The eLife—Xiaodong Liu/Zengcai Guo/Yubo Fan-Yaxiong Yang team used GCaMP-X to achieve long-term calcium imaging of neurons

Written by Geng Jinli, Liu Xiaodong

Responsible editor—Wang Sizhen, Fang Yiyi

Editor—Summer Leaf

Ca2⁺ signaling plays a vital role in the brain and is closely associated with important events such as membrane excitability, cell perception, and synaptic transmission[1].

Ca2⁺ disorders are also thought to be associated with a variety of psychiatric disorders, such as Parkinson's disease, Alzheimer's disease, epilepsy, and schizophrenia [2].

In addition to serving as an acute indicator of neuronal response (e.
g.
, firing action potentials), Ca2+ also mediates chronic/non-acute biological processes: ^Ca2+-dependent gene transcription and expression, neurite growth or pruning , LTP or LTD, learning and memory, and neurodegeneration [3].

Therefore, the development of molecular tools related to calcium imaging to monitor long-term ^Ca2+ dynamics (days/week or longer) in cells, tissues, organs, and even whole living organisms will contribute to a strong understanding
of long-term biological processes such as neuronal development and plasticity.

In fact, genetically encoded ^Ca2+ indicators, represented by GCaMP , GECIs), has been widely used in calcium fluorescence imaging of neurons and other cells [4].

GECIs are expected to avoid to some extent the chronic immune response and unstable recording quality problems often encountered by electrodes [5].

However, neurons that express GCaMP for a long time through viral infection [6] or transgenic methods [7] often develop cell damage or even apoptosis (marked by abnormal nuclear aggregation of GCaMP)
。 The author's preliminary work suggests that the main cause of GCaMP neuronal toxicity is that L-type Ca_V1 channels and others contain apoCaM Important proteins in the binding domain bind unexpectedly to GCaMP[8].

At present, the usual solution is to conduct tentative pre-experiments on expression levels/times for specific experimental conditions to avoid GCaMP damage
based on empirical data.
In this study, in view of the inherent cytotoxicity of traditional GCaMP probes, the authors use rationally designed high cytocompatibility GCaMP-X to challenge the long-term expression of calcium probes and long-term neuronal calcium imaging, providing a new solution.

Recently, Liu Xiaodong's research group of Beihang University, Guo Zengcai's research group of Tsinghua University and Professor Fan Yubo/Associate Professor Yang Yaxiong of Beihang University collaborated on the team eLife published a research paper entitled "Chronic^{Ca 2+}imaging of cortical neurons with long-term expression of GCaMP-X".
The neuronal compatibility of GCaMP-X over GCaMP was further demonstrated, and long-term GCaMP-X was realized in vivo cultured neurons (confocus) and in vivo mouse cerebral cortex (two-photon).
Imaging, quantitatively revealed the close association between spontaneous calcium oscillation (abnormality) and neuronal morphological development (injury), providing a simple and effective new method
for long-term calcium imaging in neuroscience and related fields.
(Further reading: For the relevant research progress of Liu Xiaodong's group, see the "Logical Neuroscience" report (click to read): Commun Biol.
) —Xiaodong Liu/Yubo Fan/Xiaomei Li revealed the regulatory effect of calcium channel CaV1-encoded polypeptide on neuronal activity-protrusion developmental coupling).

In cortical neurons, spontaneous calcium oscillations (calcium ion influx primarily derived from L-type calcium channels) regulate the growth of neuronal protrusions, called channel calcium dynamic-neuronal developmental coupling [9].

GCaMP accidentally binds to L-type calcium channels to interfere with the above coupling, and long-term (high) expression of GCaMP eventually causes significant neuronal damage
.
Based on the design principles of the lab's previous GCaMP-X probes, the authors "rationally designed" the jGCaMP7b-X_C and jGCaMP7b-X_N
。 To explore the different toxicities of GCaMP7 and GCaMP7b-X to cells, the authors transfected different probes into cortical neurons and found that jGCaMP7b exhibited severe nuclear accumulation and jGCaMP7b The total length and complexity of expressed neuronal protrusions were significantly reduced, while there was no significant difference
in the morphology of jGCaMP7b-X expressed neurons and YFP control groups.
To investigate the effects of jGCaMP7b and jGCaMP7b-X on L-type calcium channels, the authors used electrophysiology to explore their effect on recombination in HEK293 cells The effect of the Ca V 1.
3 channel, the results show that jGCaMP7b significantly enhances the channel activation and inactivation process of Ca V 1.
3, while jGCaMP7b-X _C It does not affect channel characteristics (Figure 1).

The above evidence shows that jGCaMP7b-X significantly attenuates neuronal toxicity and interference
with L-type channels compared with GCaMP7b.

Figure 1 Design principle and basic test of jGCaMP7-X

(Source: Geng et al.
, eLife, 2022).

To explore the effects of GCaMP-X at the in vivo level, the authors injected adult mice (> 2 months) with GCaMP6m and GCaMP6m-X viruses, respectively, in expression At different time points after 4 weeks, two-photon microscopy was used to monitor the calcium dynamics
of beard-stimulating S1 sensory cortex neurons.
The results showed that after 8-13 weeks of injection, the GCaMP6m group showed significant nuclear aggregation, while the neurons in the GCaMP6m-X_C group did not enter the nucleation phenomenon (Figure 2）
。 At 4-6 weeks after injection (generally considered the Optimal Time Window for GCaMP Expression, OTW), GCaMP-X The success rate of the calcium response of the beard stimulated neurons in the group was significantly higher than that in the GCaMP group, and the amplitude and SNR of the calcium response were also higher than those in the GCaMP group
.
At 8-13 weeks after injection (beyond the optimal time window for GCaMP expression), the success rate of beard stimulation of neuronal calcium response in mice in the GCaMP-X group was higher than that of GCaMP The amplitude and SNR of calcium response were higher than those of the GCaMP group and the nuclear aggregation neurons in the group and its nucleus aggregation (Figure 2 ）
。 The above intravital sensory response imaging shows that GCaMP-X has higher response rate and other advantages
for long-term calcium dynamic monitoring compared with GCaMP.

Figure 2: Cortical neurons infected with GCaMP-X versus GCaMP in mouse intravital sensory response imaging

(Source: Geng et al.
, eLife, 2022).

To better clarify key details, the authors decided to conduct an in-depth study of the long-term expression and imaging of GCaMP-X under in
vitro conditions.
Slow calcium oscillations have been observed in a variety of excitable and non-excitable cells in the literature [10].

In neurons, oscillating ^Ca2+ signaling can improve the efficiency and specificity of gene expression, so it plays an important role
in neuronal function and development.
The authors speculate that for long-term calcium oscillation imaging, GCaMP-X and GCaMP neurons will show significant differences
.
The same titer of GCaMP or GCaMP-X is added to the cortical neurons of DIV0 and expressed for 28 days after long-term expression with GCaMP Compared with GCaMP-X-expressed neurons, GCaMP-X expressed neurons spontaneously oscillate at a faster frequency, higher amplitude, higher synchronization, and smaller half-height width (Figure 3).

The above results preliminarily delineate the main characteristics of calcium oscillation of neurons in the cultured cortex and its variation with culture duration
.

Figure 3: Long-term imaging of calcium oscillations in cortical neurons using viral expression calcium probes

(Source: Geng et al.
, eLife, 2022).

Spontaneous ^Ca2+ oscillations, especially slow calcium oscillations, are closely associated
with important processes such as neuronal protrusion development.
To further explore the relationship between neuronal development and spontaneous calcium oscillations, the authors used virus-infected cortical neurons to express GCaMP-X for long-term expression.

The results showed that the total length of GCaMP neuronal protrusions decreased and the somal scale decreased at DIV28.
Markedly different from GCaMP, the total length of GCaMP-X neuronal protrusions increases in an S-shape with time and eventually enters the plateau phase (Figure 4)
。 Combined with the analysis results of spontaneous calcium oscillations in neurons in Figure 3, GCaMP-X imaging revealed that the amplitude of spontaneous calcium oscillations was positively correlated with neuronal protrusion development, while frequency was negatively correlated with neuronal protrusion development (Figure 4D ）
。 Further analysis showed that the development rate of neuronal protrusions and somatosomes showed a bell-shape curve (Figure 4E)
during the culture cycle (one month).

Figure 4 Ex vivo GCaMP-X long-term imaging reveals a close association between spontaneous calcium oscillations and neuronal protrusion development

(Source: Geng et al.
, eLife, 2022).

To further explore spontaneous calcium oscillations at the mouse in vivo level, GCaMP6m or GCaMP6m-X_C is expressed by viral injection in adult mouse cortical neurons and 4 weeks post-injection (OTW).
) and set two checkpoints
8-11 weeks after injection (beyond OTW).
Similar to the beard stimulation response experiment in Figure 2, GCaMP nucleated neurons exhibited abnormal calcium dynamics
even within the time window OTW.
Outside of OTW, a large number of neurons GCaMP6m are nucleated, while GCaMP6m-X_C rarely is nucleated (Figure 5C ）
。 Outside the time window, the frequency and amplitude of spontaneous calcium oscillations in GCaMP6m neurons decreased significantly, accompanied by an unusually wide half-height width and a slower rise/fall rate.

In contrast, neurons expressing GCaMP6m-XC maintained normal spontaneous calcium oscillations
throughout the experiment (11 weeks after injection).
It is worth mentioning that GCaMP6m-X_C composition is like GCaMP6m, both inside and outside the OTW There was a significant improvement in group comparison (Figure 5).

The above evidence shows that GCaMP-X can achieve long-term in vivo calcium imaging, which has advantages
that GCaMP does not have.

Figure 5: Long-term in vivo GCaMP-X imaging of spontaneous calcium oscillations of mouse cortical neurons

(Source: Geng et al.
, eLife, 2022).

In line with the idea of ex vivo analysis and corresponding to in vivo calcium oscillation imaging, the authors further investigated the effect of
GCaMP-X expression on neuronal morphological development in vivo.
Different doses of GCaMP6m or GCaMP6m-X_{C AAV} virus were injected into the adult mouse cortex, and neurons
expressing different doses of probes were compared for brain slices at different time points.
The experimental results showed that with the increase of dose, the nucleoplasm ratio of GCaMP gradually increased and the brightness of GCaMP gradually increased, while the nucleoplasm of high-dose GCaMP-X could be kept small
.
Under conditions similar to Figures 2 and 5, the authors injected high-dose GCaMP6m virus (1×10¹²v.
g.
/ml) and ultra-high doses GCaMP6m-X_C virus (1 × 10¹³v.
g.
/ml, 10 times higher) to observe S1 Temporal distribution of virus expression in the same cortical region (Figure 6C).

At 17, 55, 70 and 92 days after injection, GCaMP6m-X_C Compare
with GCaMP6m.
Observing brain sections using confocal microscopy, long-term high levels of expression of GCaMP6m-XC at 92 days did not cause nuclear aggregation at relatively low concentrations (1×10¹²v.
g.
/ml).
GCaMP6m has caused severe nuclear aggregation at 17-92 days (Figure 6D).

At the same time, from 55 to 92 days, the soma of neurons in the GCaMP6m group was significantly smaller than that in the GCaMP6m-X_C group
。 According to the results of ex vivo analysis, neuronal damage caused by in vivo expression probes is also likely to be closely related
to abnormal spontaneous calcium oscillation.
Brain slice data of long-term expression of GCaMP-X in vivo showed that GCaMP-X corresponded to the advantages of GCaMP-X in vivo imaging, GCaMP-X under long-term/high-dose in vivo expression conditions Superior to GCaMP in neuronal damage (i.
e.
, cytocompatibility).

Figure 6: Comparative analysis of long-term in vivo expression of GCaMP-X or GCaMP cortical neurons

(Source: Geng et al.
, eLife, 2022).

Compared to virus-mediated GCaMP expression, transgenic mouse-mediated GCaMP expression is considered relatively safe
.
However, recent studies have reported epileptiform activity in some transgenic mouse strains, such as Ai93 and Ai148 [11].

Based on cultured transgenic neurons, long-term imaging and analysis
were performed from both functional and morphological aspects.
For 6-month-old Rasgrf2-2A-dCre; Ai148D mouse cerebral cortex layer 2/3, using confocal fluorescence microscopy to examine the expression of GCaMP6f in brain slices (TMP-induced expression) ( Figure 7A), where significant nuclear aggregation
is observed.
At the same time, the neurite complexity and length of GCaMP6f transgenic neurons cultured in vitro were reduced compared with those in the GFP transgenic control group (Figure 7C).

Functionally, Ai148D neurons nucleated by GCaMP6f have low spontaneous calcium oscillation amplitude and frequency (10-100 mHz) energy
。 Therefore, GCaMP transgenic mice also have some cytotoxicity and should follow similar basic principles to GCaMP damage under other gene introduction methods (transient, viral).

Figure 7 Long-term imaging of cortical neurons in GCaMP transgenic mice

(Source: Geng et al.
, eLife, 2022).