Nonlinear microscopy for temporally and spatially resolved label-free imaging of living cells

The human brain consists of approximately a hundred billion of neurons that communicate through a unique series of biochemical and electrical processes. When neuronal networks are solicited, electrochemical processes happen involving millions of cells within a complex network. The morphology of the neuronal cytoskeleton is fundamental in the establishment of these complex neuronal networks and crucial for the functional integrity of electrical signaling. Traditionally, the neuronal morphology and electrical signals are measured with invasive optical probes, exogenous dyes, or by exogenous electrical recordings. To fully understand the underlying mechanisms involved in neuronal activity and for eventual clinical applications a direct label-free non-invasive optical probe is of great significance. In this thesis we demonstrated the possibility of label-free imaging of neuronal structures and electrical neuronal activity employing the unique intrinsic sensitivity of nonlinear optical techniques. We developed a 3D wide-field second harmonic (SH) imaging system that increases the SH imaging throughput for label-free elastic SH imaging by several orders of magnitude. The increase in throughput was achieved with a wide-field geometry and medium repetition rate laser source in combination with gated detection. In addition dynamic and ultrafast measurements can be performed readily with different possible polarization configurations. First, we performed label-free SH and two photon excitation fluorescence (2PEF) imaging of living cultured neurons with short acquisition time and at very low fluences. We demonstrated the use of wide-field high throughput SH microscopy for investigating dynamic changes in cytoskeletal morphology on the single cell level. The method allows real-time in vitro label-free measurements of cytoskeletal changes that can, under certain conditions, be quantified by orientational distribution or changes in the number of microtubules. Then, we investigated the changes in neuronal morphology and metabolic activity by performing label-free SH polarimetry. We calculated the coefficient of polarization, which reports on orientational irregularities in the microtubule cytoskeleton, and used endogenous 2PEF as a metabolic marker in cultured neurons throughout the stages of their morphological development. Being able to observe morphological changes in the cytoskeleton in a label-free way with clear markers of organization in combination with indicators for metabolic activity, allows us to follow neuronal differentiation in detail, and in a non-invasive way. Finally, we employed SH imaging to label-freely capture direct information of neuronal membrane potentials. We performed SH imaging of cultured neurons undergoing a depolarization by a temporary extracellular excess of K+ ions. To demonstrate the concept, we performed a patch-clamp and SH imaging comparison and showed that whole neuron membrane potential changes correlated linearly with the square root of the SH intensity. Finally, we used the nonlinear optical response of the membrane bound water to create membrane potential and ion flux maps of living cultured neurons in real time. With these results obtained with the 3D wide-field high throughput SH microscope, we demonstrated the possibilities to image in time, label-free and with low fluence, neuronal structural changes and electrical neuronal activity employing the unique intrinsic sensitivity of nonlinear phenomena.

Whole-Cell Recording of Neuronal Membrane Potential during Behavior

Carl Petersen

Neuronal membrane potential is of fundamental importance for the mechanistic understanding of brain function. This review discusses progress in whole-cell patch-clamp recordings for low-noise measurement of neuronal membrane potential in awake behaving animals. Whole-cell recordings can be combined with two-photon microscopy to target fluorescently labeled neurons, revealing cell-type-specific membrane potential dynamics of retrogradely or genetically labeled neurons. Dual whole-cell recordings reveal behavioral modulation of membrane potential synchrony and properties of synaptic transmission in vivo. Optogenetic manipulations are also readily integrated with whole-cell recordings, providing detailed information about the effect of specific perturbations on the membrane potential of diverse types of neurons. Exciting developments for future behavioral experiments include dendritic whole-cell recordings and imaging, and use of the whole-cell recording pipette for single-cell delivery of drugs and DNA, as well as RNA expression profiling. Whole-cell recordings therefore offer unique opportunities for investigating the neuronal circuits and synaptic mechanisms driving membrane potential dynamics during behavior.

Cell Press2017

Uncovering the neural substrates of communication between the brain and ventral nerve cord in Drosophila melanogaster (italic)

Florian Aymanns

Despite a long history of research in motor control, the exact mechanism of how the brain communicates with the the invertebrate ventral nerve cord (VNC) and the vertebrate spinal cord on a single neuron basis remains largely elusive.Drosophila melanogaster is an ideal model organism to address the role of individual neurons, thanks to its stereotyped nervous system.Descending neurons (DNs), interneurons in the brain that project to the invertebrate ventral nerve cord or vertebrate spinal cord, have been shown to elicit specific behaviors upon optogenetic activation in Drosophila.These results suggest that behavior is controlled by sparse sets of command neurons, each eliciting a particular behavior.However, it remains to be seen how many DNs are involved in the control of naturalistic behaviors and what other information they might encode.In order to avoid commands leading to physically impossible or destabilizing actions, the brain has to be aware of the current behavior state.Ascending neurons (ANs), interneurons in the invertebrate ventral nerve cord or vertebrate spinal cord projecting to the brain, are likely conveying this information to the brain.To which degree of fidelity and in what way ANs encode behavior state remains unclear.To address these questions, we developed a dissection approach that allows functional imaging of ANs and DNs in the cervical connective.This dissection gives us optical access to the cervical connective and parts of the VNC in behaving adult Drosophila such that a two-photon fluorescence microscope can be used to image neural activity via calcium indicators.We began by imaging a new library of sparse split GAL4 driver lines targeting ANs.Our recordings from 247 genetically identifiable ANs revealed neurons encoding walking, resting, turning, eye grooming, and foreleg movements.Anatomical characterization of these ANs showed that they predominantly project to two brain regions: the anterior ventrolateral protocerebrum (AVLP) and the gnathal ganglion (GNG).Our results suggest that ANs encoding the motion of the animal with respect to the surrounding environment (self-motion) predominantly project to the AVLP, an integrative sensory hub. ANs projecting to the GNG mostly encode discrete actions.We then proceeded to image populations of DNs.The majority of DNs we recorded encoded walking behavior, with smaller fractions encoding resting and head grooming.Some of these neurons are likely to drive walking, but we also identified neurons encoding walking speed and turning.This suggests that a core set of DNs drives a behavior whose features can be modulated by additional DNs.Besides encoding behaviors, some DNs encode sensory information, including the presence of odors and deflection of the antennae.

EPFL2022

Membrane potential dynamics of GABAergic neurons in the barrel cortex of behaving mice

Michael Avermann, Ferenc Mátyás, Carl Petersen

Computations in cortical circuits are mediated by synaptic interactions between excitatory and inhibitory neurons, and yet we know little about their activity in awake animals. Here, through single and dual whole-cell recordings combined with two-photon microscopy in the barrel cortex of behaving mice, we directly compare the synaptically driven membrane potential dynamics of inhibitory and excitatory layer 2/3 neurons. We find that inhibitory neurons depolarize synchronously with excitatory neurons, but they are much more active with differential contributions of two classes of inhibitory neurons during different brain states. Fast-spiking GABAergic neurons dominate during quiet wakefulness, but during active wakefulness Non-fast-spiking GABAergic neurons depolarize, firing action potentials at increased rates. Sparse uncorrelated action potential firing in excitatory neurons is driven by fast, large, and cell-specific depolarization. In contrast, inhibitory neurons fire correlated action potentials at much higher frequencies driven by slower, smaller, and broadly synchronized depolarization.

Elsevier2010

Uncovering the neural substrates of communication between the brain and ventral nerve cord in Drosophila melanogaster (italic)

Florian Aymanns

EPFL2022