Corticostriatal projections relying on GABA levels encode exercise-induced functional recovery in cerebral ischemic mice

doi:10.21203/rs.3.rs-1766063/v1

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Corticostriatal projections relying on GABA levels encode exercise-induced functional recovery in cerebral ischemic mice

https://doi.org/10.21203/rs.3.rs-1766063/v1

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published 29 Dec, 2022

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Stroke is a neurological disorder characterized by high disability and death worldwide. The occlusion of the middle cerebral artery (MCAO) supplying the motor cortical regions and its projection pathways regions can either kill the cortical neurons or block their projections to the spinal cord and subcortical structure. The cerebral cortex is the main striatal afferent, and the medium spiny neurons of the striatum have been identified as the major output neurons projecting to substantia nigra and pallidum. Thus, disconnection of the corticostriatal circuit often occurs in the model of MCAO. In this study, we hypothesize that striatal network dysfunction in cerebral ischemic mice ultimately modulates the activity of striatal projections from cortical neurons to improve dysfunction during exercise training. In this study, we observed that corticostriatal circuit originating from glutamatergic neurons can partially medicate improvement of motor and anxiety-like behavior in mice with exercise. Furthermore, exercise or activation of a single optogenetic corticostriatal circuit can increase the striatal gamma-aminobutyric acid (GABA) level. Using the GABA-A receptor antagonist, bicuculline, we further identified that the glutamatergic projection of the cortex in the striatum relies on activity of GABAergic synapse to modulate exercise-induced functional recovery. Overall, those results reveal that the dorsal striatum-projecting subpopulation of cortical glutamatergic neurons can influence GABA levels in the striatum, playing a critical role in modulating exercise-induced improvement of motor and anxiety-like behavior.

exercise

ischemic stroke

corticostriatal circuit

optogenetics

GABA

Cerebral ischemic stroke is a leading cause of disability and death worldwide. It can induce cell death and brain impairment, and ultimately behavioral functional deficits and long-term disability in the stroke survivors[1]. Occlusion of cerebral vasculature, such as the middle cerebral artery (MCA), which supplies the motor cortical regions and its projection pathway regions, can either kill the cortical neurons or block their projections to the spinal cord and subcortical structure [2–3]. During recovery, rewiring of neural connections is often involved in several plasticity mechanisms, including the release of neurotrophins and axonal sprouting[4–5].

Associated rehabilitative therapies, such as constraint-induced movement therapy, physical exercise training and repetitive transcranial magnetic stimulation, are commonly applied in clinical practice [6–10]. Currently, Chen Z et al. showed that exercise training can increase angiogenesis, synaptic plasticity and neurogenesis to improve motor and cognitive function following stroke partially by the caveolin-1/VEGF pathway[11]. Optogenetic intact corticospinal tract stimulation combined with exercise training can restore motor control by regionalized functional circuit formation in poststroke mice[12]. To improve dysfunction after stroke, exercise training might be an effective strategy for facilitating the rewiring of neural connections.

In the striatum, medium spiny neurons have been identified as the major output neurons projecting to the substantia nigra and pallidum. The major striatal afferents from the cerebral cortex, thalamus, and substantia nigra terminate, at least in part, on these striatal projection neurons[13]. The cortex has been reported to project to the striatal area topographically. Projections from M1 almost entirely innervate the putamen, central and dorsolateral regions. Much of the putamen receives projections from both premotor and motor regions. The dorsolateral striatum also receives overlapping projections from parietal regions related to somatosensory function[14]. Moreover, different regions in the striatum are associated with different functions: the putamen with motor control, the ventral striatum with reward; and the caudate nucleus with cognition[15]. Several studies have suggested that plasticity within the corticostriatal network exerts a crucial role in learning, adapting, psychiatric disorders and coordinating limb movement and modulating motor functions[15–18]. Thus, we hypothesize that striatal network dysfunction in cerebral ischemic mice ultimately modulates the activity of striatal projections from cortical neurons to improve dysfunction during exercise training.

Using optogenetics, we identified a dorsal striatum-projecting subpopulation of cortical glutamatergic neurons that can influence gamma-aminobutyric acid (GABA) levels in the striatum, which play a critical role in exercise-induced functional recovery. This study demonstrates a therapeutic mechanism for exercise in ischemic stroke that involves activation of the monosynaptic cortical input to the dorsal striatum.

Animals

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Fudan University (Shanghai, China). Male C57 BL/6J mice (22-25 g, 8-12 weeks) were purchased from Charles River (Beijing, China) and housed under a normal 12 h light: dark cycle in a cage with food and water available ad libitum. Additionally, the assignments of the experimental group were randomized, and the surgery and all outcome evaluations were conducted by researchers who were blinded to the assignment of the experimental group. Experimental methods and schedules are presented in Figures 1A, 2A, 3A, 4A, and 7A.

Transient MCA occlusion (MCAO)

To establish the transient MCAO model, mice were anesthetized with an intraperitoneal administration of 1 % pentobarbital sodium (1 mg/ml). Briefly, the left common carotid artery (CCA) was exposed and isolated; a monofilament was inserted into the right internal carotid artery (ICA) from the external carotid artery (ECA) to occlude the middle cerebral artery for 60 min[19]. Subsequently, the monofilament was gently pulled out, and the surgical incision was disinfected with iodine and sutured. Body temperature was monitored with a rectal thermometer and maintained at 37 °C using a heating pad throughout the surgery process. The blood flow of mice was monitored using laser Doppler anemometry (RWD Life Science, Shenzhen, China) before and after ischemia. Mice with an rCBF reduction of <70% of pre-MCAO baseline levels were excluded for further experimentation [20]. In addition, the modified neurological severity score (mNSS) was used to evaluate the dysfunction 24 hours after MCAO surgery. The score of mNSS is set from 0 points to 14 points. Mice were randomized to the Spont and EX groups when the mNSS score was more than 6 points[21]. The mice in the sham group also underwent the same operation without insertion of the monofilament and occlusion of the middle cerebral artery.

Exercise training

Male C57BL/6J mice were allowed access to a motor treadmill that started on the 3^rd day after MCAO surgery at a speed of 12 m/min for 30 min per day for 12 days (EX group). Mice without exercise were excluded from the experiment. Age-matched spontaneous recovery mice were used as a control. In the spontaneous recovery (Spont) group, C57BL/6J mice were allowed to stay on a treadmill for 30 min per day but without exercise training.

Optogenetics experiments

Adeno-associated virus (AAV) injection

After anesthetizing, mice were exposed to the skull and then were fixed in a stereotaxic frame. The 600 nl AAV2/1-CamKIIa-ChR2-eYFP (chR2, activation) or AAV2/1-CamKIIa-ArchT-eYFP (ArchT, inhibition) or AAV2/1-CamKIIa-eYFP (eYFP, control) was injected into the left cortex (anterior-posterior (AP), 2.7 mm from bregma; mediolateral (ML), 3.0 mm; dorsoventral (DV), 2.5 mm) at a flow rate of 20 nL/min by a 10 µl syringe (World Precision instruments). Before injection of the virus, the glass needle was on site for 1 min. The needle was placed in site for another 5 min after injection completion to prevent reflux and then retracted slowly.

Optical fiber implantation

An optic fiber cannula (200 um length, Newdoon Technology, Hangzhou, China) was implanted into the left cortex (AP, 2.7 mm; ML, 3.0 mm ML, DV, 2.3 mm) or dorsal striatum (AP, 0.20 mm; ML, 2.0 mm; DV, 3.5 mm) via a small hole generated with a dental drill. After that, optical fibers were mounted with a cannula and immobilized on the skull by bone cement. After wound closure, the mice were returned to their cage to recover for two weeks before MCAO surgery.

Stimulation paradigm

In a repeated optogenetic stimulation with exercise training, 473 nm blue laser light stimulation (20 ms width, firing at 10 Hz) was conducted on mice in the EX-chR2 group[22]. Each stimulation session lasted for 15 minutes per day and consisted of three cycles with 1-min stimulation and a 3-min rest phase. The 594 nm yellow laser light stimulation was performed continuously for 15 minutes per day on mice in the EX-ArchT group[23]. The power of the laser light ranged from 0.2 to 1 mW, as measured by laser power and energy meter. Both optogenetic stimulations were performed for 30 min after exercise. In the EX-eYFP and Spont-eYFP groups, mice did not receive optogenetic stimulation.

In a single optogenetic stimulation without exercise, 473 nm blue laser light stimulation (20 ms width, firing at 10 Hz) was performed on mice in the cortex (CT)-chR2, CT-eYFP, striatum (ST)-chR2 and ST-eYFP groups. A single optogenetic stimulation with open field test was conducted after a 3-min acclimatization period. The session lasted for 15 minutes and consisted of five continuous 3-min periods, with the first, third and fifth as light-OFF periods, and the second and fourth periods being light-ON periods. In the foot fault test, after a 3-min acclimatization period, the whole session consisted of three continuous periods: the first and third periods were light-OFF periods, and the second period was a light-ON period. The foot fault test in each period was conducted for 2 consecutive trials.

In a single optostimulation, the changes in striatal neurotransmitter levels were explored by HPLC analysis. On the 14th day after ischemic stroke, the stimulation (473 nm, 20 ms width, firing at 10 Hz) session consisted of three 1-min stimulations separated by a 4-min rest phase. The mice were then anesthetized immediately for HPLC analyses after a 15-min optostimulation.

Confirmation of the virus

After the final behavioral test, the cerebral regions of virus injection in mice were confirmed by a fluorescence microscope. The data of behavioral tests from mice were excluded when the regions of virus injection showed an absence of eYFP.

Cannula implantation and bicuculline injection

Two weeks before MCAO surgery, a burr hole was drilled in the left dorsal striatum in the EX+Vehicle and EX+BICU groups (n=7/group). The cannula was inserted into the dorsal striatum and fixed with dental cement. For subsequent pharmaceutical injection, Bicuculline (35 mM, 250 nl, Selleck) or vehicle (14% DMSO + 3% PEG 400 + 3% Tween 80 + 80% ddH₂O) was infused into a 5 cm-long PE50 catheter connected to a 10 µl syringe (World Precision Instruments) with a glass needle, and then a PE50 catheter was inserted into the cannula in the EX+BICU and EX+Vehicle groups, respectively. Bicuculline was injected at 30 min after exercise training at a flow rate of 25 nL/min. Bicuculline injection was performed starting on the 3^rd and ended on the 14^th day after stroke.

Behavioral testing

Behavioral tests include open field test, rotarod test, and foot fault test, and were performed 8 hours after exercise training.

Open field test: A 50 cm x 50 cm plastic box was used to explore the free movement and anxiety-likely behavior in mice. Prior to testing, 75% ethyl alcohol was used to clean the open field area. Subsequently, the mice were placed in the center of a plastic box and allowed to freely explore the area. Movement paths were captured using an infrared digital camera during a single 3- or 5-min session. Ethovison XT version 3.0 (Noldus, Wageningen, The Netherlands) software was used to analyze the movement paths of each mouse. The distance in the center was analyzed to evaluate postischemic stroke anxiety-likely behavior, and the time spent in mobility and the average speed were analyzed to evaluate postischemic stroke movement function. Data analysis was conducted by an analyzer blinded to the groups. The time schedules are shown in Figures 1A, 2A, 3A, 4A and 7A.

Rotarod test：The rotarod test was used to assess coordination as previously described [24]. Briefly, mice were placed on a 3-cm diameter and 8.5-cm width rotating drum. To establish a performance baseline, a pretraining test was performed at a linear accelerated speed for two trials per day for 3 consecutive days. The velocity of the rod was set gradually increasing from 5 rpm to 40 rpm over 5 min. The latency to fall off the rod in each trial was recorded on the 3rd, 7^th, and 14^th days after ischemic stroke. Finally, the average latency of each mouse was calculated to assess the coordination function. Each mouse underwent two trials per day within 5 min with 5-min rest intervals to avoid fatigue. The trial was terminated if the mice grabbed the rotating drum for 2 laps. The time schedules are shown in Figures 3A, 4A and 7A.

Foot fault test：The foot fault test was used to evaluate the fine motor function of mice. Briefly, mice were required to pass the horizontal stepping test apparatus for 3 consecutive trials, and the whole process was recorded by a camera positioned underneath the apparatus. The number of partial placements and the total number of steps of the affected forelimb were then recorded followed by the percentage of partial placement was analyzed. The test was performed on the 3^rd, 7^th, and 14^th days after ischemic stroke. The time schedules are shown in Figures 2A, 3A, 4A and 7A.

Preparation of tissue and immunofluorescence

Mice were excessively anesthetized with 1% pentobarbital sodium and then transcardially perfused with 0.9% normal saline and 4% paraformaldehyde by the left ventricle. Subsequently, brains were separated from the skull and were performed postfixed in 4% paraformaldehyde at 4 °C for 24 h. After being dehydrated in 30% sucrose solution, brains were cut into slices at 30-um thickness by using a freezing microtome. Immunofluorescence was conducted to identify the expression of AAV in the cortex and dorsal striatum. The cell nuclei were marked by DAPI Fluoromount-G® (Southern Biotech, Birmingham, AL, USA). A fluorescence microscope was used to observe eYPF-positive cells and fibers.

Transcriptome-wide RNA sequencing (RNA-seq)

The samples from the striatum were obtained in the EX and Spont groups on the 14^th day after stroke (n=3/group). Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol, followed by evaluating RNA purity and quantification. Subsequently, RNA sequencing libraries were generated using a TruSeq RNA Library Preparation Kit v2 (Illumina) according to the manufacturer’s protocol (Shanghai Majorbio Biopharm Technology Co., Ltd., Shanghai, China). Transcriptome sequencing was conducted by Illumina HiSeq X10 (Illumina). The data analysis was performed in the Majorbio Cloud Platform (www.majorbio. com). The Bioconductor package edgeR was used to analyze the differentially expressed genes (DEGs) among groups. A P value < 0.05 and |log2FC| ≥ 1 were considered the thresholds for significant differences. Hierarchical clustering of DEGs was analyzed by an unsupervised learning algorithm. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of DEGs were conducted. For a corrected P value < 0.05, the GO and KEGG functions were considered significantly enriched.

Western blotting

The striatal tissues were obtained in the EX and Spont groups on the 14^th day after stroke (n=4/group). Brain tissues were homogenized using RIPA lysis buffer and centrifuged at 4 °C for 15 min at 12,000 rpm. Equal amounts of samples were loaded on a 12% SDS-PAGE and run using electrophoresis at 100 V for 90 min, and then transferred to a PVDF membrane（Roche，Switzerland）at 80 V for 60 min. Subsequently, the PVDF membranes were blocked and incubated with the primary antibodies: rabbit anti-PSD 95 (1:1000, cell signal technology) and rabbit anti-Synaptophysin (1:500, Proteintech) overnight at 4 °C. Rabbit anti-Tubulin or GAPDH (1:5000; Proteintech) was considered as a loading control. Appropriate horseradish peroxidase-conjugated secondary antibodies (Proteintech) were selected to treated with membrane for 1 h at room temperature. Finally, membranes were probed with an enhanced ECL chromogenic substrates and visualized with an imaging system. The relative density of the membranes was measured by Image J software.

High-performance liquid chromatography (HPLC)

Mice from the Spont, EX, ST-chR2 and ST-eYFP groups were sacrificed with excessive 1% pentobarbital sodium on the 14^th day after ischemic stroke (n=4/group). The sample from the striatum was weighed on a balance pan and then stored at -80 °C until analyzed. Subsequently, the samples were homogenized in 0.4 N perchloric acid solution, and the supernatant was collected after centrifugation at 10000 g/min for 10 minutes. The sample supernatant was injected into HPLC equipment equipped with an Eclipse XDB C18 column at a flow rate of 0.8 mL/min. The levels of glutamate, GABA, acetylcholine (ACH), dopamine (DA) and norepinephrine (NE) were measured using HPLC (Agilent Technologies, Santa Clara, CA, USA) as previously described [25].

Statistical analysis

GraphPad Prism 8 statistical software was used to conduct the statistical analysis. Data are expressed as mean ± SD. The data from optogenetics experiments were assessed using two-way repeated-measures (RM) ANOVA followed by Sidak’s multiple comparisons test or paired Student's t tests. The effects of exercise on behavior were assessed using two-way RM ANOVA followed by Tukey’s multiple comparisons test. Neurotransmitters level and western blotting were analyzed by unpaired Student's t tests. The level of statistical significance was set at *p < 0.05, **p < 0.01, and ***p < 0.001.

1. A single optogenetic cortex-mediated circuit activation modulated improvement of dysfunction

To evaluate whether the cortical excitatory projections modulate motor function after ischemic stroke, mice received viral infection with AAV2/1-CaMKIIa-ChR2-eYFP or AAV2/1-CaMKIIa-eYFP followed by implantation with an optical fiber over the core region of the injection site (CT-chR2 or CT-eYFP mice, Figure 1B). It was observed that arborization of corticostriatal axons expressing eYFP throughout much of the striatum (Figure 1C and D, and S1). All mice received optostimulation (473 nm, 20 ms width, firing at 10 Hz) during behavioral tests on the 14^th day after ischemic stroke.

Two weeks after ischemic stroke, the mice were assessed for alterations in motor activity and anxiety-likely behavior in response to the optogenetic activation of the cortex-mediated circuit. In the open field test, the optogenetic session included five 3-min periods, with the first, third and fifth periods as the light-OFF periods and the second and fourth periods as the light-ON periods (Figure 1E). This motor-evoked response evoked was immediately following light onset. CT-chR2 mice reliably exhibited transient increases in the average speed and the distance in the center compared with the CT-eYFP group during the light-ON periods (average speed: the second period p=0.0182, the fourth period p=0.0002; distance in the center: the second period p=0.0498, the fourth period p<0.0001) (Figure 1F and G). In the fourth period during the light-ON period, the time spent on mobility within the total open field area exhibited an obvious enhancement in the ST-chR2 mice compared with that of ST-eYFP mice (p=0.0152), and there was no significant difference between the CT-chR2 and CT-eYFP groups in the second period (p=0.2492, n=6/group) (Figure 1H). Those results suggested increased motor activity and improved anxiety-likely behavior in ischemic mice with optogenetic activation of the cortex-mediated circuit.

2. A single optogenetic corticostriatal activation modulated improvement of dysfunction

Additionally, in a separate group of mice, we investigated the effect of photostimulation (473 nm, 20 ms width, firing at 10 Hz) on corticostriatal circuit by injecting virus with AAV2/1-CaMKIIa-ChR2-eYFP or AAV2/1-CaMKIIa-eYFP in the cortex and implanting an optical fiber in the dorsal striatum on the 14^th day before MCAO surgery (ST-chR2 or ST-eYFP mice, Figure 2B and C).

In the open field test, the optogenetic session included five 3-min periods. Similarly, compared with ST-eYFP mice, photostimulation of ST-chR2 mice remarkably evoked transient increases in the distance traveled in the center area during the light ON periods (the second period, p=0.0852; the fourth period, p=0.0164). This response evoked by the motor was immediately after light onset and was more evident during the fourth period, although there was no significant difference between ST-chR2 and ST-eYFP in the second period (Figure 2D). We also found that the average speed during the light-ON periods was greater than that of ST-eYFP mice (the second period, p=0.1597; the fourth period, p=0.0126) (Figure 2E). Furthermore, ST-chR2 mice also spent more time in mobility during the light-ON period than ST-eYFP mice (the second period, p=0.0090; the fourth period, p=0.0004) (Figure 2F). Surprisingly, in the fifth period (light-OFF), the time spent in mobility also exhibited a remarkable upregulation in the ST-chR2 mice compared with that of ST-eYFP mice, and it might involve repeated optostimulation (p=0.0020, n=6/group) (Figure 2F).

In the foot fault test, the optogenetic session included three periods, with the first and third periods as the light-OFF periods and the second periods as the light-ON period. Compared with the ST-eYFP mice, ST-chR2 mice also evoked transient improvement in partial placement during the light-ON period (P=0.0174) (Figure 2G). Moreover, during the light-ON period, photostimulation resulted in a decrease in the percentage of partial placement speed relative to the first period in the ST-chR2 mice (p=0.0005), whereas no significant difference was observed in the ST-eYFP mice (p=0.6449, n=6/group) (Figure 2H). Thus, a single optogenetic corticostriatal activation modulated improvements of dysfunction after ischemic stroke.

3. Corticostriatal projection regulated the GABA levels of the striatum

To evaluate the mechanism of corticostriatal projection modulating function recovery, we further assessed the levels of neurotransmitters on the 14^thday after ischemic stroke (Figure 2I and J, and Figure S2). Compared with the ST-eYFP group, optogenetic corticostriatal stimulation alone significantly reduced the glutamate levels (p=0.0002) and enhanced the GABA levels (p=0.0002) in the ST-chR2 group (Figure 2I and J). Nevertheless, there was no significant difference in ACH, NE, or DA between the ST-chR2 and ST-eYFP groups (p>0.05, n=4/group) (Figure S2). Thus, neurotransmitter changes in the striatum might exert an important role in the activation of corticostriatal circuit-modulated functional recovery.

4. Exercise training improved dysfunction.

The survival of the mice in each group was recorded daily within 14 days after stroke (Figure 3B). Mice from the sham group showed 100% survival during the 14-day observation period. No significant difference in survival was found between the Spont and EX groups at the end of the experiment (p=0.996). Within the entire experimental period, no significant differences were observed in body weight between the Spont and EX groups (p=0.6506, n=7/group) (Figure 3C).

To investigate the effect of exercise, we evaluated the recovery of motor and anxiety-likely behavior after ischemic stroke by behavioral tests, including foot fault test, open field test and rotarod test (Figure 3D-F). In the foot fault test, compared with Sham group, the partial placement of the affected forelimb in Spont and EX groups was markedly increased on the 3^th and 7^th days after stroke (p<0.0001). No significant difference was observed between the Spont and EX groups on the 3^th and 7^th day after stroke (p>0.05). After exercise, the dysfunction in the EX group was reversed compared with that of the Spont group on the 14^th day after stroke (p=0.0301, n=7/group), suggesting exercise-induced improvement of motor function (Figure 3D).

Consistently, in the rotarod test, compared with the sham group, obvious dysfunction was found in the Spont and EX groups on the 3^th and 7^th days after stroke (p<0.0001). However, on the 14^th day after stroke, better performance was found in the EX group than in the Spont group (p=0.0366), although no significant effect was observed on the 7^th day after stroke (p=0.6284, n=7/group), suggesting exercise-induced improvement of motor function (Figure 3E).

In addition, in the open field test, distance in the center area of open field was evaluated to assess the effect of exercise on anxiety-likely behavior (Figure 3F). Although there was no significant difference between the Spont and EX groups on the 7^th day after stroke (p=0.9650), a significantly increased distance in the center area was found in the EX group on the 14^th day after ischemic stroke (p=0.0125, n=7/group), suggesting exercise-induced improvement of anxiety-likely behavior.

Those results suggested that exercise training can facilitate improvement of motor function and anxiety-likely behavior, although the no significant changes in coordinated function.

5. Repeated optogenetic corticostriatal stimulation modulates exercise-induced functional recovery.

We first evaluated whether the dorsal striatum-projecting subpopulation of cortical glutamatergic neurons exerts an important role in the recovery of exercise-induced dysfunction. Mice received viral infection with AAV2/1-CaMKIIa-ChR2-eYFP (ChR2), AAV2/1-CaMKIIa-eYFP (eYFP, control) or AAV2/1-CaMKIIa-ArchT-eYFP (ArchT) followed by implantation with an optical fiber in the dorsal striatum on the 14th day before MCAO surgery (Figure 4B). The mortality of each group was recorded daily for 14 days after stroke (Figure 4C). On the 14^th day after stroke, EX-eYFP showed 80.2% survival within the whole experimental period, 73.4% survival in the EX-chR2 group and 68.6% survival in the Spont-eYFP and EX-ArchT groups. The Spont and EX-ArchT groups showed an increased mortality rate compared with the EX-eYFP and EX-chR2 groups, but the difference was not significant. In addition, for the entire experimental period, there were no significant differences in body weight among the EX-eYFP, EX-chR2, Spont-eYFP, and EX-ArchT groups (n=7/group) (Figure 4D).

In the foot fault test, compared with EX-eYFP, motor function also showed poorer recovery in the EX-ArchT group on the 14^th day after ischemic stroke (p=0.0002), which was as long as that of the Spont-eYFP group (p=0.7645). In the EX-chR2 group, repeated corticostriatal activation showed the same recovery rates as that of the EX-eYFP group on the 14^th day after ischemic stroke (p>0.999, n=7/group) (Figure 4E-F). Thus, blockade of corticostriatal projections can eliminate exercise-induced fine motor functional recovery.

In the rotarod test, exercise training induced a significant increase in latency to fall in the EX-eYFP group on the 14^th day compared with the Spont-eYFP group (p=0.0405). In the EX-eYFP group, the same recovery rate was found as that of the EX-chR2 group (p=0.9998). Nevertheless, compared with the EX-eYFP group, repeated corticostriatal inhibition failed to result in significant changes in motor function in the EX-ArchT group (p>0.999, n=7/group) (Figure 4G-H). Thus, blockade of corticostriatal projections is not sufficient to eliminate motor recovery in coordinated function after exercise intervention.

Anxiety-likely behavior in ischemic mice was evaluated using the open field test. On the 14^th day after ischemic stroke, repeated corticostriatal activation induced an increase in the distance in the center in the EX-chR2 group compared with that in the EX-ArchT (p=0.0174) and Spont-eYFP (p<0.0001) groups. No significant difference was found between the EX-chR2 and EX-eYFP groups, although EX-chR2 mice showed increased distance in the center (p=0.3711). Furthermore, anxiety-likely behavior in the EX-eYFP group was not sufficient to significantly improve in comparison to EX-ArchT groups on the 14^th day after ischemic stroke (p=0.4833, n=7/group) (Figure 4I-J). Thus, blockade of corticostriatal projections can inhibit exercise-induced anxiety-likely behavior.

Those results suggested that the glutamatergic projections from cortical neurons to the dorsal striatum are crucial to the exercise-induced improvement of motor function and anxiety-likely behavior, although the no significant changes in coordinated function.

6. Exercise training facilitated synaptic plasticity in the striatum

Transcriptome-wide RNA-seq technology was used to explore the mechanism by which exercise facilitates functional recovery. We found 703 DEGs with 300 upregulated genes and 403 downregulated genes between the EX and Spont groups (Figure 5A). We further characterized the biological functions of DEGs using GO analysis and KEGG enrichment analysis. In the top 20 functions of significant enrichment, the most biological-process-related genes were associated with “synaptic transmission, GABAergic”, “regulation neurotransmitter level”, “regulating synaptic organization”, “gamma-aminobutyric acid signaling pathway”, and “modulation of chemical synaptic transmission” in the GO analysis (Figure 6C). These results indicated that the potential mechanism of exercise-induced functional recovery was highly related to the regulation of synaptic plasticity (n=3). To evaluate the results of RNA-seq, we further assessed the levels of synaptic plasticity-associated proteins in the striatum on the 14^thday after ischemic stroke (Figure 5D-F). Compared to Spont mice, the Synaptophysin (p=0.0027) and PSD95 (p=0.0101) levels were significantly increased in the EX mice. Thus, exercise training can facilitate synaptic plasticity by enhancing expression of Synaptophysin and PSD 95.

7. Exercise training increased GABA levels of the striatum

We further assessed the levels of neurotransmitters on the 14^thday after ischemic stroke (Figures 6 and S3). After exercise, mice showed significantly decreased glutamate levels (p=0.0063) and increased GABA levels (p=0.0071) in the striatum relative to Spont group (Figure 6A and B), which is consistent with optostimulation of corticostriatal circuit. Nevertheless, there was no significant difference in ACH, NE, or DA between the Spont and EX groups(p>0.05, n=4/group) (Figure S3). These results definitively demonstrated that exercise training regulates GABA levels in the striatum which might involve activating the corticostriatal circuit. Thus, neurotransmitter changes in the striatum might exert an important role in exercise-induced functional recovery.

8. GABA receptor antagonist bicuculline eliminated exercise-induced functional recovery.

We next further evaluated the role of GABA in the dorsal striatum in exercise-induced recovery after ischemic stroke. Therefore, we selected bicuculline, a GABA-A receptor antagonist, to suppress the receptor activity in the dorsal striatum (Figure 7B). There was no significant difference in mortality or body weight among the EX, Spont, EX-BICU and EX-Vehicle groups within the whole observation period (Figure 7C and D).

In the foot fault test, the results indicated that, compared with the EX group, partial placement was significantly increased in the Spont group (p=0.0276) on the 14^th day after stroke. Even given the treatment of exercise training, mice in the EX+BICU group showed an elevation in partial placement that approached significance in comparison to the EX+Vehicle (p=0.0045) and EX (p=0.0023) groups on the 14^th day after stroke. Compared with the Spont group, the EX-Vehicle group showed obvious improvement in motor performance at the same recovery rates as the EX group on the 14^th day after ischemic stroke (p=0.9962). No difference was observed between the Spont and EX-BICU groups, suggesting the inhibitory effect of bicuculline on the recovery of fine motor skill in mice with ischemic stroke (p=0.8191) (Figure 7E and F). Thus, blockade of GABA-A receptors in the dorsal striatum can eliminate exercise-induced motor recovery.

In the rotarod test, compared with the Spont group, the latency to fall was obviously improved in the EX (p=0.0419) and EX-Vehicle (p=0.0445) groups on the 14^th day after ischemic stroke. No difference was observed between the EX and EX+Vehicle groups (p>0.999). However, bicuculline appeared to not affect coordinated function in the EX-BICU group, similar to the EX-Vehicle group (p=0.2037) (Figure 7G and H). Thus, blockade of GABA-A receptors in the dorsal striatum is not sufficient to inhibit exercise-induced motor recovery in coordinated function.

In the open field test, the results indicated significant effects of bicuculline in inhibiting exercise-induced functional recovery. The distance in center area was significantly increased in the EX group relative to Spont group, suggesting exercise-induced improvement of anxiety-likely behavior (p=0.0484). There was no significant difference between the Spont and EX-BICU groups (p=0.9997, n=7/group). Compared with the Spont group, distance in center area was also obviously increased in the EX (p=0.0484) and EX+Vehicle groups (p=0.0280). There was no significant difference between the EX and EX+Vehicle groups (p=0.9963). Mice in the EX (p=0.0612) and EX+Vehicle groups (p=0.0360) tended to have more free movement in the distance traveled in the center than the EX+BICU group on the 14^th day after ischemic stroke. (Figure 7I-J). Thus, blockade of GABA-A receptors in the dorsal striatum can eliminate exercise-induced motor recovery.

Those results suggested that the GABA-A receptors in the dorsal striatum are essential to exercise-induced motor functional recovery, although the no significant changes in coordinated function.

In this study, we found that exercise-induced functional recovery at least partially depends on activation of the corticostriatal circuit. Furthermore, corticostriatal projection modulates GABA levels in the dorsal striatum to influence the efficacy of exercise-induced functional recovery.

A previous study reported that corticostriatal fibers were increased in the dorsolateral striatum of ischemic rats with silencing of the Nogo receptor (NgR), accompanied by improved behavioral outcomes[26]. In the present study, we provided further evidence that the activation of the corticostriatal circuit is crucial for functional recovery induced by exercise treatment. Modulation of neural circuits using optogenetic technology has been considered a promising therapy in many neurological disorders, such as Parkinson’s disease[27–28], stroke[12–29], Huntington’s disease[30], and psychiatric disorders[31]. We selectively activated cortex-mediated circuit with single optogenetics at the core region of the injection site to evaluate motor function in mice with ischemic stroke. We found that motor function and anxiety-likely behavior remarkably improved immediately when optogenetics was performed (Fig. 1). We also selectively activated the corticostriatal circuit by single optogenetics, and it exhibited the same improvement in motor function and anxiety-likely behavior as the activation of the cortex-mediated circuit (Fig. 2).

Treadmill training is a simple and traditional strategy that has been performed in both animal models and human trials[32–34]. Treadmill exercise can improve short-term memory and motor function by facilitating synaptic plasticity in mice with photothrombotic stroke[35]. In our work, twelve-day exercise training improved motor function and anxiety-likely behavior in ischemic-stroke mice (Fig. 3). We used lentivirus containing the CaMKII promoter, which mainly transfected excitatory neurons in the cortex, to drive the expression of eYFP (control), ChR2 (activation) or ArchT (inhibition) in the corticostriatal circuit. Notably, motor function and anxiety-likely behavior showed obvious improvement in the EX-ChR2 and EX-eYFP groups but not in the EX-ArchT group. The inhibition of the corticostriatal circuit almost completely blocked the improvement in motor function and anxiety-likely behavior in the EX-ArchT group despite no influence on coordination (Fig. 4). There was no better recovery in motor function after activation of the corticostriatal circuit in mice with exercise, suggesting that exercise training-induced activation of the corticostriatal circuit was sufficient to evoke motor recovery. Interestingly, the anxiety-likely behavior showed an improvement but not approached significant difference, which indicated activation of the corticostriatal circuit combined with exercise might be an effective strategy to improve anxiety-likely behavior after stroke. These results demonstrated that exercise-induced recovery of motor and anxiety-likely behavior depends on corticostriatal projection but is not involved in coordinated function.

In the transcriptome-wide RNA-seq analysis, synaptic plasticity might play an important role in exercise-induced functional recovery (Fig. 5). In the top 20 functions of significant enrichment, the most biological-process-related genes were associated with “synaptic transmission, GABAergic”, “regulation neurotransmitter level”, “regulating synaptic organization”, “gamma-aminobutyric acid signaling pathway”, and “modulation of chemical synaptic transmission” in the GO analysis. In the KEGG enrichment analysis, the most significantly enriched pathways of the top 20 included “GABAergic synapse”, “Glutamate synapse”, “Serotonergic synapse”, “Axon guidance”, “Dopaminergic synapse”, and “Cholinergic synapse”. These results indicated that the potential mechanism of exercise-induced functional recovery was highly related to the regulation of synaptic plasticity (n = 3). To identify the data of RNA-seq, we further found that the synaptic plasticity-associated proteins, Synaptophysin and PSD 95, were obviously increased after exercise training (Fig. 5). The previous study has reported that corticostriatal excitatory synapses onto both striatal interneurons and projecting spiny neurons can undergo synaptic plasticity of which the main forms include long-term potentiation, long-term depression, spike timing-dependent plasticity and short-term forms of intrinsic plasticity[36]. Thus, our results further indicated that exercise training can facilitate synaptic plasticity in striatal interneurons and projecting spiny neurons.

Striatal interneurons contain Gamma-aminobutyric acid (GABA), acetylcholine, and somatostatin plus neuropeptide Y, which appears in the synapse of striatal projection neurons[13]. GABA is an inhibitory neurotransmitter. It has a bidirectional and complex effect in regulating the proliferation, migration and differentiation of neurons and neuronal network wiring during the development of the neural system[37]. We further evaluated the neurotransmitter level to explore the mechanism of corticostriatal circuit taking part in functional recovery. Strikingly, the GABA level was significantly increased and the glutamate level was decreased in the striatum of the mice with a single optogenetic activation of the corticostriatal circuit or exercise (Figs. 2 and 6). Importantly, results from exercise training consistent with the idea that alterations in neurotransmitter level in the striatum were most likely involved in functional improvement upon optogenetic activation of cortical glutamatergic neurons.

To identify the role of GABA, we used the GABA-A receptor antagonist bicuculline to explore the mechanism of exercise-induced motor recovery after stroke. Increasing evidence suggests that activation or blockade of GABA-A receptors can cause behavioral abnormalities, such as anxiety-like behavior[38], mood dysfunction[39], memory[40] and cognition[41], whereas a few studies reported abnormality of motor behavior. In the present study, we observed that injection of the GABA-A receptor antagonist bicuculline into the dorsal striatum blocked exercise-induced improvement of motor function and anxiety-likely behavior, although without a significant effect on coordinated function (Fig. 7). Consistently, He X et al. proved that suppressing striatal neurons by optogenetics can facilitate recovery following ischemic stroke in mice[42]. Our data from pharmacological blockade of the GABA-A receptor were consistent with the result that functional improvement upon exercise training were most likely involved in optogenetic activation of cortex glutamatergic neurons-evoked GABAergic neurotransmission in the striatum.

Motor neurocircuits are spread and highly complex. Cortical neurons can connect with other striatal or even basal ganglia structures that provide parallel origins of functional output from exercise training[15]. The optogenetic or pharmacological blockade of glutamatergic projection from cortical neurons to the dorsal striatum did not completely influence the exercise-elicited improvement of coordinated function. Thus, whether corticostriatal fibers from distinct regions play a complementary role remains to be investigated. Indeed, it is essential to understand how neural circuits selectively engage in motor recovery by determining the contribution of distinct corticostriatal fibers. Additionally, GABA receptors consist of GABA-A and GABA-B receptors, whereas we only investigated the GABA-A receptor to identify the role of GABA on motor function. Thus, future studies can further investigate the effect of striatal neuron subtype inhibition using the optogenetic stimulation of selective GABA neuronal subtypes during exercise.

Overall, our findings showed that cortical neurons projecting to the dorsal striatum encode the mechanisms by which exercise improves motor function and anxiety-likely behavior, and identified the potential of exercise to relieve dysfunction by regulating striatal GABA levels in mice with ischemic stroke. Therefore, the present study suggests that recruitment of the corticostriatal circuit can offer a new perspective on exploring effective rehabilitation strategies after stroke.

Acknowledgements

Funding

This study was funded by National Natural Science Foundation of China (grant number 81871841).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Study design and conception: Ying Xing, Zhanzhuang Tian, Yulong Bai; major experimental implementation: Ying Xing, Anjing Zhang; behavioral tests and data analysis: Congqin Li, Jing Han, Xue Chun Chang; manuscript writing: Ying Xing, Jun Wang, Lu Luo. All authors approved the final manuscript before submission for publication.

Data Availability

All data in the current study are available from the corresponding authors on reasonable request

Ethics Approval

The present study was approved by the Institutional Animal Care and Use Committee of Fudan University (2020 Huashan hospital JS-152)

Consent to participate

Not applicable.

Consent for publication

Not applicable.

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Corticostriatal projections relying on GABA levels encode exercise-induced functional recovery in cerebral ischemic mice

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