Dataset Information

Chronic Ca²⁺ imaging of cortical neurons with long-term expression of GCaMP-X.

ABSTRACT: Dynamic Ca²⁺ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca²⁺ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca²⁺ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca²⁺ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca²⁺ fluctuations, in comparison with GCaMP. Chronic Ca²⁺ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca²⁺ transients progressively developed into autonomous/global Ca²⁺ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca²⁺, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca²⁺ oscillations became discernible around 2-3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca²⁺ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca²⁺ imaging applications involving long-time and/or high-level expression of Ca²⁺ probes.

SUBMITTER: Geng J

PROVIDER: S-EPMC9699699 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X.

Geng Jinli J Tang Yingjun Y Yu Zhen Z Gao Yunming Y Li Wenxiang W Lu Yitong Y Wang Bo B Zhou Huiming H Li Ping P Liu Nan N Wang Ping P Fan Yubo Y Yang Yaxiong Y Guo Zengcai V ZV Liu Xiaodong X

eLife 20221005

Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design i ...[more]

PMID: 36196992

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Project description:The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca2+ indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca2+ fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f+/ChAT+) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and β4 subunits, and most express ionotropic serotonin receptors (5-HT3Rs). Cholinergic MG neurons also display small, spontaneous Ca2+ transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na+ channel dependent impulses, presynaptic Ca2+ channels and postsynaptic receptor function reveal that the Ca2+ transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT3Rs. Electrical stimulation of axonal connectives to MG evoked Ca2+ responses in the neurons that similarly depended on nAChRs or/and 5-HT3Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca2+ transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca2+ transients and stimulus evoked Ca2+ responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT3Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca2+ imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.

Dataset Information

Chronic Ca²⁺ imaging of cortical neurons with long-term expression of GCaMP-X.

Publications

Chronic Ca<sup>2+</sup> imaging of cortical neurons with long-term expression of GCaMP-X.

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