Elsevier

Cell Calcium

Volume 89, July 2020, 102225
Cell Calcium

GCaMP as an indirect measure of electrical activity in rat trigeminal ganglion neurons

https://doi.org/10.1016/j.ceca.2020.102225Get rights and content

Highlights

  • Genetically encoded Ca2+ indicators (GECIs) are used to assess neuronal activity.

  • GCaMP6 s, m, and f, and jGCaMP7 s and f were assessed for this purpose in sensory neurons.

  • Single spikes could only be resolved at a low (<3 Hz) frequency of activity.

  • Magnitude or transient rate of rise were not reliable measures of activity either.

  • GECIs should not be used in sensory neurons to assess changes in activity.

Abstract

While debate continues over whether somatosensory information is transmitted via labeled line, population coding, frequency coding, or some combination therein, researchers have begun to address this question at the level of the primary afferent by using optical approaches that enable the assessment of neural activity in hundreds to even thousands of neurons simultaneously. However, with limited availability of tools to optically assess electrical activity in large populations of neurons, researchers have turned to genetically encoded Ca2+ indicators (GECIs) including GCaMP to enable the detection of increases in cytosolic Ca2+ concentrations as a correlate for neuronal activity. One of the most widely used GECIs is GCaMP6, which is available in three different versions tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance of the two (GCaMP6m). In order to determine if these issues were unique to GCaMP6 itself, or if they were inherent to more than one generation of GCaMP, we also characterized jGCaMP7. In the present study, we sought to determine the utility of the three GCaMP6 isoforms to detect changes in activity in primary afferents at frequencies ranging from 0.1–30 Hz. Given the heterogeneity of sensory neurons, we also compared the performance of each GCaMP6 isoform in subpopulations of neurons defined by properties used to identify putative nociceptive afferents: cell body size, isolectin B4 (IB4) binding, and capsaicin sensitivity. Finally, we compared results generated with GCaMP6 with that generated from neurons expressing the next generation of GCaMP, jGCaMP7s and jGCaMP7f. A viral approach, with AAV9-CAG-GCaMP6s/m/f, was used to drive GECI expression in acutely dissociated rat trigeminal ganglion (TG) neurons, and neural activity was driven by electrical field stimulation. Infection efficiency with the AAV serotype was high >95 %, and the impact of GCaMP6 expression in TG neurons over the period of study (<10 days) on the regulation of intracellular Ca2+, as assessed with fura-2, was minimal. Having confirmed that the field stimulation evoked Ca2+ transients were dependent on Ca2+ influx secondary to the activation of action potentials and voltage-gated Ca2+ channels, we also confirmed that the signal-to-noise ratio for each of the isoforms was excellent, enabling detection of a single spike in>90% of neurons. However, the utility of the GCaMP6 isoforms to enable an assessment of the firing frequency let alone changes in firing frequency of each neuron was relatively limited and isoform specific: GCaMP6s and 6m had the lowest resolution, enabling detection of spikes at 3 Hz in 15% and 32% of neurons respectively, but it was possible to resolve discrete single spikes up to 10 Hz in 36% of GCaMP6f neurons. Unfortunately, using other parameters of the Ca2+ transient, such as magnitude of the transient or the rate of rise, did not improve the range over which these indicators could be used to assess changes in spike number or firing frequency. Furthermore, in the presence of ongoing neural activity, it was even more difficult to detect a change in firing frequency. The frequency response relationship for the increase in Ca2+ was highly heterogeneous among sensory neurons and was influenced by both the GCaMP6 isoform used to assess it, the timing between the delivery of stimulation trains (inter-burst interval), and afferent subpopulation. Notably, the same deficiencies were observed with jGCaMP7s and 7f in resolving the degree of activity as were present for the GCaMP6 isoforms. Together, these data suggest that while both GCaMP6 and jGCaMP7 are potentially useful tools in sensory neurons to determine the presence or absence of neural activity, the ability to discriminate changes in firing frequency ≥ 3 Hz is extremely limited. As a result, GECIs should probably not be used in sensory neurons to assess changes in activity within or between subpopulations of neurons.

Introduction

The development of optical approaches that enable monitoring the activity in hundreds, if not thousands of sensory neurons simultaneously has allowed researchers to address fundamental questions about how sensory information is conveyed to the central nervous system [[1], [2], [3], [4], [5]]. In theory, these optical approaches may not only be used to characterize how a stimulus is encoded within a subpopulation of neurons, but across subpopulations of neurons innervating the same tissue, as well as how this encoding changes with injury. Because neural activity (i.e., number of action potentials, firing frequency, and/or duration of firing) is a critical variable in these studies, it would be ideal, if not most appropriate, to use techniques to monitor changes in membrane potential. However, despite significant improvements in multi-electrode arrays [6,7], as well as both the signal-to-noise ratio and the temporal dynamics of fluorescent voltage sensors [8,9], there remain significant barriers to the widespread use of these technologies, especially when applied with single cell resolution. Researchers have therefore turned to an indirect measure of neural activity, changes in intracellular Ca2+, based on the premise that there is a sufficient number of voltage gated Ca2+ channels (VGCCs) activated when an action potential enters the sensory neuron cell body to drive a detectable increase in intracellular Ca2+ concentration [10]. The major advantage of using intracellular Ca2+ as a proxy for neural activity is the signal-to-noise ratio, which is improved by the facts that there are significant changes in intracellular Ca2+ concentration, which are amplified by a number of Ca2+ regulatory processes, and that the signal arises from the cytosol which constitutes a considerably larger signal source than the cell membrane. Furthermore, the temporal dynamics of a Ca2+ transient initiated by an action potential are up to three orders of magnitude slower than that of the action potential itself, thereby making it possible to record action potential-evoked Ca2+ transients with widely-available imaging technology [11].

While genetically encoded Ca2+ indicators (GECIs) have been used in a number of high profile studies of primary afferents in which novel coupling mechanisms [12], inflammatory mediator-induced shifts in population coding [1], unique afferent subpopulations [13], and stimulus encoding properties [1], have been described, it is still important to consider the limitations of this approach and the conclusions that can be drawn from these studies. Potentially most problematic, Ca2+ transients may be initiated in sensory neurons via a number of mechanisms independent of VGCC activation [[14], [15], [16]], and therefore do not necessarily reflect neural activity. Similarly, because of the array of cellular mechanisms involved in the regulation of intracellular Ca2+, the magnitude and duration of Ca2+ transients in sensory neurons are largely independent of the number of VGCCs activated [16,17]. As a result, the correlation between magnitude or duration of the Ca2+ transient and neural activity is likely to be low. Use of intracellular Ca2+ as a surrogate for neural activity may be particularly problematic in sensory neurons if a goal of the study is to assess changes such as firing frequency or spike number between neurons because of the heterogeneity in the relative contribution of Ca2+ regulatory processes controlling the magnitude and duration of evoked Ca2+ transients [18]. Similarly, interpretation of injury-induced changes in the magnitude and duration of evoked Ca2+ transients may be confounded by the fact that Ca2+ regulatory mechanisms change in response to injury or inflammation [17,[19], [20], [21]]. The relatively slow temporal dynamics of Ca2+ transients may further dissociate the relationship between intracellular Ca2+ and neural activity [15]. Finally, given the heterogeneity of sensory neurons with respect to both the action potential duration [22,23] and the density and distribution of VGCCs [18,24], there are likely to be subpopulations of neurons in which a single action potential, or even a short burst of action potentials, is not sufficient to drive a detectable increase in intracellular Ca2+ [4].

To address at least some of the potential limitations that accompany the use of Ca2+ as a proxy for neural activity, investigators have continued to optimize GECIs, including GCaMP, created from the fusion of green fluorescent protein, calmodulin and myosin light chain kinase, which is optimized for signal-to-noise and speed. At present, the most widely used GCaMP is GCaMP6, given its wide availability in genetic mouse lines and its superior expression levels as compared with its successor, jGCaMP7. For these reasons, the purpose of the present study was to determine the extent to which the GCaMP6 isoforms (s, m, and f) could be used to detect changes in activity across subpopulations of sensory neurons. To do this, we infected dissociated rat trigeminal ganglia (TG) neurons with either AAV9-CAG-GCaMP6s/m/f or AAV1/9-Syn-jGCaMP7s/f and measured changes in fluorescence in vitro. Our results suggest that while all three GCaMP6 and both jGCaMP7 isoforms may be used for the detection of a single action potential in putative nociceptive afferents, none of the isoforms can be reliably used to assess changes in firing frequencies greater than 3 Hz without a detailed characterization of the neuron’s stimulus response properties. These results suggest that GCaMP6 and jGCaMP7 can be used as a dichotomous variable (i.e. the presence or absence of action potentials), as well as an indicator of change in spike number or frequency below 3 Hz. However, neither GCaMP6 nor jGCaMP7 can be used to quantify activity at higher frequencies.

Section snippets

Animals

Adult male Sprague Dawley rats (Envigo, Indianapolis, IN) weighing between 200−400 g were used for experiments. Rats were pair housed with a 12:12 light:dark cycle with food and water ad libitum at the University of Pittsburgh. Experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and followed the National Institutes for Health guidelines for laboratory animal use.

TG cell dissociation and culture

Prior to sacrifice, rats were anesthetized with a ketamine (55 mg/kg), xylazine (5.5

AAV9-CAG-GCaMP6s/m/f-WPRE-SV40 infection efficiency of trigeminal ganglia neurons

The infection efficiency in TG neurons was estimated from the ratio of neurons in which a stimulation-induced increase in GCaMP fluorescence was detected relative to the total number of live neurons, where a neuron was considered live if it was possible to detect a stimulation-induced increase in fura-2 fluorescence. An increase in GCaMP fluorescence (ΔF/F0) was considered a response to stimulation if the increase was time locked to the stimulus and greater than two standard deviations above

Discussion

The purpose of this study was to determine the utility of GCaMP as an indirect measure of TG neuronal activity. In this regard, the level of expression of GCaMP6(s/m/f) was sufficient to detect increases in intracellular Ca2+ in virtually all TG neurons with AAV9-CAG-GCaMP6(s/m/f)-WPRE-SV40. GCaMP6 transients were dependent on Ca2+ influx through VGCCs. Importantly, the influence of AAV9 and GCaMP6s/6f expression on either resting or evoked Ca2+ concentration after an expression duration of up

Funding sources

This work was supported by the United States National Institues of Health (R01NS083347 and R01DK107966 (MSG), F32NS103231 and T32NS073548 (JEH) and a grant from the Bloch Foundation (MSG).

Credit author statement

Drs. Jane Hartung and Michael Gold contributed to the design, analysis, and writing of the manuscript, while Dr. Hartung also collected the data..

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank members of the Gold lab, the Pittsburgh Center for Pain Research, and in particular Dr. Charles Warwick for their helpful feedback on this study.

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