neurons URX and BAG are also depicted. Scale bar is 15 μm. DC, dichroic mirror; OPA, optical parametric amplifier; sCMOS, scientific complementary metal–oxide–semiconductor.

Wide-field temporal focusing (WF-TEFO) is a two-photon imaging technique which is based on light-sculpting. It effectively decouples the parameters governing lateral size of a light beam and its axial resolution. Thus, this technique allows for exciting a large area in the lateral dimension while retaining exceptional resolution in the axial direction. In an actual setup, this is akin to creating a thin ‘disc’ of excitation light. WF-TEFO is well suited for fast volumetric imaging, as its scanning is reduced to one dimension only.

In our latest experiments, we tailored the properties of our WF-TEFO to record, with high temporal and spatial resolution, the activity of neurons in the head ganglia of C. elegans in vivo using Ca2+ imaging. Pivotal to our results was the use of a nuclear-localized, genetically encoded calcium indicator (NLS-GCaMP5K) that permits unambiguous discrimination of individual neurons within the densely-packed head ganglia of C. elegans. We demonstrate near-simultaneous recording of activity of up to 70% of all head neurons. In combination with a lab-on-a-chip device for stimulus delivery, this method provides an enabling platform for establishing functional maps of neuronal networks.

Figure 2: Brain-wide WF-TEFO Ca2+-imaging in C. elegans: (A) Schematics of the microfluidic PDMS device. (I-II) Red and blue sketches indicate worm immobilization and gas delivery channel, respectively. (II) Cross-section (not to scale). (III) Phase contrast image of immobilized worm inside the device. The head (tail) is at the bottom (top). Ventral is shown by the vulva’s location (blue arrow). (B) WF-TEFO imaging of head region of a Punc-31::NLS-GCaMP worm. (I) Maximum intensity projection of 14 z-planes. Dashed lines outline the locations of head ganglia as shown in (II). Scale bar represents 10 μm and refers to panels I,III-V. (II) Schematic of the left anterior head ganglia modified from ref. 9. Black lines outline neuronal nuclei. Grey lines outline the anterior ganglia as indicated. Green area indicates pharynx. (III) Single z-plane (z = 2 μm). Dashed lines indicate y-z and x-z cross-sections shown in (IV) and (V) respectively. Arrows in (III-V) indicate example neurons, each seen in two projections. The crosses indicate worm orientation (A – anterior, P – posterior, D – dorsal, V – ventral, L – left, R - right).

To aid in cell segmentation, we use transgenic worms expressing the Ca2+-sensor GCaMP5K in a pan-neuronal and nucleus-bound fashion. We also used custom-designed microfluidic devices to restrain the worms while applying chemosensory stimuli. Using this approach, we demonstrate brain-wide and near-simultaneous Ca2+- imaging of 70% of the neurons contained in the head ganglia.

The spatial and temporal resolution of our imaging technique is adequate for investigating global properties of the C. elegans nervous system, such as correlated activity among groups of neurons and their responses to sensory stimuli. This paves the way for future investigations on how sensory information is processed at the level of the whole brain, and for establishing a functional map of C. elegans’ nervous system and also potentially in other model organisms.

Figure 3: Data analysis of WF-TEFO Ca2+-imaging in worms during chemosensory stimulation: (A) Activity of 69 neurons under consecutively changing O2- concentrations. Rows are heat-plots of NLS-GCaMP5K fluorescence time series. Colour indicates percent fluorescence changes (∆F/F0). Colourbar indicates scaling. X-axis represents elapsed recording time. O2- concentrations shifted as indicated by dashed lines. (B) Matrix showing correlation coefficient (R) calculated from data in (A). Colour indicates the degree of correlation. Colourbar indicates scaling. The data in A-B are grouped by agglomerative hierarchical clustering.

Selected Publications

  • Schrödel, T., Prevedel, R., Aumayr, K., Zimmer, M., Vaziri, A. (2013). Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat Methods. 10(10):1013-20
  • Vaziri, A., Emiliani, V. (2012). Reshaping the optical dimension in optogenetics. Curr Opin Neurobiol. 22(1):128-37 
  • Andrasfalvy, BK., Zemelman, BV., Tang, J., Vaziri, A. (2010). Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc Natl Acad Sci U S A. 107(26):11981-6 
  • Losonczy, A., Zemelman, BV., Vaziri, A., Magee, JC. (2010). Network mechanisms of theta related neuronal activity in hippocampal CA1 pyramidal neurons. Nat Neurosci. 13(8):967-72 
  • Vaziri, A., Tang, J., Shroff, H., Shank, CV. (2008). Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci U S A. 105(51):20221-6

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