Low-intensity contralesional electrical theta burst stimulation modulates ipsilesional excitability and enhances stroke recovery

Experimental Neurology 323 (2020) 113071

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Research Paper

Low-intensity contralesional electrical theta burst stimulation modulates ipsilesional excitability and enhances stroke recovery

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Laura J. Boddingtona, Jason P. Graya, Jan M. Schulzb, John N.J. Reynoldsa,

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a b

Department of Anatomy and the Brain Health Research Centre, Brain Research New Zealand, University of Otago, Dunedin 9054, New Zealand Department of Biomedicine, University of Basel, Basel 4056, Switzerland

ARTICLE INFO

ABSTRACT

Keywords: Motor cortex In vivo Electrophysiology Photothrombotic Contralesional Excitability

Targeting interhemispheric inhibition using brain stimulation has shown potential for enhancing stroke recovery. Following stroke, increased inhibition originating from the contralesional hemisphere impairs motor activation in ipsilesional areas. We have previously reported that low-intensity electrical theta burst stimulation (TBS) applied to an implanted electrode in the contralesional rat motor cortex reduces interhemispheric inhibition, and improves functional recovery when commenced three days after cortical injury. Here we apply this approach at more clinically relevant later time points and measure recovery from photothrombotic stroke, following three weeks of low-intensity intermittent TBS (iTBS), continuous TBS (cTBS) or sham stimulation applied to the contralesional motor cortex. Interhemispheric inhibition and cellular excitability were measured in the same rats from single pyramidal neurons in the peri-infarct area, using in vivo intracellular recording. A minimal dose of iTBS did not enhance motor function when applied beginning one month after stroke. However both a high and a low dose of iTBS improved recovery to a similar degree when applied 10 days after stroke, with the degree of recovery positively correlated with ipsilesional excitability. The final level of interhemispheric inhibition was negatively correlated with excitability, but did not independently correlate with functional recovery. In contrast, contralesional cTBS left recovery unaltered, but decreased ipsilesional excitability. These data support focal contralesional iTBS and not cTBS as an intervention for enhancing stroke recovery and suggest that there is a complex relationship between functional recovery and interhemispheric inhibition, with both independently associated with ipsilesional excitability.

1. Introduction Interhemispheric inhibition is the mechanism by which each cerebral hemisphere inhibits the other (Ferbert et al., 1992) and is thought to be engaged in the brain during the process of initiating a unilateral movement (Meyer et al., 1995; Di Lazzaro et al., 1999). Stroke has been shown to alter the normal balance of interhemispheric inhibition, with the contralesional hemisphere exerting excessive inhibition onto the ipsilesional hemisphere (Murase et al., 2004; Duque et al., 2005). In this scenario, the ‘interhemispheric imbalance’ model posits that the ipsilesional hemisphere is ‘doubly-disabled’ where stroke-induced ipsilesional damage is coupled with excessive inhibition originating from the contralesional hemisphere (Nowak et al., 2009; Di Pino et al., 2014). Targeting this abnormal contralesional excitability with non-invasive neuromodulation techniques such as repetitive transcranial magnetic stimulation (rTMS) has shown promise for enhancing functional outcomes after stroke (Mansur et al., 2005; Takeuchi et al., 2005; Avenanti

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et al., 2012; Koch et al., 2012; Sung et al., 2013; Wang et al., 2014; Du et al., 2016), but is yet to show sufficient benefit to be recommended for clinical practice (Lefaucheur et al., 2014). Non-invasive neuromodulation studies using rTMS and transcranial direct current stimulation (tDCS) are often designed on the premise that ‘turning down’ an excessively disinhibited contralesional hemisphere or ‘turning up’ an over–inhibited ipsilesional cortex will ‘rebalance’ the hemispheres and enhance functional return (Au-Yeung et al., 2014; Lefebvre et al., 2014; Rocha et al., 2016). However, reports of the benefit gained from rebalancing the hemispheres are inconsistent (Ackerley et al., 2010; Seniów et al., 2012; Talelli et al., 2012; Stinear et al., 2015; McDonnell and Stinear, 2017; Xu et al., 2019). Of note, the effectiveness of conventional neuromodulation is usually assessed using motor evoked potentials (MEPs), which are an indiscriminate indicator of changes in the efficacy of monosynaptic brain connections versus intrinsic excitability of the activated circuit (Bestmann and Krakauer, 2015; Matheson et al., 2016). In addition, these extracranial approaches

Corresponding author. E-mail address: [email protected] (J.N.J. Reynolds).

https://doi.org/10.1016/j.expneurol.2019.113071 Received 14 March 2019; Received in revised form 20 September 2019; Accepted 25 September 2019 Available online 24 October 2019 0014-4886/ © 2019 Elsevier Inc. All rights reserved.

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are likely to affect the excitability and synaptic connectivity globally of many different neural elements in the activated area. Hence an alternative approach to global neuromodulation after stroke could be to more selectively target the abnormally functioning interhemispheric circuitry (Boddington and Reynolds, 2017). In order to investigate the modulation of interhemispheric inhibition at the cellular level in the motor cortex, we previously (Barry et al., 2014) developed an in vivo electrophysiological recording system, modeled on the human double-coil TMS paradigm (Ferbert et al., 1992). By recording intracellularly in the healthy motor cortex of anesthetized rats, we demonstrated that an excitatory synaptic input to a single pyramidal neuron could be inhibited by intracranial electrical stimulation applied to the contralateral motor cortex at specific interstimulus intervals. Most importantly, the contralateral stimulation only elicited this interhemispheric inhibition when set to a low intensity, just below that which elicited a simultaneous excitatory response through the recruitment of converging transcallosal excitatory circuits. Moreover, when electrical stimulation at the same low intensity was applied to the contralateral hemisphere as a single treatment of intermittent theta-burst stimulation (iTBS) (Huang et al., 2005), long-term depression was induced in the crossed pathway, effectively ‘switching off’ interhemispheric inhibition for the remainder of the experiment (Barry et al., 2014). Presumably, contralateral stimulation below the threshold for eliciting an excitatory response preferentially recruits lower threshold circuits that interact with inhibitory elements synapsing onto the recorded neuron. Barry et al. (2014) found that it was iTBS that induced long-term depression and reduced interhemispheric inhibition following electrical stimulation. These findings suggest that targeting specific neural elements with more focused stimulation may provide improved outcomes in neuromodulation studies. Consistent with studies that have shown that modulation of interhemispheric inhibition can correlate with improvements in motor function after stroke (Takeuchi et al., 2005; Avenanti et al., 2012), Barry et al. (2014) also found that low-intensity electrical iTBS, which down-regulated interhemispheric inhibition in normal animals, enhanced functional recovery in animal models of cortical injury and stroke. However, stimulation was commenced during the acute phase after injury. In contrast, only a limited therapeutic window remains in humans after stroke, in which this invasive stimulation could be harnessed to enhance recovery (Vallone et al., 2016). In the present study we investigated whether low-intensity intracranial electrical iTBS, and its functional ‘opposite’ cTBS, could modulate recovery when applied at later time points after stroke. By making intracellular recordings from pyramidal neurons in the ipsilesional cortex at the end of the stimulation period, we also aimed to determine the relationship between functional recovery, interhemispheric inhibition and cortical excitability at the level of individual neurons following stroke.

administered at all incision sites. The skull was exposed and a 3 mm diameter craniotomy was made over the left forelimb motor cortex (centered at 1.5 mm anterior and 4.0 mm lateral to Bregma). A cold light source (KL 1500 LED, Schott, USA) was positioned directly over the craniotomy and the brain was illuminated for twenty minutes. The photosensitive dye Rose Bengal (1.3 mg/kg, Sigma Life Science, USA) was infused intravenously via the jugular vein over the first two minutes of illumination. After illumination, the skullcap was replaced and secured to the skull using dental cement. Wounds were then sutured and sterilized with Betadine. The analgesic carprofen (5 mg/kg s.c.) and saline (8–10 mL) for hydration was administered, followed by the anesthetic reversal agent antisedan (2.5 mg/kg, s.c.). 2.3. Electrode implantation surgical procedures Following a period of stroke recovery (either 7 or 28 days) rats were anesthetized as per above. Under surgical anesthesia, rats were securely head-fixed, the skull was re-exposed and a cortical stimulating electrode (MS303/2-B/SP, stainless steel, two-channel twisted electrode; Plastics One, United States) implanted into layer 5 of the contralesional hemisphere (2.5 mm lateral and in line with Bregma, to a depth of 1.5 mm below the cortical surface). A silver wire extradural EEG electrode was also secured 1–2 mm posterior to the stroke lesion. The stimulating electrode was tested and the minimal intensity at which contralesional electrical stimulation could induce an evoked response in the EEG recording from the peri-lesional neural tissue was determined. All electrodes were secured in place with skull screws and dental cement, and wounds were sutured before the administration of pain relief, saline, and anesthetic reversal as above. 2.4. Theta burst stimulation schedule and dose On each stimulation day, freely-moving awake animals were connected to the stimulator and theta burst stimulation (15 pulses per second on average; 3 pulses applied per burst at 50 Hz, bursts repeated at 5 Hz for a total of 600 pulses; monophasic; 500 μs pulse width) applied to the contralesional stimulating electrode in a continuous (cTBS) or intermittent pattern (iTBS; 2 s on followed by an 8 s break; Huang et al., 2005). In either case, stimulation began three days after the contralesional stimulating electrode was implanted. Rats received either no stimulation (sham) or one of two different doses of stimulation: a minimal dose (one set of 600 pulses of iTBS or cTBS, twice a week, ~2–3 days apart, five sessions total), or an intensive dose (five sets of 600 pulses of iTBS or cTBS, each set spaced by 10 min, five days a week, 15 sessions total). Rats were all randomly allocated to treatment groups after electrode implantation. On the first day of testing, the resting motor threshold was determined by increasing the stimulation intensity until a motor twitch in the contralateral (ipsilesional) forelimb was evoked. The final TBS intensity was set to half-way between the resting motor threshold, and the evoked-EEG response threshold intensity determined during the electrode implantation surgery. This final calculated intensity (average across all rats 0.31 ± 0.05 mA) was almost identical to the contralateral cortex intensity set in Barry et al. (2014), i.e. just below that which elicited an excitatory response in the recorded neuron (0.33 ± 0.05 mA).

2. Materials and methods 2.1. General experimental details All animal experimentation was performed in accordance with the ethical approvals granted by the University of Otago Animal Ethics Committee (approval number 75–14). Rats were individually housed under a 12-h reversed light/dark cycle, and had access to both food and water ad libitum.

2.5. Grid-walking task behavioral assessments and data analysis

2.2. Photothrombotic stroke lesion surgical procedures

To assess motor function, rats were placed on the center of an elevated wire mesh grid (height 70 cm, area 40 × 40 cm, mesh size 2.5 × 2.5 cm, wire thickness 1.5 mm, Emtech, University of Otago, NZ) and allowed to explore the area for two minutes (Schaar et al., 2010). As rats traversed the grid, they would either make correct or incorrect forelimb paw placements which were video recorded from below for later offline analysis. An incorrect paw placement or ‘foot fault’ was defined as a misplaced step whereby the forelimb slipped completely

All survival surgical procedures were performed under aseptic conditions. Male Wistar rats (6–9 weeks old, n = 67) were anesthetized with ketamine (75 mg/kg, s.c.) and domitor (0.5 mg/kg, s.c.) under cover of the systemic antibiotic amphoprim (30 mg/kg, s.c). Once a surgical plane of anesthesia was reached, rats were head-fixed in a stereotaxic frame and local anesthetic (bupivicaine; 2.5 mg/kg) was 2

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through the grid (either in full, or by slipping off after the initial placement), or the step was incorrectly positioned (supported solely by the wrist region of the forelimb). To normalize for different activity levels between rats, foot faults were expressed as a percentage of the total steps (correct and incorrect) taken in each session. Rats received both a pre- and post-stimulation grid-walking task assessment on each day of stimulation. No acute effect of stimulation was noted in the gridwalking task performance between pre- and post-stimulation assessments therefore data from these two assessments were pooled for each testing day. Data are presented as percentage improvement scores, which represent the improvement in foot fault scores on the final testing day compared to the most impaired foot fault score recorded prior to the first day of TBS application. Animals were excluded from further behavioral analyses if their baseline foot faults were 9% or greater, or if they did not exhibit an increase in foot faults when tested following the stroke, up to the first day of stimulation. 9 animals were excluded by these criteria: two from the Early minimal sham group, one from the Early minimal iTBS group, one from each of the Early intensive iTBS, cTBS and sham groups, and one from each of the Delayed minimal iTBS, cTBS and sham groups, leaving a sample of 58 rats for behavioral analysis.

individually trigger two stimulators (NL800A) to deliver monophasic electrical stimulation to either the ipsilesional ‘test’ electrode or the contralesional ‘conditioning’ stimulating electrodes to elicit post-synaptic potentials (PSPs) in the recorded cell. For interhemispheric inhibition experiments, the contralesional electrode was stimulated at specific inter-stimulus intervals (ISIs; −10 ms to +10 ms; 1 ms steps) relative to stimulating the ipsilesional electrode, to determine the influence of contralesional activation on ipsilesionally-evoked PSP properties (see Fig. 2a). Contralesionally-evoked PSPs were elicited using near threshold-intensity stimulation (as previously described in Barry et al., 2014). Ipsilaterally-evoked PSPs were elicited using the stimulation intensity that produced a PSP at approximately 70% of the maximum PSP amplitude. 2.7. Electrophysiological data analysis All analysis was performed offline using Spike2 and Axograph X for Windows. All PSP measurements were made using an average PSP trace made up of at least three individual replicate traces. The amplitude (mV) of the PSP was defined as the absolute difference between the PSP peak and the down-state membrane potential just prior to the stimulus. For interhemispheric inhibition experiments, in order to determine the extent of the influence of contralesional stimulation on the ipsilesionally-evoked response, the maximum slope of the conditioned PSP was measured and normalized to an unconditioned PSP (test pulse alone) evoked at the same stimulation intensity. At specific ISIs when the conditioning stimulation preceded the test stimulation, the normalized PSP slope was reduced, indicating the contralesional hemisphere inhibited the ipsilesional response. Since the specific interstimulus interval (ISI) that evoked the strongest interhemispheric inhibition differed between experiments, the ISI that produced the maximum reduction in slope was aligned across all cells in order to average the responses between experiments (see Fig. 2d for average responses and Fig. 2e for individual cells).

2.6. In vivo electrophysiology, surgical and recording methods Within a week following the final application of TBS, rats were anesthetized with urethane (1.6–2.0 g/kg body weight i.p.) and prepared for in vivo intracellular recording as previously described (Barry et al., 2014). Once anesthetized, rats were securely head-fixed in a stereotaxic frame with ear bars and an incisor bar. Core body temperature was maintained between 36 and 37 °C for the duration of the experiment. The skull was exposed and overlying connective tissue was bluntly dissected away from the skull and headpiece. Muscles were deflected away from the side and rear of the skull and the cisterna magna punctured to reduce brain pulsations. An additional cortical stimulating electrode was implanted and cemented in place in layer 5 of the ipsilesional hemisphere (approximately 1.5 mm lateral to and in line with Bregma, on a 30° angle from vertical, to a depth of 1.5 mm below the cortical surface). A further craniotomy was made anteromedial to the stroke lesion and a ‘well’ was fashioned from dental cement around this craniotomy, forming the site for intracellular recording. A small incision was carefully made in the dura of the recording craniotomy, to expose the brain prior to advancing the micropipette through the cortex. Sharp micropipettes (50–90 MΩ resistance) were pulled from borosilicate glass capillary tubes (3 mm O.D, 1.62 mm I.D, Harvard Apparatus Ltd., Kent) using a vertical puller (Model PE-21, Narishige, Japan) and were filled with a 1 M solution of potassium acetate and in some cases biocytin (1%) for later neuronal recovery. The micropipette was advanced through the brain (from layer 2/3 to deep layer 5) in 1 μm steps using a 3-axis micromanipulator (Model IVM, Scientifica, United Kingdom) until a neuron was impaled, or to a maximal depth of 1.8 mm from the cortical surface. Impalement of a neuron was indicated by a rapid hyperpolarization of the recorded membrane potential and fluctuations between hyperpolarized down-states and relatively more depolarized up-states with occasional action potential firing. Signals were recorded using a Neurolog headstage (NL102G; Digitimer, United Kingdom) and preamplifier (Module NL102), digitized at 20 kHz with a CED Micro1401 (Cambridge Electronic Design, United Kingdom) and recorded using Spike2 (Version 7.12c, Cambridge Electronic Design, United Kingdom). After successful impalement, the neuron was left to stabilize for five minutes and membrane potential fluctuations were recorded. Cellular properties and intrinsic excitability were determined through the injection of hyperpolarizing to depolarizing current steps (200pA steps) until current injection induced action potential firing. Custom software (Stimulator Control, Dr. John Reynolds and SCL Limited) was used to

2.8. Histological analyses After completion of electrophysiological experiments, deep urethane anesthesia was maintained and the rat was either transcardially perfused with 4% paraformaldehyde in 0.01 M phosphate buffered saline (PBS), or brains were extracted and post-fixed in 4% paraformaldehyde, before transfer into 0.01 M PBS. Brains were processed and serially sectioned on a vibratome (Vibratome Co., USA) before staining with cresyl violet to determine stroke lesion size and position, and correct electrode placement. In brains with a filled neuron, the anterior portion of the brain containing the filled neuron was instead processed using standard histological procedures for neuronal recovery (Horikawa and Armstrong, 1988). 2.9. Statistical analyses Residuals for all data sets were plotted to assess normality. Welch's ANOVA with Dunnett's post hoc comparison was used to compare functional improvement in the pooled early treatment groups. A twoway repeated measures ANOVA with Tukey's multiple comparisons tests was used to identify differences between treatment-group foot faults over time. For foot faults measured before and after the effects of stroke and treatment, Wilcoxon's signed rank test for paired samples was used. Pearson's correlation coefficient was computed to assess the various relationships between functional recovery, PSP amplitude, down to up-state amplitude, and interhemispheric inhibition. All data described in the text are means ± SEM, unless otherwise stated. Data are plotted as means ± SEM, or box and whisker plots showing, median, quartiles and ranges. Level of significance was set to P < 0.05, using two-tailed tests. 3

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Fig. 1. Theta burst stimulation influences functional recovery on the grid-walking task after stroke. (a) Box and whisker plots showing the percentage improvement of rats within each experimental group. (b) Functional improvement for the early stimulation groups of each intensity combined. Triangles indicate intensive; circles minimal stimulation. (c) Foot fault scores across early stimulation groups for each treatment type: pre-stroke (purple), post-stroke (gray), and after three weeks of stimulation (green/ sham, orange/cTBS, blue/iTBS). *, **, and *** indicate significant differences of P < 0.05, P < 0.01, and P < 0.001 between bar means, respectively. Horizontal line indicates the average baseline foot faults across all groups. (d) Representative examples of the cortical photothrombotic stroke lesions induced in the left motor cortex from each experimental group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the recovery of those rats most severely affected by stroke. When pooling data from individual animals in the normal and intense groups of each early treatment paradigm (Fig. 1b), this pattern becomes clearer, with iTBS enhancing recovery by 54 ± 4.5%, compared to 39.7 ± 6.3% for sham and 32.6 ± 7.3% for cTBS treated rats. Notably all iTBS-treated rats recovered by at least 26%, whereas some rats in both sham and cTBS treatment groups showed minimal change or even worsening of function following treatment. The early pooled groups differed from each other (P < 0.05; F (2, 23.7) = 3.759 Welch's ANOVA), however due to the degree of overlap of improvement of individuals in each group, only a trend in improvement is apparent for iTBS compared to sham (Dunnett's multiple comparisons test against sham: iTBS, t = 1.89, P = 0.14; cTBS, t = 0.73, P = 0.72). Rodents post stroke commonly recover a significant proportion of function spontaneously, hence to investigate further whether iTBS significantly modified this tendency towards improvement and enhanced recovery, we compared the change in foot faults for the combined early groups over the course of the experiment (Fig. 1c). Considering foot faults pre-stroke, post-stroke, and after all stimulation sessions, a significant effect of treatment (P < 0.05; F (2, 37) = 3.797 repeated measures two-way ANOVA) and of timepoint was detected (P < 0.0001; F (1.796, 66.44) = 117.9)). Importantly, the groups did not differ by number of foot faults at baseline, or after stroke induction (P > 0.05; Tukey's multiple comparisons). However, at the end of 3 weeks of stimulation, iTBS had significantly reduced foot faults compared to both sham (P < 0.05) and cTBS treatments (P < 0.01). Adding further support to the beneficial effects of contralesional iTBS applied 10 days after stroke, there was no difference between baseline and post stroke foot faults for the iTBS group (Wilcoxon signed rank test, P = 0.2), suggesting that the stimulation had returned the foot

3. Results 3.1. Effects of contralesional TBS on functional recovery after stroke We previously reported that a minimal dose of low-intensity iTBS enhanced functional recovery when stimulation began three days after cortical injury (Barry et al., 2014). We wished to determine whether this same small dose of stimulation would also enhance recovery if applied at later time points (ten days; ‘Early’, and thirty-one days; ‘Delayed’) following stroke. Delayed minimal stimulation of either type beginning thirty-one days following stroke trended towards marginally enhancing functional recovery (see Fig. 1a, left three groups; n = 6 per stimulation group), however the large variability in recovery compared to sham suggested that a minimal dose of TBS was insufficient to alter recovery at this chronic stage after stroke. In contrast, early minimal stimulation with iTBS on average appeared to enhance the degree and spread of functional improvement compared to sham or cTBS (Fig. 1a, middle three groups; iTBS: 59.8 ± 6.5%, sham: 51.4 ± 6.8%, cTBS: 39.2 ± 8.4%, n = 7–9 per stimulation group). Since repeated applications of TBS may produce significantly enhanced or prolonged aftereffects on functional outcomes (Nyffeler et al., 2009; Cazzoli et al., 2012; Nettekoven et al., 2014), we next applied a more intensive stimulation regimen at this early time point. We found an intensive dose of iTBS-treatment very consistently improved recovery compared to a similar number of behavioral sessions of cTBS or sham treatment, with all iTBS rats demonstrating at least 26% improvement in function (range: 26.5 to 61.0%; average ± SD, 47.7 ± 5.4%; Fig. 1a; n = 5–6 per stimulation group). Comparison with the range of recovery in the sham group (range − 3.6 to 53.9%; 26.0 ± 8.6%; Fig. 1a) suggests that intensive iTBS strongly influenced 4

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faults to baseline levels, whereas a residual deficit remained for the sham (Wilcoxon signed rank test, P < 0.001) and cTBS (Wilcoxon signed rank test, P < 0.01) groups. There were no differences in lesion volumes (Early stimulation: 18.7 ± 2.3 mm3; Delayed stimulation: 18.2 ± 2.2 mm3; see Fig. 1d for example stroke lesions) or electrode placements between treatment groups (data not shown) underlying the group differences in functional recovery. Therefore, the application of contralesional iTBS at an early time point enhances functional recovery following stroke, particularly for more severely affected individuals. 3.2. Intracellular recordings from pyramidal neurons in the peri-lesional area following photothrombotic stroke At the completion of the behavioral experiments described above, the animals were anesthetized with urethane and pyramidal neurons in the cortex adjacent to the stroke area impaled, where possible. Out of 58 animals, 21 animals yielded 24 neurons, all within 950 μm from the edge of the infarct (average 438 ± 79 μm; estimated from histology of recovered cells; see Fig. 2c). Of the 10 biocytin-filled cells that could be recovered, the gross morphology (Fig. 2b) did not differ from pyramidal neurons we have recorded previously from intact animals (Barry et al., 2014). Electrophysiological recordings showed that neurons exhibited a comparable down-state membrane potential (−74.4 ± 9.1 mV, n = 24; mean ± SD) and input resistance (22.8 ± 8.0 MΩ; mean ± SD) to neurons recorded in non-lesioned animals (down-state membrane potential: −78.9 ± 9.3 mV, input resistance: 26.3 ± 10.9 MΩ, n = 9, mean ± SD; unpublished data from Barry and Reynolds). All neurons exhibited transitions between a hyperpolarized down state and a relatively depolarized up state due to synchronized corticothalamic activity (Steriade et al., 1993, 2001). Maximal inhibition across all cells was elicited at conditioning-to-test-pulse intervals between 2 and 8 ms, (12.5 ± 2.9% for all groups combined, Fig. 2a and d; see Fig. 2e for individual cell examples). Interhemispheric inhibition measured in cells from sham-treated rats in all experimental groups (17.1 ± 5.8%, n = 6; mean ± SD) compared well with that previously reported by Barry et al. (2014) in healthy rats (17.2 ± 5.2%, n = 12; mean ± SD). It was noted that the PSP amplitude of the iTBS early stimulation group was larger than both the sham and cTBS cells (Fig. 2f and g, iTBS: 22.1 mV ± 1.0 n = 3; sham: 11.3 mV ± 3.6, n = 3; cTBS: 10.6 mV ± 4.2, n = 4). This same pattern was seen in the amplitude of the spontaneous membrane potential fluctuations for the iTBS treatment group (Fig. 2h, iTBS: 21.6 mV ± 0.8; sham: 10.0 mV ± 3.6; cTBS: 8.4 mV ± 0.9). 3.3. Relationship between electrophysiological measures and functional improvement Our previous work had identified that iTBS could modulate interhemispheric inhibition in the healthy brain, and that this same stimulation pattern could enhance recovery after stroke, however the relationship between the two measures was not empirically determined (Barry et al., 2014). The present study, however, presented the opportunity to determine whether changes in functional recovery after stroke were indeed related to interhemispheric inhibition and indices of synaptic input strength, as inferred from measures taken at the completion of the behavioral procedures. For each rat in which an intracellular recording was successfully obtained, the electrophysiological measurements were compared to the percentage of functional improvement obtained by that rat during the period of recovery and stimulation. When considering cells from either all animals, or the early stimulation animals alone, there was no correlation between interhemispheric inhibition and functional improvement on the grid walking task (Fig. 3a and b). There was however a significant correlation between interhemispheric inhibition and PSP amplitude (Fig. 3c; r = −0.65,

Fig. 2. Recording interhemispheric inhibition in the ipsilesional hemisphere. (a) Diagram of stroke lesion, recording micropipette, and stimulation electrode position (left). Example inter-stimulus intervals where the conditioning stimulus (orange lightning bolt) occurs at different times relative to the test stimulus (blue lightning bolt) (left). (b) An example of a biocytin-filled pyramidal neuron. (c) A filled pyramidal neuron from a different experiment, showing the cell soma located within 0.26 mm of the border of the stroke lesion. (d) Normalized conditioned PSP slope (%) for all sham treated cells (n = 8) with inter-stimulus intervals (ISIs) aligned to the interval where the normalized slope was most reduced. (e) Normalized conditioned PSP slope (%) for cells from two individual rats. In the first cell (top panel), the conditioned response is maximally reduced 5 ms prior to the test pulse whereas in the second cell (bottom panel), the largest reduction is seen 2 ms prior to the test pulse. (f) Representative PSP traces elicited by ipsilateral single stimuli recorded from cells in the early stimulation groups. (g) Combined average PSP trace overlay for early stimulation iTBS (blue), cTBS (orange) and sham (green) treatment groups. (h) Representative traces of spontaneous membrane potential fluctuations from cells from the early stimulation groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 5

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functional recovery directly with cellular excitability and interhemispheric inhibition. We demonstrated a relationship between synaptic activity in the ipsilesional hemisphere and interhemispheric inhibition, however no direct relationship between functional recovery and interhemispheric inhibition. Notably, however there was a significant positive correlation between synaptic input strength following stimulation and functional recovery for the early stimulation groups. Therefore, low-intensity iTBS of the contralesional hemisphere may improve functional recovery through its effects on synaptic transmission of the ipsilesional hemisphere. 4.1. Cortical reorganization after stroke A low dose of stimulation was insufficient to enhance functional outcomes at later chronic phases of recovery, however in this study and in our earlier work the same low dose of stimulation enhanced recovery during the sub-acute and acute stage of stroke respectively (Barry et al., 2014). It is well established that over the days and weeks following stroke, the neighboring brain regions undergo a period of heightened plasticity allowing for substantial reorganization of lost functions into the surviving tissue, however this stabilizes over time, alongside a reduction in the gains made with rehabilitation (Carmichael et al., 2001; Murphy and Corbett, 2009; Krakauer et al., 2012). Our two studies therefore likely demonstrated this difference in the high capacity for plasticity in the acute and sub-acute stage manifesting as rapid improvements in function, compared with the ongoing reduction in plasticity during the chronic stages alongside reduced recovery. Indeed previous animal studies have demonstrated that over these same stages of recovery, changes in indicators of plasticity, such as axonal sprouting and peri-neuronal net expression (Carmichael, 2003; Wang and Fawcett, 2012), occur in the peri-lesional tissue, the extent of which is associated with the degree of functional recovery achieved (Carmichael et al., 2001; Carmichael and Chesselet, 2002; Dijkhuizen et al., 2003; Carmichael et al., 2005). Specifically, over the first few days following stroke the level of axonal sprouting is high and the level of peri-neuronal net protein expression is dramatically reduced, creating an environment favoring the formation of new synaptic connections (Carmichael et al., 2005). Over days fourteen to twenty-eight poststroke the presence of peri-neuronal nets gradually increases, alongside the upregulation of growth-inhibiting proteins, preventing further axonal sprouting (Carmichael et al., 2005). In our previous study, we applied iTBS coinciding with the acute period after stroke (Barry et al., 2014), when axonal sprouting is high and when iTBS had the potential to facilitate new synapse formation and consequently enhance recovery. In contrast, the present study applied iTBS during the sub-acute and later chronic phases of recovery, coinciding with periods where axonal sprouting was decreasing and alongside the steady up-regulation of peri-neuronal nets working to prevent new synapse formation (Berardi et al., 2003; Rhodes and Fawcett, 2004; Carmichael et al., 2005). At the later delayed time point, the low dose of stimulation may have been insufficient to perturb these processes. Further work should determine whether a more intensive dose of stimulation would be able to enhance recovery at the more delayed time point. Whilst rTMS has been trialed extensively due to the relative ease and non-invasive nature of application, large equipment and the presence of a skilled operator is required in order to prime rehabilitation, which likely precludes rTMS as an efficient long-term tool for use alongside at-home rehabilitation programs. An implantable stimulating system like that used for conditions such as Parkinson's disease, depression, or pain (Benabid, 2003; Mayberg et al., 2005; Falowski, 2015) could allow for more frequent or even continuous stimulation protocols to be applied during both rehabilitation and activities of daily life. A multi-contact extradural stimulating electrode could be implanted over the hand/distal upper limb motor cortical area contralateral to the lesioned hemisphere, with the stimulation intensity set to be just sufficient to activate low-threshold crossed corticocortical axons (Barry

Fig. 3. Correlation between cellular measures of interhemispheric inhibition and excitability with functional improvement. There was no correlation between interhemispheric inhibition and functional improvement, whether all animals (a) or only the early stimulation animals (b) were considered. Interhemispheric inhibition and cell excitability, measured as the amplitude of ipsilesionally-evoked PSPs, were negatively correlated when cells from all groups were considered (c) but not significantly, when limiting the comparison to the early stimulation animals only (d). Functional improvement in the early stimulation animals was strongly correlated with measures of cell excitability, namely (e) the amplitude of ipsilesional PSPs and (f) the amplitude of Up-todown state membrane potential fluctuations.

P < 0.01, n = 15) with all rats considered together. The same trend remained for the early stimulation group alone, however with fewer animals this relationship was no longer significant (P = 0.17; r = −0.53, n = 8). For the early stimulation groups the measures of synaptic input strength (3e and f) and functional gain were found to be strongly and significantly correlated (PSP amplitude: r = 0.84, P < 0.01 n = 8; Up-to-down state amplitude: r = 0.76, P < 0.05, n = 8). Therefore, our data suggest that the early application of contralesional stimulation may lead to functional improvement through a process related to positive changes in ipsilesional excitability, which is independently associated with interhemispheric inhibition. 4. Discussion Our data showed that low-intensity electrical iTBS applied to the contralesional hemisphere enhanced functional recovery at the subacute/early chronic stage after stroke. We also made in vivo intracellular recordings at the end of the experiment from neurons located within 1.0 mm of the border of the ischemic stroke, which provided us with the unprecedented opportunity to associate changes in 6

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et al., 2014), and stimulation applied in conjunction with a structured rehabilitation programme. Our data suggest that as time after stroke increases, a more intensive combined stimulation and therapy regimen may be required to produce a therapeutic benefit; thus an implanted system may represent a feasible treatment option for certain stroke patients (Levy et al., 2008, 2016).

interhemispheric inhibition (Barry et al., 2014) by using low intensity electrical stimulation and not TMS (Xu et al., 2019) as the means of targeting transcallosal crossed inhibitory circuits. 5. Conclusion In summary, in line with our previous work, this study continues to support the use of low-intensity contralesional iTBS applied using electrical stimulation as an intervention for enhancing stroke recovery. Further experiments are required to determine whether more intensive stimulation can extend the window in which invasive stimulation of this nature is beneficial to chronic stroke, and to determine the precise role that the complex interaction between changes in ipsilesional excitability and interhemispheric inhibition plays in recovery of function following stroke.

4.2. Relationship of synaptic effects of TBS to functional outcomes Our study presented us with the unique opportunity to associate our changes in functional motor ability after stroke with interhemispheric inhibition and ipsilesional synaptic activity. While interhemispheric inhibition as measured in this study did not relate to functional recovery directly, we found a strong correlation between functional improvement and ipsilesional synaptic input strength, which reinforces the importance of directing therapies towards increasing ipsilesional synaptic activity. Similarly in humans it has been shown that irrespective of the method of modulation, good functional outcomes rely on the return of pre-stroke ipsilesional excitability (Rehme et al., 2012). The strength of our finding is that ipsilesional excitability was measured across two independent measures, one being an evoked synaptic response, and the other being synaptic-driven membrane potential fluctuations recorded during synchronized cortico-thalamic activity (Steriade et al., 1993, 2001). Since stimulation was delivered contralesionally, this indicates that TBS must influence peri-lesional activity through some interhemispheric mechanism. This is consistent with work in humans which has demonstrated that contralesional rTMS can modulate ipsilesional excitability in humans (Ackerley et al., 2010). Previous work in rats demonstrated that activation of callosal axonal processes using single pulses of TMS primarily activates dendrites targeting upper layer interneurons (Murphy et al., 2016). It is possible that the intracranial activation of the same transcallosal axons in the present study may have modulated the activity of these local inhibitory microcircuits to influence excitability in the ipsilesional hemisphere. Additionally, direct callosal activation by iTBS-rTMS has been shown to reduce parvalbumin expression in the rat cortex, indicating altered GABAergic signaling and a disinhibited cortical state (Benali et al., 2011). It is important to note that our measure of interhemispheric inhibition is static (recorded at rest during no movement activation) and measured at a single time point following three weeks of stimulation. Since in vivo intracellular recording is only possible during the terminal procedure, this precludes us from making similar measures of ipsilesional excitability and interhemispheric inhibition during behavioral testing. It is plausible that the chronic application of intermittent thetaburst stimulation did indeed induce changes in interhemispheric inhibition that were not detectable through a single measurement at the terminal procedure. It is also important to consider that interhemispheric inhibition varies depending on the movement stage at which it is measured in. During rest or sustained isometric contraction, no abnormalities in interhemispheric inhibition are detected after stroke (Butefisch et al., 2007; Cassidy et al., 2015; Stinear et al., 2015), whereas interhemispheric inhibition has consistently been shown to be disrupted after stroke during the initiation phase of voluntary movement (Murase et al., 2004; Duque et al., 2005; Boddington and Reynolds, 2017). It is therefore not surprising that interhemispheric inhibition after stroke did not differ compared to our previous study conducted in healthy animals, since in both cases interhemispheric inhibition was measured under anesthesia (Barry et al., 2014). To address these important issues, future studies should explore the measurement of interhemispheric inhibition dynamically during movement, and compare this directly at the beginning and end of an intervention with changes in function. To obtain a reliable measure of the relationship of changes in interhemispheric inhibition and stroke recovery over time, it would be better to minimize the co-activation of crossed excitatory callosal circuits that can mask changes in

Funding This work was supported by the Royal Society of New Zealand Rutherford Discovery Fellowship (to J.N.J.R), the W & B Miller Postgraduate Scholarship from the Neurological Foundation of New Zealand with support from the HB Williams Turanga Trust, New Zealand (to L.J.B), a University of Otago Research Grant, and a Postgraduate publication bursary award from the University of Otago, New Zealand (to L.J.B). Declaration of Competing Interest The Authors declare no conflicts of interest. Acknowledgements We would like to thank Mr. Andrew Gray for statistical advice. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.expneurol.2019.113071. References Ackerley, S.J., Stinear, C.M., Barber, P.A., Byblow, W.D., 2010. Combining theta burst stimulation with training after subcortical stroke. Stroke 41, 1568–1572. Au-Yeung, S.S.Y., Wang, J., Chen, Y., Chua, E., 2014. Transcranial direct current stimulation to primary motor area improves hand dexterity and selective attention in chronic stroke. Am. J. Phys. Med. Rehabil. 93, 1057–1064. Avenanti, A., Coccia, M., Ladavas, E., Provinciali, L., Ceravolo, M.G., 2012. Low-frequency rTMS promotes use-dependent motor plasticity in chronic stroke: a randomized trial. Neurology 78, 256–264. Barry, M.D., Boddington, L.J., Igelström, K.M., Gray, J.P., Shemmell, J., Tseng, K.Y., Oorschot, D.E., Reynolds, J.N.J., 2014. Utility of intracerebral theta burst electrical stimulation to attenuate interhemispheric inhibition and to promote motor recovery after cortical injury in an animal model. Exp. Neurol. 261, 258–266. Benabid, A.L., 2003. Deep brain stimulation for Parkinson’s disease. Curr. Opin. Neurobiol. 13, 696–706. Benali, A., Trippe, J., Weiler, E., Mix, A., Petrasch-Parwez, E., Girzalsky, W., Eysel, U.T., Erdmann, R., Funke, K., 2011. Theta-burst transcranial magnetic stimulation alters cortical inhibition. J. Neurosci. 31, 1193–1203. Berardi, N., Pizzorusso, T., Ratto, G.M., Maffei, L., 2003. Molecular basis of plasticity in the visual cortex. Trends Neurosci. 26, 369–378. Bestmann, S., Krakauer, J.W., 2015. The uses and interpretations of the motor-evoked potential for understanding behaviour. Exp. Brain Res. 233, 679–689. Boddington, L.J., Reynolds, J.N.J., 2017. Targeting interhemispheric inhibition with neuromodulation to enhance stroke rehabilitation. Brain Stimul. 10, 214–222. Butefisch, C.M., We ling, M., Netz, J., Seitz, R.J., Homberg, V., 2007. Relationship between interhemispheric inhibition and motor cortex excitability in subacute stroke patients. Neurorehabil. Neural Repair 22, 4–21. Carmichael, S.T., 2003. Plasticity of cortical projections after stroke. Neuroscientist 9, 64–75. Carmichael, T., Chesselet, M.-F., 2002. Synchronous neuronal activity is a signal for axonal sprouting after cortical lesions in the adult. J. Neurosci. 22, 6062–6070. Carmichael, S.T., Wei, L., Rovainen, C.M., Woolsey, T.A., 2001. New patterns of intracortical projections after focal cortical stroke. Neurobiol. Dis. 8, 910–922.

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