Impact of parietal damage and ensuing visuospatial spontaneous recovery
In accordance with prior studies, lesions targeting both banks of the feline right posterior parietal cortex (known as pMS) induced a complete contralesional visuospatial orienting deficit in all tasks. These deficits were present immediately after the lesion (only 24 h post-injury) and started to improve spontaneously shortly thereafter. The basis of this improvement is likely to be a combination of network modulation vicariation (Rushmore et al., 2010) and reduction in acute effects such as inflammation, lesion-induced depolarization and cortical spreading depression events (see reviews by Cramer, 2008; Nudo, 2011).
For the high-contrast moving task (Moving 1), subjects regained function in the contralesional visual hemispace within 5–10 days, and exhibited complete and stable recovery 30 days thereafter (Moving 1, 30 days post-injury 93 ± 4% vs. 98 ± 1% pre-lesion, P = 0.05; data not shown in figure form) which remained unaltered across the follow-up period. In contrast, recovery for static or laser-based moving targets (Day 70: Static pre-rTMS, 39 ± 7% vs. pre-lesion, 82 ± 3%; P = 0.00; and Moving 2 pre-rTMS, 62 ± 9% vs. pre-lesion 88 ± 3%; P = 0.02 correct performance) operated at a slower pace and reached plateau levels of incomplete recovery between 40 and 60 days after the injury (see Fig. 2).
Figure 2. General group visuospatial orienting performance levels for static and motion tasks. Group average levels of correct visual detection performance (n = 12) for static (upper panel, STATIC) and or moving LED targets (bottom panel, MOVING 2) presented in the ipsilesional (white circles) or the contralesional (black circles) visual hemispace respectively, across the three follow-up phases (pre-lesion, post-lesion (from days 1 to 70 post-injury; D1–D70) and rTMS treatment phase (from R1 to R7). Error bars represent the SE of the group mean. Gray shaded portion represents the rTMS treatment phase across rTMS rounds (from R1 to R7). Gray arrows placed above each panel highlight key time periods of the follow-up which are summarized in the histogram panel on the right (Prelesion, Post-lesion Day 1, Pre-rTMS treatment, post-rTMS-R7). *P < 0.05 for values at rTMS R7 vs. pre-rTMS levels. Notice the effects of the lesion on contralesional visual detection leading to hemispatial neglect, its spontaneous recovery across the 70 days that follow damage and subsequent mild rTMS-induced improvements for contralesional detection, particularly for static visual targets.
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Unilateral lesions not only induced the expected pattern of contralesional visuospatial defects, but significantly affected detection performance for visual targets presented in the ipsilesional hemispace. Such effects were particularly noticeable for the Static detection task (Static: drop from 72 ± 2% to 58 ± 3%; P = 0.00). The drop in ipsilesional performance was significant in Moving 2 task but negligible for Moving 1 (Moving 2, from 78 ± 4% to 70 ± 4%, P = 0.01; see Fig. 2; and Moving 1, from 98 ± 1% pre-lesion to 93 ± 5% Day 70, P = 0.05; data not shown in figure form) and remained unaltered across the follow-up.
Effects of the rTMS regime on visuospatial recovery
Once plateau levels of pre-rTMS were achieved, animals started a daily rTMS regime consisting of a total of 70 consecutive sessions delivered across 14 weeks of treatment. In agreement with published observations (Rushmore et al., 2010), the sham group demonstrated a complete absence of improvement, and those effects endured beyond pre-rTMS levels for both the Static (from 20 ± 9% to post-sham rTMS 22 ± 12% correct performance; P = 0.68) and Moving 1 tasks (from pre-TMS 77 ± 20% to post-sham rTMS 70 ± 13%, P = 0.55; data not shown in figure form).
As for the 12 subjects assigned to sessions of real 10-Hz rTMS, a significant three-way interaction between follow-up phase, task, and visual hemispace was found (F13,130, P = 0.01). As a group improvements reached statistical significance over time for the Static task (pre-rTMS, 39 ± 7% to post-rTMS, 53 ± 7%; P = 0.00; Fig. 2). Overall, results accounted for variable levels of contralesional correct performance ranging from improvements of +67% to losses of -15% with respect to individual subject's pre-rTMS treatment levels.
According to statistical criteria for minimal neglect recovery (see 'Material and methods' section), the groups of active rTMS-treated animals were classified into the categories of Responders (n = 6) and Non-responders (n = 6). Overall the rTMS regime generated two groups of equally treated animals, which thus far had performed equivalently in the Static task (Pre-rTMS: Responders, 36 ± 6% vs. Non-responders, 42 ± 14% correct performance; P = 0.89). An initial decrease in performance characterized the Non-responders in the Static task, and in any case active rTMS treatment failed to influence correct performance levels (rTMS R7, 38 ± 12% vs. pre-rTMS, 40 ± 14%; P = 0.70). In contrast, within the contralesional hemispace Responders exhibited progressive increases in visuospatial orienting with the accrual of active rTMS sessions, and reached their performance peak after seven rounds of rTMS (rTMS R7, 68 ± 4% vs. pre-rTMS, 42 ± 6%; P = 0.01; Fig. 3). This recovery appeared, however, to be task-dependent and accordingly Responders experienced little improvements with respect to pre-rTMS treatment levels in the Moving 2 task (pre-rTMS, 70 ± 13% vs. post-rTMS, 79 ± 6%; P = 0.67; Fig. 3).
Figure 3. Contralesional visuospatial orienting performance for Responders and Non-responders in static and motion tasks. Group average levels of correct contralesional detection performance for the Static (upper panel) and Moving 2 (lower panel) tasks for the Responders (black squares, n = 6) and Non-responders (white squares, n = 6), throughout the three-phase follow-up. Error bars represent the SE of the group mean. The gray shaded portion represents the rTMS treatment phase from R1 to R7. Gray arrows placed above each panel highlight key time periods which are summarized in the histogram panel on the right. *P < 0.05 for values at rTMS R7 vs. pre-rTMS levels. Notice in Responders the rTMS-induced improvements in contralesional detection performance levels, particularly for the Static task. In contrast, Non-responders showed a stabilization of performance for these two tasks during the same period of time. Similar patterns, although not reaching significance, were found in contralesional and ipsilesional performance for the Moving 2 task.
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For the Static task, the rTMS regime did not significantly alter performance in the Responders group for ipsilesional targets (Pre-rTMS, 60 ± 3% vs. rTMS R7, 67 ± 8%; P = 0.45; Fig. 4). Interestingly, in the Non-responders group, while rTMS treatment failed to positively influence contralesional detection it did produce decreases in correct performance for ipsilesional targets (Static task pre-rTMS, 58 ± 5% vs. rTMS R7, 43 ± 2%; P = 0.03). Similar effects were observed for the Moving 2 task (Pre-rTMS, 68 ± 6% vs. rTMS R7, 47 ± 3%; P = 0.01; Fig. 4). Taken together, these data strongly suggest that in a specific subpopulation of participants the rTMS treatment could have modulated cortical function in an unexpected manner, impairing an ipsilateral function which should had remained otherwise unaffected.
Figure 4. Ipsilesional visuospatial orienting performance for Responders and Non-responders in static and motion tasks. Average levels of correct ipsilesional detection performance for the Static (upper panel) and Moving 2 (lower panel) tasks for the Responders (black squares) and Non-responders (white squares). Error bars represent SE of the group mean. The gray shaded portion represents the rTMS treatment phase from R1 to R7. Gray arrows placed above each panel highlight key time periods of the follow-up, which are summarized in the histogram panel on the right. *P < 0.05 for values at rTMS R7 vs. pre-rTMS levels. Notice Responders' ipsilesional performance showed no differences between the rTMS pre and post-treatment. In contrast, Non-responders displayed maladaptive response to rTMS treatment that significantly reduced their ability to detect and orient to ipsilesional targets in both tasks.
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Prior to lesion all subjects displayed nearly complete correct performance for the detection of static contralesional pericentral targets corresponding to the binocular portions (15–45°) of the visual field (Static 15°, 98 ± 1%; 30°, 96 ± 2%; 45°, 93 ± 4% correct detection performance). In contrast, peripheral targets presented at monocular visual field eccentricities (60–90°) were detected at more moderate performance rates (Fig. 5; Static 60°, 82 ± 7%; 75°, 69 ± 8%; 90°, 42 ± 10%). A gradient evolving from pericentral to periphery and extending to the contralesional 15o, 30o, and 45o eccentric locations characterized the spontaneous recovery phase for all visuospatial paradigms (Static 15o, 83 ± 8%; 30o, 58 ± 10%; and 45o, 44 ± 11%). Ipsilesionally, a paradoxical expansion of the visuospatial attention span towards the periphery (60°, from 78 ± 6% to 96 ± 0%; 75°, from 45 ± 8% to 83 ± 0%; and 90°, from 14 ± 4% to 75 ± 0%) was followed by a progressive return to pre-injury correct performance levels (60°, 52 ± 10%; 75°, 19 ± 8%; and 90°, 12 ± 5%) by the end of the spontaneous recovery period (Fig. 5). Very similar findings were also obtained for the Moving 2 task (data not shown in figure form).
Figure 5. General group eccentricity-specific recovery effects of orienting to static visual targets. Radial maps indicating percentage correct performance in static target detection at each visual eccentricity for the entire population (n = 12) of participants. Bar length represent the correct performance for the ipsilesional (right) or contralesional (left) visual hemispace. Data are shown (top to bottom) for  pre-lesion,  postlesion day 1,  end of spontaneous recovery phase or pre-rTMS phase and  at the end of the seventh round of rTMS treatment. Concentric half circles represent steps of 20% performance, from the inner (15°) to the outermost (90°) eccentricity. Gray shaded portions display the space area of correct visual detection and orienting performance. Note that felines performed better for pericentral and mid-peripheral than for far peripheral eccentricities pre-lesion, and that unilateral parietal damage mainly affected orienting response towards targets in the contralesional visual hemispace. Spontaneous recovery of function progressed from pericentral to mid peripheral locations (0–60°) and rTMS driven recovery increased performance levels for some of those same locations (30–60°).
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Our analysis shows that, prior to rTMS, the spontaneous recovery patterns for Static contralesional targets were not significantly different between Responders and Non-responders. This occurred regardless of the contralesional visual space in either binocular (15°, Responders 97 ± 2% vs. Non-responders 70 ± 13%, P = 0.10; 30°, 68 ± 10 vs. 48 ± 18%, P = 0.40; 45°, 42 ± 1% vs. 47 ± 19%, P = 0.73) or monocular (60°, 17 ± 11% vs. 40 ± 18%, P = 0.18; 75°, 20 ± 16% vs. 17 ± 11%, P = 0.89; 90°, 10 ± 8% vs. 13 ± 13%, P = 0.58; Fig. 6) vision. Very similar findings were also observed for the Moving 2 task (Fig. 7).
Figure 6. Eccentricity-specific recovery of orienting to static visual targets for Responders and Non-responders. Radial maps displaying the percentage correct performance in static target detection at each visual eccentricity for the subsets of Responders (n = 6) and Non-responders (n = 6) in the Static task. Data are shown (top to bottom) for four stages of the study. Bar length represents the correct performance for the ipsilesional (right) or contralesional (left) visual hemispace. Concentric half circles each represent steps of 20% performance, from the most pericentral (15°) to the most peripheral (90°) eccentricity. Gray shaded portions represent the area of correct visual detection and orienting performance. Notice that in both groups of subjects, Responders and Non-responders, post-lesion spontaneous recovery progressed at the contralesional space from 0 to 45°, at uneven levels of performance, while suffering ipsilesional peripheral losses (90–60°). Only Responders showed rTMS-driven ameliorations, which extended to the further contralesional periphery (60°), while increasing performance levels at the prior eccentricities. In contrast, Non-responders showed paradoxical losses in mid peripheral ipsilesional eccentricities (45°).
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Figure 7. Eccentricity-specific recovery of moving stimuli for Responders and Non-responders. Radial maps displaying the percentage correct performance in motion target detection at each visual eccentricity for the groups of Responders (n = 6) and Non-responders (n = 6) for the Moving 2 task. Data are shown (top to bottom) for four stages of the study. Bar length represent the percentage correct performance for the ipsilesional (right) or contralesional (left) visual hemispace. Concentric half circles represent steps of 20% correct performance, from the inner (15°) to the outermost (90°) eccentricity. Gray shaded portions represent the area of correct visual detection and orienting performance. Notice that mainly Responders and, at lower rates, also Non-responders, recovered contralesional orienting spontaneously in a pericentral to mid-peripheral gradient (from 0 to 45°). Both groups had ipsilesional peripheral losses (from 90 to 75°). Responders improved performance from pericentral to far peripheral eccentricities (30–75°). Non-responders showed paradoxical losses in specific ipsilesional eccentricities (75–45°).
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After seventy sessions of rTMS treatment significant differences between the two subgroups of rTMS-treated animals emerged. Responders showed significant gains in the detection of Static targets presented in pericentral locations (Static: 45°, pre-rTMS 42 ± 11% vs. rTMS R7 92 ± 4%, P = 0.01; and 60°, 17 ± 11 vs. 61 ± 10% correct detections, P = 0.04), but not for eccentricities in the periphery (Fig. 6). Similar patterns of eccentricity-dependent ameliorations, mainly involving binocular visual locations in the Moving 2 task were also found, although they failed to reach statistical significance (Moving 2: 15°, pre-rTMS 94 ± 3% vs. rTMS R7 100%, P = 0.09; 30°, 82 ± 11% vs. 97 ± 3%, P = 0.20; 45°, 73 ± 16% vs. 89 ± 7%, P = 0.39; 60°, 70 ± 18% vs. 83 ± 8%, P = 0.37; Fig. 7). In contrast, in the Non-responders group the rTMS treatment resulted in a pattern of degraded performance for monocular targets (Static: 60°, pre-rTMS 40 ± 18% vs. rTMS R7 28 ± 16%, P = 0.06; 75°, 17 ± 11 vs. 7 ± 5%, P = 0.25; 90°, 13 ± 13% vs. 0%, P = 0.36; Moving 2: 45°, pre-rTMS 66 ± 20% vs. rTMS R7 50 ± 18%, P = 0.37; 60°, 64 ± 19% vs. 43 ± 19%, P = 0.14; 75°, 44 ± 17% vs. 27 ± 16%, P = 0.37; 90°, 18 ± 8% vs. 4 ± 4%, P = 0.14).
Interestingly, Responders and Non-responders also showed different patterns for ipsilesional performance. More precisely, with rTMS Non-responders exhibited a reduction in performance for the detection of targets at monocular eccentricities with significance only found at Static 45° and some Moving 2 targets (Static: 90°, pre-rTMS 17 ± 7% vs. rTMS R7 0%, P = 0.05; 75°, 23 ± 11% vs. 6 ± 6%, P = 0.09; 60°, 39 ± 14 vs. 21 ± 14%, P = 0.41; 45°, 94 ± 3% vs. 68 ± 8%, P = 0.04; Moving 2: 90°, pre-rTMS 19 ± 9% vs. rTMS R7 0%, P = 0.01; 75°, 45 ± 17% vs. 0%, P = 0.04; 60°, 68 ± 14% vs. 9 ± 4%, P = 0.09).
Behavioral predictors of rTMS-induced recovery
The behavioral data derived from this study indicate that rTMS significantly improved contralesional performance in a subset of animals. Interestingly, the single most contributing predictor of positive rTMS-induced recovery for the whole group was found to be the plateau levels of spontaneous recovery achieved prior to the onset of neurostimulation. In other words, the greater the levels of spontaneous levels an animal exhibited the greater the potential rTMS-induced recovery (correlation coefficient of r = 0.74, P = 0.03). Finally, the eccentricities of the contralesional visual hemispace that appeared most highly correlated with final recovery levels were the 15° (r = 0.85, P = 0.00), 30° (r = 0.72, P = 0.00), and 45° (r = 0.60, P = 0.04) visual targets.
Durability of achieved recovery in absence of rTMS treatment
Six weeks after the discontinuation of the rTMS regime, recovery rates for contralesional detection in the Responders group remained at similar levels to those reached after the last round of treatment (Static: rTMS R7 68 ± 5% vs. post-rTMS 65 ± 5% correct performance, P = 0.21) and this long-lasting performance was most apparent in the mid-periphery targets (Fig. 8). Interestingly, for Non-responders the discontinuation of rTMS sessions induced significant gains in performance, which had progressively degraded during the neurostimulation phase. Those effects were particularly significant for the detection of motion targets (Moving 2: ipsilesional targets, post-rTMS R7 46 ± 3% vs. rTMS R7 58 ± 3%, P = 0.01; contralesional targets, 41 ± 15% vs. 65 ± 10%, P = 0.01) whereas it did not influence the detection of static targets (Static ipsilesional targets R7, 42 ± 5% vs. post-rTMS 48 ± 3%, P = 0.10; and contralesional post- rTMS R7, 38 ± 3% vs. post-rTMS 45 ± 12%, P = 0.56). These effects reverted to pre-rTMS values particularly for mid-central ipsilesional eccentricities (Moving 2: 45°, post-rTMS 50 ± 18% vs. rTMS R7 81 ± 19%, P = 0.24; 60°, 43 ± 19% vs. 67 ± 23%, P = 0.26; Fig. 8). Overall, the restoration of performance in Non-responders proved to be reversible once the rTMS regime ended, which further supports the role of neurostimulation as being responsible for the maladaptive effects observed in this subset of animals.
Figure 8. Durability of rTMS-induced effects on visuospatial performance. Summary graphs of post-lesion correct performance recovery for contralesional (left column) or ipsilesional (right column) visuospatial detection in the Static (upper panel) and Moving 2 (bottom panel) tasks. Data recorded pre-rTMS (end of the spontaneous recovery phase), post-rTMS R7, and 6 weeks post-rTMS are compared to demonstrate the durability of the adaptive and maladaptive rTMS-driven effects in our population of Responders (black squares) and Non-responders (white squares). Error bars represent SEM. *P < 0.05 for values vs. pre-rTMS treatment; #P < 0.05 for 6 weeks post-rTMS vs. rTMS R7 levels. Notice that the rTMS recovery achieved by Responders in the Static task remained and did not show any signs of wearing off 6 weeks after the discontinuation of the rTMS regime. Interestingly, the unexpected and maladaptive ipsilesional (and more moderately also contralesional) decreases in performance in Non-responders wore off once the rTMS was discontinued. The latter effects proved particularly significant for the detection of static and laser-based motion stimuli presented in the ipsilesional hemispace.
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Lesion analysis and anatomical–behavioral correlations
The intention of the experiment was to damage the homologue of the human posterior parietal cortex, known as pMS, and to later apply rTMS on the rostrally adjoining aMS cortex, which is known for its ability to adequately compensate lost function after lesion (see Fig. 1 for details on the anatomy). A comprehensive lesion analysis indicated that, for all animals, the majority of the injured cortical area was removed. Nonetheless, areas of incomplete damage were found extending 1–3 mm rostrally in some subjects (n = 3 in Responders and n = 3 in Non-responders), impinging into the aMS cortex (stereotaxic levels A9–A11) or 1 mm caudally into the ventral posterior suprasylvian and the dorsal posterior suprasylvian regions (stereotaxic level P3; n = 2 in Responders and n = 3 in Non-responders). In addition, all 12 subjects showed very minor collateral damage to the pMS-adjacent visual areas such as primary visual area A19 and the splenial visual area, due to a minor but unpreventable diffusion of the neurotoxin. This spread appears to be consistent with other studies using the same methods (also see Rudolph & Pasternak, 1996; Huxlin et al., 2008; Rushmore et al., 2010; Das et al., 2012; Supporting Information Figs S1 and S2).
Quantification of injured area (mm2) showed no significant differences in the amount of lesion between groups, either for the medial (pMLS) or the lateral (pLLS) bank of the posterior parietal (pMS) cortex along the length of both pMS and aMS visual areas. Overall, the amount of spared tissue between Responders and Non-responders in both the injured pMS cortex (pMLS: 21 ± 8% vs. 14 ± 6%, P = 0.2; pLLS: 18 ± 6% vs. 15 ± 6%, P = 0.60) and the rTMS-stimulated aMS cortex (aMLS, 79 ± 7% vs. 58 ± 13%, P = 0.10 and aLLS, 79 ± 7% vs. 64 ± 13%, P = 0.10; data not shown in figure form) was not statistically different across groups. Responders and Non-responders also did not show significant differences in spared cortex at any specific coordinates across the rostral–caudal extent from pMS through aMS (medial bank, F4,32, P = 0.32; lateral bank, F4,32, P = 0.60). The final step was to determine whether the amount of spared tissue correlated to behavioral measures in the pMS cortex, and we found no relationship of anatomy with spontaneous (r = 0.35, P = 0.24) or rTMS-induced recovery (r = 0.15, P > 0.05). Overall, this observation suggests that lesion size was not the main determinant of the observed discrepancies between Responders and Non-responders.