In vivo calcium imaging reveals functional rewiring of single somatosensory neurons after stroke

Abstract

Functional mapping and microstimulation studies suggest that recovery after stroke damage can be attributed to surviving brain regions taking on the functional roles of lost tissues. Although this model is well supported by data, it is not clear how activity in single neurons is altered in relation to cortical functional maps. It is conceivable that individual surviving neurons could adopt new roles at the expense of their usual function. Alternatively, neurons that contribute to recovery may take on multiple functions and exhibit a wider repertoire of neuronal processing. In vivo two-photon calcium imaging was used in adult mice within reorganized forelimb and hindlimb somatosensory functional maps to determine how the response properties of individual neurons and glia were altered during recovery from ischemic damage over a period of 2-8 weeks. Single-cell calcium imaging revealed that the limb selectivity of individual neurons was altered during recovery from ischemia, such that neurons normally selective for a single contralateral limb processed information from multiple limbs. Altered limb selectivity was most prominent in border regions between stroke-altered forelimb and hindlimb macroscopic map representations, and peaked 1 month after the targeted insult. Two months after stroke, individual neurons near the center of reorganized functional areas became more selective for a preferred limb. These previously unreported forms of plasticity indicate that in adult animals, seemingly hardwired cortical neurons first adopt wider functional roles as they develop strategies to compensate for loss of specific sensory modalities after forms of brain damage such as stroke.

Figure 1.

Imaging protocol and experimental timeline. A, Vibrotactile stimulation (1 s, 100 Hz) of the cFL or cHL during imaging of IOSs was used to define S1FL and S1HL, respectively. The time course and regional distribution of the IOS response to cFL (top) and cHL (bottom) stimulation are shown in A. Each frame in the time course shows the percentage change in light intensity across the IOS image over a 0.5 s period before, during, or after limb stimulation. IOS response maps (see Materials and Methods) were thresholded at 50% of peak response amplitude and merged with an image of the surface vasculature to create color-coded regional maps of cFL and cHL activation, as shown in A (far right). The membrane-permeant Ca²⁺ indicator OGB-1 AM and astrocyte marker SR101 were applied to facilitate two-phton Ca²⁺ imaging within areas defined by IOS imaging. B, Two-photon images, acquired in vivo, of OGB-1 and SR101 labeling in S1HL in the region demarcated by the dashed black box in the thresholded map in A (depth, 147 μm). Ca²⁺ imaging in both S1FL and S1HL during vibrotactile stimulation was used to assess sensory-evoked single-cell responses. C, Mean Ca²⁺ signals, averaged from 10 trials, from the neurons (n1 and n2), astrocyte (a1), and region of the neuropil (np) denoted in B. cHL stimulation evoked strong Ca²⁺ transients in n1, n2, and the neuropil, but not in a1. Imaging, targeted stroke, and recovery were performed according to the experimental timeline shown in D.

Figure 2.

Regional and cellular organization of forelimb and hindlimb somatosensory cortex. A, IOS response maps in a control mouse before and after the sham procedure reveal a consistent topography of cHL- and cFL-activated cortex, with cHL-activated tissue posterior and medial to the cFL representation. Mechanical stimulation of the ipsilateral limbs did not elicit a clear region of activated cortex. B, The borders of the presham and postsham IOS response maps from cHL and cFL stimulation, thresholded at 50% of peak response amplitude (light red/green lines demonstrated presham borders; dark red/green lines demonstrated postsham borders). Despite different imaging conditions and anesthesia, IOS response maps collected before and after sham procedures revealed consistent patterns. C, Two-photon Ca²⁺ imaging was performed at three locations (1–3, demarcated by dashed boxes) spanning the border between cHL- and cFL-activated cortex (three depths per location: 120, 138, and 156 μm below the cortical surface). A threshold analysis of somatic responses was performed for these image planes, and the locations of neurons with above-threshold responses to limb stimulation are superimposed on the map of the IOS response borders. Green dots show the locations of neurons with above-threshold responses to cFL stimulation, and red dots show the locations of cHL-activated neurons. Only two neurons responded to stimulation of any of the other limbs: one neuron responded to iFL stimulation alone (yellow dot), and a second neuron responded to both forelimbs (green dot, yellow border). Importantly, two-photon imaging revealed an extremely sharp border at the cellular level, with no spatial overlap between neurons sensitive to cHL or cFL stimulation. D, E, Representative image field traces from image planes (D, depth, 120 μm; E, depth, 138 μm) adjacent to the border in cHL- and cFL-activated cortex, respectively (depth, 120 and 128).

Figure 3.

Limb selectivity in somatosensory cortex of control mice. A, Two-photon data from layer 2/3 of the S1HL of a naive mouse, from the region defined by the black box in Figure 1A. Mean Ca²⁺ signals (average of 10 trials of stimulation per limb) from three neurons (1–3, labeled in the OGB-1/SR101 image on the left) and the mean neuropil (np) response from six areas of the np (white boxes) are shown. Sensory-evoked Ca²⁺ transients were only elicited by stimulation of the cHL [gray shading denotes period of vibrotactile stimulation (1 s, 100 Hz)]. Difference image montages (bottom) show the responding neuronal somatic Ca²⁺ signal, and were created by binning in groups of five frames (by averaging) and subtracting the mean baseline image from each frame. The difference images in A illustrate the change in fluorescence (ΔF/F_o) from baseline fluorescence over 0.75 s before, during, and after cHL stimulation. B, Two-photon traces and difference images from S1FL (depth, 114 μm) of a control mouse. Again, only stimulation of the preferred limb (cFL) elicited a strong Ca²⁺ transient. Threshold criteria (see Materials and Methods) were used to define “strong” somatic responses in neurons in S1HL and S1FL. Graphs in C and E show the prevalence of above-threshold responses to limb stimulation in S1HL (2282 neurons) and S1FL (1498 neurons), respectively, in control animals. Selectivity for the preferred limb and a paucity of responses to mechanical stimulation of the other limbs is apparent. D, F, The mean image field Ca²⁺ transient records across all control animals (S1HL, 9 mice; S1FL, 10 mice), analogous to a local sensory-evoked field recording. Representative image field signals in response to stimulation of each limb were acquired from each animal and averaged (error bars show the SEM). Again, nearly complete selectivity for the preferred limb is apparent.

Figure 4.

Disrupted forelimb representation and preserved hindlimb somatosensory processing 2 weeks after targeted stroke. A, Prestroke and poststroke IOS response maps reveal disruption of the cFL representation 2 weeks after photothrombosis of S1FL. The thresholded IOS response map at the top right of A shows the limb representation before stroke, with the prestroke and poststroke maps merged onto a single image immediately below. No clear cFL-evoked activity was observed after stroke (in the original FL area), although the cHL representation was preserved (truncated laterally by the infarct). Weak cFL activation posterior to S1HL may represent a shifted pattern of cFL-evoked activity, but its proximity to the edge of the craniotomy makes this difficult to discern. B, Two-photon Ca²⁺ imaging in layer 2/3 of the preserved cHL representation revealed normal limb selectivity in neurons and the surrounding neuropil in this animal. Representative data from individual neurons (1, 2) and the mean neuropil (np) Ca²⁺ signal (average of 10 trials per limb, depth of 126 μm, from the region demarcated in A) demonstrate normal sensory-evoked transients and preserved limb selectivity in S1HL. The color-coded neuronal response map (determined by threshold analysis of somatic responses, with color representing above-threshold responses to stimulation of the corresponding limb) demonstrates highly limb-selective responses to cHL stimulation in this image plane.

Figure 5.

Functional plasticity 1–2 months after stroke. A, B, Prestroke and poststroke maps of the sensory-evoked IOSs in mouse Str22, imaged 1 month after targeted photothrombotic infarction of the S1FL. Before stroke, the IOSs evoked by cFL and cHL stimulation had typical topography with a sharp border between the limb representations. After stroke, a new pattern of cFL-evoked IOS activity is observed, whereas cHL-evoked activity is preserved. Considerable overlap exists between cHL- and cFL-evoked IOS activity. C–E, Representative two-photon Ca²⁺ imaging data from the region demarcated in B. Ca²⁺ traces (averaged from 10 trials) for two neurons (labeled in E) and the mean neuropil (np) response (also from the image plane shown in E) and neuronal response maps (showing above-threshold somatic Ca²⁺ responses to different limbs, coded by color) at two depths are illustrated. Note the aberrant (nonselective) response to nonpreferred limbs (red arrowheads) in the representative traces, and the reduced limb selectivity in the neuronal response maps. F, G, In mouse Str25, 1 month after stroke, IOS imaging revealed a cFL-evoked pattern of activity with a posterior and medial shift and significant overlap with the cHL-evoked representation. Neuronal response maps in the core of S1HL and in regions of IOS overlap demonstrated reduced limb selectivity (most prominent in regions of IOS overlap). H, I, Mean Ca²⁺ records (error bars show the SEM) of the image field responses from all animals for S1HL and S1FL, respectively. These mean traces show that local network activity was altered in both the preserved S1HL and reorganized S1FL, exhibiting reduced limb selectivity (increased amplitude of the image field Ca²⁺ transient elicited by stimulation of nonpreferred limbs) that peaks 1 month after targeted stroke.

Figure 6.

Posterior S1FL reorganization. A, B, Mouse Str34, imaged 2 months after photothrombosis: IOS response maps show increased overlap between the cFL- and cHL-evoked IOSs, and a posterior shift in cFL activation. C, Ca²⁺ imaging (depth, 141 μm, from the labeled region in the IOS map) demonstrated that the posterior regions of the reorganized cFL representation contained neurons (e.g., 1, 2, labeled in D) with strong, limb-selective responses to cFL stimulation. Aberrant responses (red arrowheads) were observed in some neurons (e.g., 3). D, A difference image illustrating the strong Ca²⁺-induced fluorescence signal (ΔF/F_o) over ∼3 s after cFL stimulation onset in the image section from which the representative neurons in C were selected [shown below the corresponding 2-photon image of OGB-1 (green) and SR101 (red) labeling]. E, IOS maps from a naive animal showing the cFL- and cHL-evoked representations. F–I, Difference images illustrating the ΔF/F_o in S1FL and regions of the cortex posterior to S1FL and lateral/posterior to S1HL during cFL stimulation (over ∼3 s after stimulus onset) from the mouse shown in E (borders of the image sections denoted on the IOS map in E). Importantly, cFL stimulation evoked little or no Ca²⁺ signal in the posterior and lateral regions in control animals. J, Mean records of the Ca²⁺ image field activity elicited by cFL stimulation in the S1FL (n = 2*) and posterior/lateral (Post/Lat) cortex (n = 4) of control mice and the same regions in animals imaged 1–2 months after stroke (n = 7). The cFL-evoked activity revealed by two-photon Ca²⁺ imaging in these posterior and lateral regions was significantly stronger in stroke animals compared with controls (ANOVA, p = 0.009), confirming the posterior S1FL reorganization suggested by the IOS maps. *In two mice, OGB-1 AM was microinjected in S1FL and the control regions posterior and lateral to S1FL/S1HL, whereas it was injected into S1HL and the control regions in two other controls.

Figure 7.

Mean changes in limb selectivity after stroke. A, Left, The average prevalence of above-threshold neuronal Ca²⁺ responses to different limbs for neurons in the S1HL for all control and stroke animals. Multivariate analysis revealed a significant effect of stroke on limb selectivity (p = 0.003) in S1HL, and univariate test showed that this effect was significant for all limbs (p = 0.045; cFL, p = 0.007; iFL, p < 0.001; iHL, p = 0.015). The graph on the right compares the prevalence of above-threshold somatic responses in the core of S1HL with limb selectivity in regions of overlap between the cFL- and cHL-evoked intrinsic signal (Fig. 5) in animals imaged 1 or 2 months after stroke. Multivariate analysis confirmed that the limb selectivity differed between these two regions (p < 0.001), and univariate tests revealed a significant increase in cFL responses in overlapping regions (p = 0.044). B, Multivariate analysis of the prevalence of above-threshold somatic Ca²⁺ transients in neurons in S1FL (left) revealed a weak effect of stroke on limb selectivity in reorganized S1FL (p = 0.051). Univariate tests revealed a significant increase in cHL-evoked responses in reorganized S1FL (p = 0.043). Right, Comparing anterior (preserved) and posterior (reorganized) S1FL did not reveal a significant difference in limb selectivity between the regions (MANOVA, p = 0.194). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 8.

Astrocyte signaling after stroke. A, Pooled data examining above-threshold Ca²⁺ responses from all astrocytes imaged in S1FL and S1HL suggested a weak effect of stroke on astrocyte responses to preferred and nonpreferred limbs (p = 0.112). Univariate analysis confirmed that sensory-evoked Ca²⁺ signals in response to stimulation of the preferred limb (cFL and cHL for S1FL and S1HL, respectively) were more common after stroke. B, In S1HL, above-threshold responses to cHL stimulation were more common after stroke (ANOVA, p = 0.043), although the overall effect on limb selectivity was relatively weak (MANOVA, p = 0.109). C, As was the case for neuronal somata, limb selectivity of astrocytes differed in the S1HL core regions compared with regions of overlap between the cFL- and cHL-evoked intrinsic signal (MANOVA, p = 0.033), because above-threshold responses to the nonpreferred (aberrant) limbs were more common in areas of overlap (p = 0.031). D, The prevalence of sensory-evoked Ca²⁺ signals in response to stimulation of the different limbs was not significantly altered in S1FL (MANOVA, p = 0.271). *p < 0.05.