Resolution of High-Frequency Mesoscale Intracortical Maps Using the Genetically Encoded Glutamate Sensor iGluSnFR

Abstract

Wide-field-of-view mesoscopic cortical imaging with genetically encoded sensors enables decoding of regional activity and connectivity in anesthetized and behaving mice; however, the kinetics of most genetically encoded sensors can be suboptimal for in vivo characterization of frequency bands higher than 1-3 Hz. Furthermore, existing sensors, in particular those that measure calcium (genetically encoded calcium indicators; GECIs), largely monitor suprathreshold activity. Using a genetically encoded sensor of extracellular glutamate and in vivo mesoscopic imaging, we demonstrate rapid kinetics of virally transduced or transgenically expressed glutamate-sensing fluorescent reporter iGluSnFR. In both awake and anesthetized mice, we imaged an 8 × 8 mm field of view through an intact transparent skull preparation. iGluSnFR revealed cortical representation of sensory stimuli with rapid kinetics that were also reflected in correlation maps of spontaneous cortical activities at frequencies up to the alpha band (8-12 Hz). iGluSnFR resolved temporal features of sensory processing such as an intracortical reverberation during the processing of visual stimuli. The kinetics of iGluSnFR for reporting regional cortical signals were more rapid than those for Emx-GCaMP3 and GCaMP6s and comparable to the temporal responses seen with RH1692 voltage sensitive dye (VSD), with similar signal amplitude. Regional cortical connectivity detected by iGluSnFR in spontaneous brain activity identified functional circuits consistent with maps generated from GCaMP3 mice, GCaMP6s mice, or VSD sensors. Viral and transgenic iGluSnFR tools have potential utility in normal physiology, as well as neurologic and psychiatric pathologies in which abnormalities in glutamatergic signaling are implicated.

Significance statement: We have characterized the usage of virally transduced or transgenically expressed extracellular glutamate sensor iGluSnFR to perform wide-field-of-view mesoscopic imaging of cortex in both anesthetized and awake mice. Probes for neurotransmitter concentration enable monitoring of brain activity and provide a more direct measure of regional functional activity that is less dependent on nonlinearities associated with voltage-gated ion channels. We demonstrate functional maps of extracellular glutamate concentration and that this sensor has rapid kinetics that enable reporting high-frequency signaling. This imaging strategy has utility in normal physiology and pathologies in which altered glutamatergic signaling is observed. Moreover, we provide comparisons between iGluSnFR and genetically encoded calcium indicators and voltage-sensitive dyes.

Keywords: calcium; cortex; glutamate; in vivo imaging; membrane potential; optogenetic.

Figure 1.

Setup and surgical preparation for wide-field imaging with genetically encoded sensors. A, A CCD camera was used to image cortical iGluSnFR signals (λ = 530 nm) excited by blue LED (λ = 473 nm). B, A chronic window was implanted on the top of mouse skull after removing skin. The chronic window is relatively transparent, covering ∼ 8 mm × 8 mm field of view on the cortex, including the motor, somatosensory, visual, and association cortices. M1, Primary motor; CG, anterior segment of cingulate cortex; HLS1, hindlimb area of the primary somatosensory cortex; FLS1, forelimb area of the primary somatosensory cortex; BCS1: primary barrel sensory cortex; V1, primary visual cortex; V2_LM, lateromedial visual cortex; M2, secondary motor cortex; RS, retrosplenial cortex; PTA, parietal association area; V2L, lateral secondary visual cortex; S2, secondary somatosensory cortex; A1, auditory cortex; V2_MM: mediomedial secondary visual cortex.

Figure 2.

Virally transduced iGluSnFR robustly reports sensory-evoked responses in the cortex. A, AAV1.hSyn.iGluSnFR was injected into the forelimb, barrel, and visual cortex ∼4 weeks before implanting the chronic window. B, Expression of virally transduced iGluSnFR in half of the hemisphere across the motor, somatosensory, and visual cortex. C, Brain slice sections show that the virally transduced iGluSnFR is expressed in the superficial layers of the cortex. D, Multiple sensory-evoked cortical responses in virally transduced iGluSnFR mouse show sensitive, specific, and rapid event-related kinetics at under 1.5% isoflurane anesthesia. Representative montage shows the iGluSnFR responses in the cortex after sensory stimulation and is averaged from 20 trials. E, Time course plots of sensory-evoked responses (Ea–Ed are whisker, visual, forelimb, and hindlimb stimulation, respectively; mean ± SEM) measured from the respective primary sensory region [region of interest (ROI): 667 × 667 μm, n = number of animals]. F, A 1 ms flash light visual stimulation induces an initial rapid response, followed by a clearly separated, delayed secondary response in the awake mouse. Fa, Scheme illustrating the experimental setup of visual stimulation in the awake mouse. Fb, Representative montage of cortical iGluSnFR responses after visual stimulation (mean of 20 trials). Fc, Plot of light flash-evoked visual responses shown as mean ± SEM from 667 × 667 μm boxes placed within V1 (red box shown in Fb) (n = number of animals).

Figure 3.

Ai85 transgenic iGluSnFR mouse reveals the sensitive, specific, and rapid kinetics of iGluSnFR by detecting sensory stimulation-evoked regional responses in the cortex. Ai, Specific expression of iGluSnFR in cortical excitatory neurons is achieved by crossing Emx-Cre mouse line and Cre-dependent iGluSnFR to the CaMKII gene locus, Aii, Emx-CaMKII-iGluSnFR mouse exhibits uniform cortical expression of iGluSnFR at the mesoscopic level. Image shows the wide-field green fluorescence from the cortex. B, Coronal brain sections show dense expression of iGluSnFR in cortex and hippocampus in Emx-CaMKII-iGluSnFR mouse. Coronal sections (Ba–Bf) correspond to locations indicated in schematic diagram. C, Brief sensory stimulation evokes large and fast cortical responses of iGluSnFR signals in Emx-CaMKII-iGluSnFR mouse. Representative montage of 20 trials averaged iGluSnFR responses after sensory stimulation. D, Plots of sensory-evoked responses are mean ± SEM from 667 × 667 μm boxes placed within the corresponding primary sensory cortices denoted by the red boxes in C (n = number of animals). E, A 1 ms flash light visual stimulation induces an initial fast response followed by a clearly separated secondary response in awake Emx-CaMKII-iGluSnFR mouse. Ea, Eb, Representative montage of iGluSnFR responses after visual stimulation in the same animal 4 months apart. Representative montage of 20 trials averaged iGluSnFR responses after visual stimulation. Plot of visual responses shown as mean ± SEM from 667 × 667 μm boxes placed within V1 (red boxes in Ea and Eb, n = 20 trials).

Figure 4.

Interhemispheric connections are revealed by iGluSnFR spontaneous activity. Aa, Spontaneous iGluSnFR activity from 333 × 333 μm boxes placed in the left (red) and right hindlimb cortex (blue) as well as the right visual cortex (green) from an isoflurane-anesthetized (1.5%) Emx-CaMKII-iGluSnFR (Ai85) mouse. Raw iGluSnFR imaging was recorded at 150 Hz and the signals were corrected with global signal regression (GSR) and band-pass filtered to 0.1–12 Hz. Ab, Power spectral density (PSD) analysis indicates that iGluSnFR signals contain signal power likely contributed to by breathing and heartbeat-induced hemodynamic responses (arrows). GSR removes most of the heartbeat-related signals without significantly affecting the other signals. B, Seed-pixel-based correlation maps of iGluSnFR signals show interhemispheric connections of left and right hindlimb cortex within 0.1–30 Hz frequency range in 1.5% isoflurane anesthetized Emx-CaMKII-iGluSnFR mouse. Asterisks indicate seed locations. C, Seed-pixel-based correlation maps of 0.1–12 Hz iGluSnFR activity illustrate the interhemisphere connections for different sensory modalities and also reveal long-range intrahemisphere connections (e.g., barrel cortex–motor cortex, visual cortex–cingulate cortex) in 3 animals <1.5% isoflurane anesthesia and quiet wakefulness. Asterisks indicate seed locations. The maps are similar under anesthesia and quiet wakefulness in the same animal.

Figure 5.

The glutamate transporter blocker TBOA amplifies 1 ms flash light visual stimulation-evoked responses and spontaneous activity in Ai85 iGluSnFR transgenic mice. A, An open skull craniotomy (∼ 3 × 3 mm) was made over the visual cortex. 500 μm TBOA in HEPES-buffered saline was incubated over the exposed cortex after baseline recording. B, Representative image showing iGluSnFR responses to visual stimulation with 1 ms flash light visual stimulation in an isoflurane-anesthetized (1%) Emx-CaMKII-iGluSnFR mouse (left), which is amplified in the presence of 500 μm TBOA (right). Images show visual responses averaged from stimulation onset to 2 s in one mouse in the presence or absence of TBOA (40 trials). C, Plot of visual responses shown as mean ± SEM from 667 × 667 μm region of interest placed within the V1 (show in red in B, n = 4 mice). Insert shows higher magnification of visual responses within the first 140 ms from the onset of light flash. Da, Representative image demonstrating that 500 μm TBOA increases the SD of spontaneous activity within the craniotomy compared with baseline recording. SD (stdev) map of iGluSnFR spontaneous activity after 500 μm TBOA application was calculated over the 133 s recording, and was normalized to fold increase over baseline. Db, Raw traces show iGluSnFR spontaneous activity with or without 500 μm TBOA application.

Figure 6.

2P imaging of sensory-evoked and spontaneous iGluSnFR signals in the visual cortex of Ai85 iGluSnFR transgenic mice. A, 2P imaging reveals dense expression of iGluSnFR in the visual cortex of Emx-CaMKII-iGluSnFR transgenic mice. Images show the depth series of iGluSnFR expression from brain surface to −300 μm depth and the maximal intensity projection of iGluSnFR expression from the first 300 μm (3 μm interval for Z-scanning). B, A 1 ms light flash visual stimulation evokes a similar profile of iGluSnFR responses in the visual cortex of 1% isoflurane-anesthetized mouse in wide-field and two photon imaging. Ba, Representative wide-field imaging shows iGluSnFR responses in the visual cortex (left, brain image; right, averaged map of iGluSnFR responses within the first second from the stimulation onset). Bb, Plots of visual responses are shown as mean ± SEM from 667 × 667 μm box placed within the V1 (red box in a-right; 20 trials). Bc, Representative 2P imaging shows iGluSnFR responses in the visual cortex (red box in Ba, left) at a depth of −150 μm (left, structural image; right, averaged map of iGluSnFR responses within the first second from stimulation onset). Bd, Plots of visual responses are shown as mean ± SEM from the entire imaging field (red box in Bc, right, 20 trials). C, A representative trace of iGluSnFR spontaneous signals (blue) is shown. Ca, ROI for extracting spontaneous iGluSnFR signals. Cb, Raw trace of iGluSnFR signals. Cc, Higher magnification of iGluSnFR signals from the box in Cb.

Figure 7.

iGluSnFR is a fast regional activity reporter compared with other recombinant neuronal activity sensors. A, Comparison of seed-pixel correlation maps seeded within the hindlimb somatosensory cortex among various neuronal activity sensors within delta-, theta-, and alpha-frequency band indicates that iGluSnFR preserves the most high-frequency components for generating maps showing interhemispheric connections. All maps were generated from 20,000 frame sequences recorded at 150 Hz and were processed with global signal regression. Asterisks indicate seed locations. B, Plot of the visual-stimulation-evoked responses measured from visual cortex showing rapid kinetics of iGluSnFR signals that are comparable to VSD signals and are faster than GCaMP3 and GCaMP6s. Representative traces are shown as mean ± SEM of signals within V1 from the indicated sensors in individual animals (20 trials).