Calcium imaging

Two-photon imaging of calcium signaling in the brain of a living mouse. Neurotar image.

Calcium imaging in awake, behaving mice

At Neurotar, we study calcium signaling in the most physiologically relevant model: the brain of awake, behaving mice. Our assays are ideal for longitudinal assessments of efficacy or mode of action (MoA).

Calcium signaling as a proxy for neuronal activity

Calcium imaging is a gold standard for assessing neuronal activity and studying neuronal networks, thanks to the strong correlation between neuronal activity and intracellular Ca2+ fluctuations. Impairments of calcium homeostasis result in profound functional alterations. Multiple neurodegenerative disorders, as well as the healthy aging process, are characterized by disruption of calcium homeostasis and signaling. This makes calcium imaging an invaluable tool in understanding and addressing the challenges of calcium dysregulation.

Calcium imaging in the intact and awake brain

Wide-field imaging of the intact mouse brain

The development of calcium indicators, like the genetically encoded GCaMP, has greatly advanced calcium imaging studies. GCaMP is especially useful because it rapidly switches between non-fluorescent and fluorescent states in response to changing Ca2+ levels, making it ideal for measuring these fluctuations.

While calcium signaling can be imaged in cultured neuronal cells or acute brain slices, these in vitro and ex vivo preparations do not capture the complexity of an intact living brain. Transgenic mice expressing GCaMP in excitatory neurons allow for calcium imaging in live animals, preserving this complexity.

Calcium signals are sensitive to common anesthetics, ketamine, and isoflurane, so imaging in awake mice enhances the physiological relevance of these experiments. Neurotar is the only CRO offering calcium imaging in non-anesthetized, head-fixed mice to avoid the confounding effects of anesthetics on calcium signaling dynamics. The gif above illustrates cortex-wide calcium signaling (dF/F) in a living, behaving mouse expressing GCaMP in excitatory neurons. The image was obtained at Neurotar using wide-field calcium imaging on the Invigilo setup.

Why use two-photon microscopy to study calcium signaling?

Two-photon microscopy allows us to measure neuron activity at the cellular level in specific brain regions, providing valuable insights into the efficacy and mode of action (MoA) of drugs targeting select neuronal populations. This technique captures detailed images up to 1 mm deep with remarkable spatial resolution down to the organelle level. Thanks to low phototoxicity, we can perform longitudinal studies, following the same brain region in the same mouse over several months, gaining invaluable insights into long-term changes.

sample calcium signaling data from neurotar

Using two-photon microscopy, the activity of individual neurons can be tracked over extended periods. Neurotar data are shown on the left.

Until recently, our two-photon imaging studies focused on tracking calcium dynamics in the sensorimotor, visual, or prefrontal cortex. Thanks to pioneering work by Michael Goard’s lab at UCSB, we have extended our imaging capabilities to the hippocampus.

Calcium imaging in the hippocampus of awake mouse

Neurotar’s team successfully replicated the protocol of implanting microperiscopes to assess calcium transients in this deeper brain region. The gif on the left shows two-photon calcium imaging in the hippocampus of an awake, freely-moving mouse performed at Neurotar.

Access to the hippocampus opens new possibilities for studying learning and memory in health and disease, offering great promise for investigating memory impairments associated with neurodegenerative disorders.

To study the effects of trauma on neuronal activity, Neurotar’s team combines two-photon imaging with laser injury models. Two-photon imaging is crucial for stroke and traumatic brain injury (TBI) studies as it allows us to observe cellular processes in their natural environment, track changes over time, and gain insights into the mechanisms underlying neuronal damage and recovery.

Calcium signaling after large (diameter 150 µm, left panel) or small (diameter 50 µm, right panel) laser injury showing an increase in neuronal activity. Neurotar images.

Why use wide-field imaging to study calcium signaling?

The brain functions through coordinated activity between regions, making wide-field microscopy ideal for visualizing and quantifying network connectivity across the entire cortex. With its large field of view and high temporal resolution (< 50 ms), it is perfect for assessing the efficacy or mechanism of action (MoA) of compounds affecting cortical networks.

Wide-field imaging in awake mice

Neurotar’s experience primarily involves exploring the effects of psychedelics on calcium signaling. However, wide-field imaging can reveal changes in calcium signaling across various brain disorders, including schizophrenia, epilepsy, stroke, traumatic brain injury, ischemic stroke, Alzheimer’s, and Huntington’s disease.

For wide-field imaging studies, Neurotar utilizes imaging equipment and data analysis methods developed in-house. Our Invigilo wide-field imaging system is optimized for experiments in awake, head-fixed mice, integrating imaging with behavioral observation.

Magnetic locomotion tracking facilitates mouse behavior analysis, segmenting imaging data into active locomotion and volitional immobility subsets. This segmentation is crucial for distinguishing the effects of therapeutic interventions from those of locomotion on neuronal activity. Using Neurotar’s automated analysis method, we can analyze long imaging periods (up to one hour), essential for detecting the effects of slow-acting therapeutic substances.

Calcium imaging studies offered by Neurotar

Our most popular calcium imaging study options include:

Visualizing calcium dynamics within the sensorimotor, visual, or prefrontal cortex at cellular resolution using GCaMP6 two-photon calcium imaging to study treatment effects.
Combining two-photon imaging with laser injury models to examine trauma impacts or neuroprotective drug effects on neuronal activity.
Utilizing two-photon imaging of the hippocampus with glass micro periscopes to study calcium transients in GCaMP6 mice.
Employing wide-field imaging of the entire cortex to investigate network effects of therapeutic compounds and interventions.
Conducting two-photon or wide-field imaging studies that correlate calcium signaling with behavior (such as locomotion, whisker stimulation, and pupil tracking) to enhance the translational relevance of preclinical assays.

How can Neurotar facilitate your research using calcium signaling?

  • First, we’ll have an in-depth discussion about your research questions. Together, we will select the most suitable calcium imaging modality and design an optimally powered in vivo imaging study to test your hypothesis.
  • We can assist in selecting calcium mouse lines and/or transgenic mouse models and sourcing the required licenses and mice from reputable service providers.
  • Our team can advise on using viral vectors to introduce GCaMP expression in transgenic disease models.
  • We’ll help you weigh the pros and cons of different drug administration routes, such as oral (gavage), i.p., s.c., i.v., i.t. (intrathecal) injection, or injection into the Cisterna Magna for experiments in awake mice.
  • For trauma studies, we will work with you to select the most appropriate injury model.
  • We can help identify meaningful behavioral readouts that complement calcium imaging data.
  • At the end of the study, you will receive a comprehensive analysis and interpretation of the results, including recommendations for future studies or adjustments to current protocols based on your compound’s effects.
  • If necessary, we can harvest and preserve tissues for further analysis by your in-house team or a dedicated in vitro CRO partner.
  • Finally, we are happy to assist with your publications, poster abstracts, investor presentations, and other communications.

Other two-photon brain imaging services offered by Neurotar

Neurodeg. Disease
(e.g. AD, PD)
Stroke and TBINeuropathic Pain
and Migraine
(e.g. Schizophrenia)
Blood-brain barrier integrity
Trans-BBB pharmacokinetics
Dendritic spine turnover
Microglial dynamics or response to injury
Calcium signaling
Abeta Plaque or Tau Tangle dynamics
Mitochindria dysfunction
Ischemic Stroke model
Regeneration of peripheral neurons

Publications by Neurotar

Kislin M., et al. (2014) Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents.  J Vis Exp.(88):e51869.  DOI: 10.3791/51869

Fedotova A., et al. (2023) Dissociation Between Neuronal and Astrocytic Calcium Activity in Response to Locomotion in Mice. Function (Oxf). 4(4):zqad019. DOI: 10.1093/function/zqad019

Other relevant publications

  • Akerboom J., et al. (2012) Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci. 32(40):13819-40. DOI: 10.1523/JNEUROSCI.2601-12.2012.
  • Chen T., et al. (2013) Ultra-sensitive fluorescent proteins for imaging neuronal activity. Nature. 499(7458): 295–300. DOI: 10.1038/nature12354.
  • Cramer J., et al. (2020) In vivo widefield calcium imaging of the mouse cortex for analysis of network connectivity in health and brain disease. Neuroimage. 199:570-584. DOI: 10.1016/j.neuroimage.2019.06.014.
  • Grienberger C., et al. (2022) Two-Photon Calcium Imaging of Neuronal Activity. Nature Reviews Methods Primers 2, 67. DOI: 10.1038/s43586-022-00147-1
  • Hamm J., et al. (2017) Altered cortical ensembles in mouse models of schizophrenia. Neuron. 94(1): 153–167.e8. DOI: 10.1016/j.neuron.2017.03.019.
  • Kawamoto E., et al. (2012) Physiology and Pathology of Calcium Signaling in the Brain. Front Pharmacol. 3: 61. DOI: 10.3389/fphar.2012.00061.
  • Lisek M., et al. (2020) Ketamine and Calcium Signaling-A Crosstalk for Neuronal Physiology and Pathology. Int J Mol Sci. 21(21):8410. DOI: 10.3390/ijms21218410.
  • Lu J., et al. (2021) An analog of psychedelics restores functional neural circuits disrupted by unpredictable stress. Molecular Psychiatry 26:6237–6252. DOI: 10.1038/s41380-021-01159-1.
  • Nakai J., et al. (2001) A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol. 19(2):137-41. DOI: 10.1038/84397.
  • Redman W., et al. (2022) Long-term transverse imaging of the hippocampus with glass microperiscopes. Elife. 11:e75391. DOI: 10.7554/eLife.75391.
  • Seshadri S., et al. (2020) Calcium Imaging in Drug Discovery for Psychiatric Disorders. Front Psychiatry. 11: 713. DOI: 10.3389/fpsyt.2020.00713.
  • Wang H., et al. (2020) Frequency-Dependent Block of Excitatory Neurotransmission by Isoflurane via Dual Presynaptic Mechanisms. J Neurosci. 40 (21) 4103-4115. DOI:10.1523/JNEUROSCI.2946-19.2020.