skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.


Title: Tracking calcium dynamics from individual neurons in behaving animals
Measuring the activity of neuronal populations with calcium imaging can capture emergent functional properties of neuronal circuits with single cell resolution. However, the motion of freely behaving animals, together with the intermittent detectability of calcium sensors, can hinder automatic monitoring of neuronal activity and their subsequent functional characterization. We report the development and open-source implementation of a multi-step cellular tracking algorithm (Elastic Motion Correction and Concatenation or EMC 2 ) that compensates for the intermittent disappearance of moving neurons by integrating local deformation information from detectable neurons. We demonstrate the accuracy and versatility of our algorithm using calcium imaging data from two-photon volumetric microscopy in visual cortex of awake mice, and from confocal microscopy in behaving Hydra , which experiences major body deformation during its contractions. We quantify the performance of our algorithm using ground truth manual tracking of neurons, along with synthetic time-lapse sequences, covering a wide range of particle motions and detectability parameters. As a demonstration of the utility of the algorithm, we monitor for several days calcium activity of the same neurons in layer 2/3 of mouse visual cortex in vivo , finding significant turnover within the active neurons across days, with only few neurons that remained active across days. Also, combining automatic tracking of single neuron activity with statistical clustering, we characterize and map neuronal ensembles in behaving Hydra , finding three major non-overlapping ensembles of neurons (CB, RP1 and RP2) whose activity correlates with contractions and elongations. Our results show that the EMC 2 algorithm can be used as a robust and versatile platform for neuronal tracking in behaving animals.  more » « less
Award ID(s):
1822550
PAR ID:
10353869
Author(s) / Creator(s):
; ; ; ;
Editor(s):
Gutkin, Boris S.
Date Published:
Journal Name:
PLOS Computational Biology
Volume:
17
Issue:
10
ISSN:
1553-7358
Page Range / eLocation ID:
e1009432
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    The neural code relates the activity of the nervous system to the activity of the muscles to the generation of behavior. To decipher it, it would be ideal to comprehensively measure the activity of the entire nervous system and musculature in a behaving animal. As a step in this direction, we used the cnidarian Hydra vulgaris to explore how physiological and environmental conditions alter simple contractile behavior and its accompanying neural and muscle activity. We used whole-body calcium imaging of neurons and muscle cells and studied the effect of temperature, media osmolarity, nutritional state, and body size on contractile behavior. In mounted Hydra preparations, changes in temperature, nutrition state, or body size did not have a major effect on neural or muscle activity, or on contractile behavior. But changes in media osmolarity systematically altered contractile behavior and foot detachments, increasing their frequency in hypo-osmolar media solutions and decreasing it in hyperosmolar media. Similar effects were seen in ectodermal, but not in endodermal muscle. Osmolarity also bidirectionally changed the activity of contraction burst (CB) neurons, but did not affect the network of rhythmic potential (RP) neurons in the ectoderm. These findings show osmolarity-dependent changes in the activity of CB neurons and ectodermal muscle, consistent with the hypothesis that CB neurons respond to media hypo-osmolarity, activating ectodermal muscle to generate CBs. This dedicated reflex could serve as an excretory system to prevent osmotic injury. This work demonstrates the feasibility of studying an entire neuronal and muscle activity in a behaving animal. 
    more » « less
  2. Perceptual experiences may arise from neuronal activity patterns in mammalian neocortex. We probed mouse neocortex during visual discrimination using a red-shifted channelrhodopsin (ChRmine, discovered through structure-guided genome mining) alongside multiplexed multiphoton-holography (MultiSLM), achieving control of individually specified neurons spanning large cortical volumes with millisecond precision. Stimulating a critical number of stimulus-orientation-selective neurons drove widespread recruitment of functionally related neurons, a process enhanced by (but not requiring) orientation-discrimination task learning. Optogenetic targeting of orientation-selective ensembles elicited correct behavioral discrimination. Cortical layer–specific dynamics were apparent, as emergent neuronal activity asymmetrically propagated from layer 2/3 to layer 5, and smaller layer 5 ensembles were as effective as larger layer 2/3 ensembles in eliciting orientation discrimination behavior. Population dynamics emerging after optogenetic stimulation both correctly predicted behavior and resembled natural internal representations of visual stimuli at cellular resolution over volumes of cortex. 
    more » « less
  3. Abstract Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500–600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5–6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells. 
    more » « less
  4. Abstract Miniaturized fluorescence microscopes (miniscopes) enable imaging of calcium events from a large population of neurons in freely behaving animals. Traditionally, miniscopes have only been able to record from a single fluorescence wavelength. Here, we present a new open-source dual-channel Miniscope that simultaneously records two wavelengths in freely behaving animals. To enable simultaneous acquisition of two fluorescent wavelengths, we incorporated two CMOS sensors into a single Miniscope. To validate our dual-channel Miniscope, we imaged hippocampal CA1 region that co-expressed a dynamic calcium indicator (GCaMP) and a static nuclear signal (tdTomato) while mice ran on a linear track. Our results suggest that, even when neurons were registered across days using tdTomato signals, hippocampal spatial coding changes over time. In conclusion, our novel dual-channel Miniscope enables imaging of two fluorescence wavelengths with minimal crosstalk between the two channels, opening the doors to a multitude of new experimental possibilities. TeaserNovel open-source dual-channel Miniscope that simultaneously records two wavelengths with minimal crosstalk in freely behaving animals. 
    more » « less
  5. Abstract Traumatic brain injury (TBI) affects neural function at the local injury site and also at distant, connected brain areas. However, the real‐time neural dynamics in response to injury and subsequent effects on sensory processing and behaviour are not fully resolved, especially across a range of spatial scales. We used in vivo calcium imaging in awake, head‐restrained male and female mice to measure large‐scale and cellular resolution neuronal activation, respectively, in response to a mild/moderate TBI induced by focal controlled cortical impact (CCI) injury of the motor cortex (M1). Widefield imaging revealed an immediate CCI‐induced activation at the injury site, followed by a massive slow wave of calcium signal activation that travelled across the majority of the dorsal cortex within approximately 30 s. Correspondingly, two‐photon calcium imaging in the primary somatosensory cortex (S1) found strong activation of neuropil and neuronal populations during the CCI‐induced travelling wave. A depression of calcium signals followed the wave, during which we observed the atypical activity of a sparse population of S1 neurons. Longitudinal imaging in the hours and days after CCI revealed increases in the area of whisker‐evoked sensory maps at early time points, in parallel to decreases in cortical functional connectivity and behavioural measures. Neural and behavioural changes mostly recovered over hours to days in our M1‐TBI model, with a more lasting decrease in the number of active S1 neurons. Our results in unanaesthetized mice describe novel spatial and temporal neural adaptations that occur at cortical sites remote to a focal brain injury. 
    more » « less