Spatio-temporal parameters for optical probing of neuronal activity

Abstract

The challenge to understand the complex neuronal circuit functions in the mammalian brain has brought about a revolution in light-based neurotechnologies and optogenetic tools. However, while recent seminal works have shown excellent insights on the processing of basic functions such as sensory perception, memory, and navigation, understanding more complex brain functions is still unattainable with current technologies. We are just scratching the surface, both literally and figuratively. Yet, the path towards fully understanding the brain is not totally uncertain. Recent rapid technological advancements have allowed us to analyze the processing of signals within dendritic arborizations of single neurons and within neuronal circuits. Understanding the circuit dynamics in the brain requires a good appreciation of the spatial and temporal properties of neuronal activity. Here, we assess the spatio-temporal parameters of neuronal responses and match them with suitable light-based neurotechnologies as well as photochemical and optogenetic tools. We focus on the spatial range that includes dendrites and certain brain regions (e.g., cortex and hippocampus) that constitute neuronal circuits. We also review some temporal characteristics of some proteins and ion channels responsible for certain neuronal functions. With the aid of the photochemical and optogenetic markers, we can use light to visualize the circuit dynamics of a functioning brain. The challenge to understand how the brain works continue to excite scientists as research questions begin to link macroscopic and microscopic units of brain circuits.

Keywords: Calcium Imaging; Microscopy; Multi-photon microscopy; Neuronal activity; Neuronal circuits; Voltage Imaging.

Fig. 1

Spatio-temporal parameters and neurotechnologies for understanding the brain

Fig. 2

Adapted and modified with permission from Larkum and Nevian (2008). a Image of a biocytin-stained layer 5 pyramidal neuron annotated with positions of three electrodes at the soma (gray); the nexus of the apical tuft dendrites (blue); and a basal dendrite (orange). b–g Neuronal responses where black traces show somatic recordings while orange and blue traces correspond to dendritic recordings from electrodes identified in a. b An EPSP initiated at the basal dendrite and corresponding recording at the soma. c An EPSP initiated close to the soma and recorded at the basal dendrite. d An NMDA spike with extracellular synaptic stimulation. An EPSP-like current injection at the basal dendrite and apical nexus can generate a sodium spike (e) and a Ca²⁺ spike (f), respectively. g A current pulse injection at the soma produces an action potential, which can backpropagate to the dendrites

Fig. 3

a Energy diagram and excitation spectra for single-photon (1P), two-photon (2P), and three-photon (3P) absorption and corresponding fluorescence emission spectrum (green). b 3D point spread function of the normalized fluorescence intensity via 1P, 2P, and 3P excitation

Fig. 4

a A 1P epifluorescence microscope. Relative axial discrimination is shown in dashed lines for a and c. Inset shows the representative 2D image output. b Adapted with permission from Antic (2003). (Left) A 1P image of a layer 5 pyramidal neuron loaded with JPW-3028 showing the soma and proximal apical dendrites. (Middle) Somatic whole-cell recording (0) and optical recording at the (1) soma and (2) apical trunk. (Right) Scaled optical recordings at the soma (gray) and apical dendrite (black) for direct comparison of the timing and shape of the signals. c A 1P laser scanning confocal microscope with de-scanned detection. Inset shows the representative 3D image output

Fig. 5

a Nipkow disk confocal microscope. Relative axial discrimination is shown in dashed lines. Inset shows the representative 3D image output. b Adapted with permission from Takahashi et al. (2010). (Left) Twenty neurons were monitored at 2000 frames/s. (Right) Spontaneous ΔF/F traces of individual neurons; the locations were shown in the left image

Fig. 6

a A 1P light-sheet microscope. Relative axial discrimination is shown in dashed lines. Inset shows the representative 3D image output. b Adapted with permission from Holekamp et al. (2008). (Left) Image of vomeronasal sensory neurons. (Right) Time courses of the fluorescence intensity neurons in the area imaged. Intensity traces from the subset of cells marked in (A) are coded by color

Fig. 7

a, b Adapted with permission from Bouchard et al. (2015). SCAPE microscopy: planar excitation using a single objective lens. b Orthogonally aligned illumination and detection point spread function for SCAPE microscopy. c Adapted with permission from Hillman et al. (2018). Images of 5 neurons in the whisker barrel cortex. (Bottom) Calcium response from the 5 neurons

Fig. 8

a A 2P microscope using different modes of axial scanning by the sample, objective lens, or via an acousto-optic modulator (AOM). 2P microscope can either use beam scanners to scan along the focal plane via galvanometer mirrors, resonant mirrors, and AOMs. Relative axial discrimination is shown in dashed lines. Inset shows the representative 3D image output. b Adapted with permission from Reddy et al. (2008). AOM-based 3D scanner. c Adapted with permission from Nadella et al. (2016). (Left) Images of layer 2/3 pyramidal neurons of the visual cortex expressing GCAMP6f. (Right) Corresponding calcium responses

Fig. 9

a, b Adapted with permission from Wu et al. (2020). a FACED technique to split a femtosecond pulsed laser into multiple sub-pulses to form a spatially separated and temporally delayed multiple foci at the sample plane. b Voltage traces from visual cortex (V1) neurons showing orientation selectivity. Preferred orientations (black traces) show more sub- and suprathreshold activity than nonpreferred orientations (gray traces)

Fig. 10

a Adapted with permission from Ouzounov et al. (2017). (Top left) 3D reconstruction of 3P images of GCaMP6s-labeled neurons in the mouse cortex and the hippocampus (green, fluorescence, magenta, third-harmonic generated signal). (Right top and middle) Selected XY frames at various depths in a. (Bottom left) Image of the stratum pyramidale layer of the hippocampus and (bottom right) corresponding spontaneous activity recorded from the labeled neurons. b Adapted with permission from Wang et al. (2018), where they used a 3P microscope to achieve functional calcium imaging through an intact skull of a rodent. c Adapted from Yildirim et al. (2019). (Left) Three-dimensional rendering of a sequence of 450 lateral 3P images. (Middle) Selection of lateral images from layers 2/3, 4, 5, and 6. (Right) Average calcium responses (ΔF/F) for representative cells in each layer over 10 trials

Fig. 11

a Patterned illumination via a holographic multi-foci 2P microscope. Relative axial discrimination is shown in dashed lines. Inset shows the representative 3D image output. b Adapted from Castanares et al. (2020). A flattened z-stack image showing the proximal dendritic tree of a L5 pyramidal neuron loaded with Alexa-488 and Cal-520. The scale bar is 50 μm. c Holographically projected multiple foci incident on the dendrites sites and fluorescence recorded using an EMCCD camera. d An image of L5 pyramidal neuron with holographically projected foci at the oblique branches. e Calcium responses during a train of two APs at different frequencies with 70-Hz train evoking a dendritic spike. f Adapted from Bovetti et al. (2017). (Top Left) Image via scanning 2P microscope showing the GCaMP6f-expressing neurons. (Top middle and right) The red crosses indicate the positions of the spots used for scanless imaging. (Bottom) Fluorescence signals recorded with the camera during scanless multipoint illumination of the neurons

Fig. 12

Summary of optical technologies and their spatio-temporal properties