In vivo imaging of calcium and glutamate responses to intracortical microstimulation reveals distinct temporal responses of the neuropil and somatic compartments in layer II/III neurons
Introduction
While brain-machine interface technology has been successfully translated to the clinic, a persistent challenge for users is a lack of somatosensory feedback to guide movements [[1], [2], [3], [4]]. These interfaces often require implantation of biomaterials into the brain to detect signals from nearby cells. More recently, intracortical microstimulation (ICMS) through implanted biomaterials has enabled tactile and proprioceptive feedback for brain-machine interfaces in both non-human primate [5,6] and human [[7], [8], [9]] studies. Although there are new stimulation modalities such as optogenetics [10], single photon optogenetics's spatial selectivity is limited by a large diffusion radius of photon scatter in tissue, genetic manipulation requirement, lack of spatially selective promoters, trade-offs in virus diffusion radius and transduction rates, and poor uniform expression patterns due to issues with multiplicity of inserted genes and position effects from random insertion location caused by promoter enhancers and inhibitors [11]. Though other new stimulation modalities demonstrate potential for driving neural activity with greater spatial resolution [12], the most common feedback modality remains intracortical microstimulation (ICMS), which uses extracellular electrodes to deliver current to neuronal populations within the somatosensory cortex.
Biocompatibility issues that can impact the 50–150 μm microenvironment of passive recording microelectrodes such as glial scarring [[13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]], vascular injury [13,16,[29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41]], metabolic stress [29], and neurodegeneration [13,30,[42], [43], [44]] can also impact ICMS microelectrodes. Particularly, many studies have shown that ICMS implants and subsequent stimulation can generate neural loss and changes to vascularization [[45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55]]. In non-human primate models, neural loss and single unit recordings with ICMS electrodes are similar in magnitude as passive recording devices [56,57]. The corresponding increase of distance to the nearest excitable neural element due to neurodegeneration is thought to cause an increase in the stimulation threshold [50,58,59]. In turn, this means an increase in stimulation amplitude or charge injection is necessary to achieve similar levels of neural response, which can ultimately damage both the device and the tissue [11,43,59]. This has led to substantial biomaterial research to safely inject greater charge into the tissue without damaging the electrode or the tissue [11,[59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75]]. The conventional hypothesis is that, up to the stimulation safety limit [68], increasing stimulation amplitude will allow activation of more distant neurons from the electrode. This hypothesis is sometimes interpreted to suggest that the tissue response is only problematic for recording electrodes, since stimulating electrodes can always be “turned up” to offset the tissue response. However, empirical evidence suggests increasing amplitude will not always improve performance. Namely, increasing amplitude can alter the nature of perceived sensations in ICMS in a highly variable way from one electrode to another, instead of simply increasing the perceived intensity of the stimulus [8,76]. The biological correlates of this phenomena are not fully understood nor how this amplitude response is altered by neural loss and glial scarring surrounding the electrode [51,56,[77], [78], [79], [80]]. This work is focused on understanding the biological correlates of the amplitude response prior to the formation of the glial scar with respect to one of the most common intracortical microelectrode biomaterials, silicon and iridium oxide. In order to better inform next-generation ICMS technologies, it is necessary to decouple the biomaterial and biological aspects of the amplitude response of neurostimulation.
There is a long history of research on understanding the biology of the amplitude response of neurostimulation. Early electrophysiology and modeling studies established a current-distance relationship for ICMS, in which increasing the amplitude of stimulation increases the radius of suprathreshold depolarization, recruiting a wider range of neurons [[81], [82], [83], [84], [85]]. More recent modeling studies have shown that this current-distance relationship can accurately predict ICMS discrimination in non-human primates [86]. Slab models that consider distinct recruitment properties of Pyramidal neurons and interneurons suggest that interneuron recruitment is dense around the electrode while Pyramidal cells are more sparsely recruited [87]. The field's ability to understand the biology of neurostimulation was revolutionized by the development of voltage-sensitive dyes, calcium sensors, and optical intrinsic imaging methods, which all have been used to corroborate the current-distance relationship [[88], [89], [90]]. Using neuronal calcium imaging, Histed et al. showed that ICMS in layer II/III of cortex engages antidromic activation of horizontal fibers within the layer, resulting in sparse, distributed activation of neurons that does not rely upon synaptic transmission [76]. This result stands in contrast to other in vivo imaging studies that show that ICMS results in activation densely localized around the stimulation source, and recruits abundant orthodromic, trans-synaptic activity [88,91,92]. Resolving these conflicting reports is essential to understanding the localization of clinical ICMS.
The temporal extent of ICMS has also been explored. Studies using in vivo electrophysiology [93], optical intrinsic imaging [94], and voltage and calcium imaging [88,89,95,96] suggest that pulses of ICMS result in initial excitation of local neural populations followed by a brief period of loss of neural activity. For trains of pulses, neurons close to the electrode can follow the train at high (≤500 Hz) and low (≤10 Hz) frequency stimulation, while distant neurons can fail to follow the train [88,93,95,96]. This is possibly due to conduction failure [92,97], inhibitory interneuron inputs [88,89], virtual anode formation [95], or potassium build-up [98]. Recent modeling efforts of antidromic activation in Deep Brain Stimulation suggest failure at high frequencies is driven by hyperpolarizing afterpotentials in the cell body [99]. Elucidating the mechanisms that determine whether a cell will fail or not could help to guide how train duration and frequency are used to maximized clinical efficacy of ICMS.
The present study therefore seeks to address several contentious aspects of the current ICMS model: 1) spatially, does stimulation amplitude increase recruitment of somas and neuropil density around the electrode, 2) temporally, does increasing stimulation amplitude drive some neurons to fail over long trains of stimulation, and 3) does increasing stimulation amplitude increase the area and distance of recruited neural elements? To answer these questions, we use two-photon microscopy imaging of mice expressing either the calcium sensor GCaMP6s or the glutamate sensor iGluSnFr in neurons to measure the somatic and neuropil response to ICMS. We show that somas, neuropil, and glutamate release exhibit greatest activation within the first 20–40 μm of the electrode with rapid spatial drop off, indicative of a dense activation pattern [81,95]. In contrast, somas more distant to the electrode are less likely to sustain calcium activity during long-train ICMS, however, the probability of sustained calcium activity can be increased with higher amplitude stimulation. During 15–20 μA long-train stimulation, glutamate release at the electrode site is sustained for at least 10s post-stimulation, revealing disruption of glutamate re-uptake. Finally, we correlate neuropil calcium activity to somatic activity of cells that do and do not show activity drop off. The neuropil is 3-fold more correlated with somas that sustain activity, with stronger correlations for higher amplitudes of stimulation. Thus, while increasing the amplitude of stimulation may elevate the spatial extent of neural recruitment, it also modulates the temporal response and glutamate neurotransmission. This suggests that increasing ICMS device amplitude may have unintended effects, which has implications for using amplitude to improve the ICMS effect or to compensate for glial scarring.
Section snippets
Animals and virus injection
All subjects were mature (>8 weeks and >25 g) male mice. Animals were housed in 12 h light/dark cycles with free access to food and water. For experiments measuring neuronal calcium activity, transgenic mice expressing the calcium sensor GCaMP6S under the control of the Thy-1 promoter were used (4 animals; C57BL/6J-Tg(Thy1-GCaMP6s)GP4.3Dkim/J; Jackson Laboratories, Bar Harbor, ME) [100]. For experiments measuring glutamate release, C57BL/6J mice were used (3 animals; Jackson Laboratories).
Mesoscale imaging shows dense and dynamic neuronal calcium activity during ICMS
Our objective in the present study was to understand how increasing ICMS amplitude affected the spatial and temporal calcium activity of somas and neuropil. To accomplish this, we implanted single shank, planar electrodes into the somatosensory cortex of transgenic mice that preferentially express the calcium sensor GCaMP6s in excitatory neurons [100,[113], [114], [115]] (Fig. 1A). Subsequently, we applied a 30s train of 100 Hz, asymmetric square waves with a 100 μs, 15 μA cathodic phase, a 50
Discussion
Given that implanting electrode biomaterials (for stimulation and recording) can result in scar tissue formation within 50–150 μm of the electrode that alters the electrical connectivity of the electrode and neural tissue, it is essential to determine the spatial and temporal extent of neural activation during ICMS. Using two-photon microscopy of the Thy1 cortical neural calcium response to electrical stimulation, we show that neural calcium activity is densely recruited within 40 μm around the
Conclusions
Using in vivo two-photon imaging of calcium and glutamate activity, we have shown that ICMS densely recruits neurons and causes excitatory glutamate release within 20–40 μm of the electrode. The neural and glutamate response rapidly falls off as a function of distance from the electrode. In all cases, the response is stronger with higher amplitude. During long trains of stimulation, there is a population of more distant neuronal somas that fails to follow the stimulation. Despite this, the
Acknowledgments
We would like to thank Alberto L. Vazquez for assistance and training for the viral injections and Kevin C. Stieger for critical review of the manuscript. This work was supported by NIH R01NS094396, R01NS105691, and R21NS108098.
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