Calcium imaging

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

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.

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.

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.

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.

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:

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 TBI	Neuropathic Pain and Migraine	Neuropsychiatry (e.g. Schizophrenia)	Epilepsy
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.