Dense dopaminergic innervation of the peri‐infarct cortex despite dopaminergic cell loss after a pure motor‐cortical stroke in rats

After ischemic stroke, the cortex directly adjacent to the ischemic core (i.e., the peri‐infarct cortex, PIC) undergoes plastic changes that facilitate motor recovery. Dopaminergic signaling is thought to support this process. However, ischemic stroke also leads to the remote degeneration of dopaminergic midbrain neurons, possibly interfering with this beneficial effect. In this study, we assessed the reorganization of dopaminergic innervation of the PIC in a rat model of focal cortical stroke. Adult Sprague–Dawley rats either received a photothrombotic stroke (PTS) in the primary motor cortex (M1) or a sham operation. 30 days after PTS or sham procedure, the retrograde tracer Micro Ruby (MR) was injected into the PIC of stroke animals or into homotopic cortical areas of matched sham rats. Thus, dopaminergic midbrain neurons projecting into the PIC were identified based on MR signal and immunoreactivity against tyrosine hydroxylase (TH), a marker for dopaminergic neurons. The density of dopaminergic innervation within the PIC was assessed by quantification of dopaminergic boutons indicated by TH‐immunoreactivity. Regarding postsynaptic processes, expression of dopamine receptors (D1‐ and D2) and a marker of the functional signal cascade (DARPP‐32) were visualized histologically. Despite a 25% ipsilesional loss of dopaminergic midbrain neurons after PTS, the number and spatial distribution of dopaminergic neurons projecting to the PIC was not different compared to sham controls. Moreover, the density of dopaminergic innervation in the PIC was significantly higher than in homotopic cortical areas of the sham group. Within the PIC, D1‐receptors were expressed in neurons, whereas D2‐receptors were confined to astrocytes. The intensity of D1‐ and DARPP‐32 expression appeared to be higher in the PIC compared to the contralesional homotopic cortex. Our data suggest a sprouting of dopaminergic fibers into the PIC and point to a role for dopaminergic signaling in reparative mechanisms post‐stroke, potentially related to recovery.

Sprague-Dawley rats either received a photothrombotic stroke (PTS) in the primary motor cortex (M1) or a sham operation.30 days after PTS or sham procedure, the retrograde tracer Micro Ruby (MR) was injected into the PIC of stroke animals or into homotopic cortical areas of matched sham rats.Thus, dopaminergic midbrain neurons projecting into the PIC were identified based on MR signal and immunoreactivity against tyrosine hydroxylase (TH), a marker for dopaminergic neurons.The density of dopaminergic innervation within the PIC was assessed by quantification of dopaminergic boutons indicated by TH-immunoreactivity. Regarding postsynaptic processes, expression of dopamine receptors (D1-and D2) and a marker of the functional signal cascade (DARPP-32) were visualized histologically.Despite a 25% ipsilesional loss of dopaminergic midbrain neurons after PTS, the number and spatial distribution of dopaminergic neurons projecting to the PIC was not different compared to sham controls.Moreover, the density of dopaminergic innervation in the PIC was significantly higher than in homotopic cortical areas of the sham group.Within the PIC, D1-receptors were expressed in neurons, whereas D2-receptors were confined to astrocytes.The intensity of D1-and DARPP-32 expression appeared to be higher in the

| INTRODUC TI ON
In human stroke survivors, increase in functional connectivity and activation of the peri-infarct cortex (PIC) are considered to be predictors of good motor recovery (Cramer et al., 2006;van Assche et al., 2021).In rat models of ischemic stroke, the PIC is defined as the zone surrounding the ischemic scar with a width of approximately 1 mm (Brown et al., 2007).Here, inhibitory perineuronal networks degrade because of inflammatory processes, and growthpromoting genes are up-regulated (Carmichael, 2006;Carmichael et al., 2005), allowing axonal sprouting and the formation of novel networks (Carmichael, 2003).This process is accompanied by dendritic remodeling and increased spine turnover (Brown et al., 2008(Brown et al., , 2010)).Preventing these changes by performing local protein synthesis inhibition interfered with motor rehabilitation, arguing for a functional role of the PIC for successful motor recovery (Schubring-Giese et al., 2016).
Post-stroke plasticity further depends on growth factors (Ma et al., 2021), like the neurotrophin family, with the glial cell linederived neurotrophic factor (GDNF) as a prominent member (Skaper, 2012).GDNF promotes neuronal survival because of its anti-oxidative and anti-inflammatory effects (Zhang et al., 2021(Zhang et al., , 2022) ) and supports axonal sprouting after CNS injury (Batchelor et al., 2002).The synthesis of GDNF within the PIC is regulated by dopaminergic signaling; here, GDNF expression in reactive astrocytes is triggered in response to dopamine 1 (D1)-receptor activation (Kuric et al., 2013).In turn, levodopa treatment after stroke increased GDNF levels within the lesioned hemisphere.In line with a beneficial effect of dopamine-dependent GDNF expression on recovery, continuous infusion of D1 and D2 dopamine receptor antagonists into the PIC interfered with motor rehabilitation in a rat model of motor-cortical photothrombotic stroke (Vitrac et al., 2022).
Levodopa treatment, on the other hand, supported recovery of sensorimotor function after transient occlusion of the middle cerebral artery (MCAO) in rats (Ruscher et al., 2012).
Despite its apparently beneficial role for PIC reorganization and functional recovery, the dopaminergic system is threatened after stroke: ipsilesional loss of dopaminergic neurons is a well-described phenomenon in mice (Kronenberg et al., 2012), rats (Nagasawa & Kogure, 1990), and humans (Forno, 1983;Winter et al., 2015), even if ischemic lesions are in distant (i.e., supratentorial) locations.In general, such a selective loss of remote neuronal populations may occur with a delay of days to a few weeks post-stroke and is termed exo-focal neurodegeneration (Baron et al., 2014).Although this phenomenon is commonly investigated in the MCAO-model, we have recently reported an unexpected widespread secondary loss of dopaminergic midbrain neurons after a photothrombotic stroke (PTS) applied within the primary motor cortex (M1) of rats (Hosp et al., 2020).
Following these observations, we aimed to characterize the dopaminergic innervation of the PIC in a PTS model of rats.
Whereas loss of dopaminergic midbrain neurons was displayed histologically, the quantity and spatial distribution of dopaminergic neurons projecting from the midbrain toward the PIC were assessed by a retrograde tracing approach.Moreover, the density of dopaminergic presynaptic boutons within the PIC was measured.The resulting findings were further compared to precisely matched sham-treated animals.To capture the postsynaptic side, expression patterns of dopamine receptors within the PIC as well as markers of a functional intracellular signaling cascade were assessed histologically.

| Animals and experiments
Adult 8-12 week-old male Sprague-Dawley rats (n = 15; 280-320 g; Charles River, RRID:RGD_737891) were used for this study.Animals were housed in cages in groups of three with a 12/12-h light/dark cycle, and interventions were performed during the daytime.Behavioral assessments were performed at the beginning of the light phase.Food and water were available ad libitum.Animal experiments were carried out in accordance with the ARRIVE guidelines, the EU Directive 2010/63/EU for animal experiments and were approved by the state of Baden-Württemberg under license number G-18/14.Sample size calculations were performed based on previous studies (Frase et al., 2022;Hosp et al., 2020) and an a priori biometrical evaluation performed by an institutional statistician.Estimating an effect size of 1.8 (Cohen's d) and referring to unpaired two-tailed t-tests at a significance level of 0.05 with a power of 80%, a sample size of 6 had to be chosen to indicate significant differences.Chemicals were purchased from Sigma-Aldrich, unless noted otherwise.
Experiments were conducted in line with the RIGOR criteria (Lapchak et al., 2013): animals were randomly assigned to groups.Three animals had to be killed because of perioperative complications.No further animal had to be excluded, and all data is reported in the manuscript.With respect to behavioral assessments, tissue processing, staining procedures, and histological analyses, researchers were blinded and unaware of group identities.
PIC compared to the contralesional homotopic cortex.Our data suggest a sprouting of dopaminergic fibers into the PIC and point to a role for dopaminergic signaling in reparative mechanisms post-stroke, potentially related to recovery.

K E Y W O R D S
dopamine, neurodegeneration, peri-infarct cortex, rodent model, stroke

| Surgical procedures
The experimental protocol is summarized in Figure 1a.Individual animals could be identified by a code of colored rings that were applied at the base of the tail.Before surgery, animals were randomly assigned to two groups by drawing lots: one group received a PTS (PTS group; n = 6), whereas the other group underwent a sham procedure (sham group; n = 6).During all surgical procedures, blood oxygenation and heart rate were constantly monitored (MouseSTAT Pulse Oximeter for mice and rats, Kent Scientific Corporation), body temperature was controlled using a heating pad (Temperature Controller TC-1000, CWE Inc.).Carprofen (5 mg/kg, s.c.; Norbrook, Cat. No. ANADA 200-520) was given on five consecutive days after each surgery for pain relief.
The head was fixed in a stereotaxic frame (Stoelting Co.).After a median skin incision, preparation, and cleaning of the skull, a PTS was induced through the intact bone.Rose Bengal dye (Cat.No. 632-69-9 10 mg/kg body weight; 7.5 mg/mL in sterile saline) was injected into the tail vein using a 24G venous line (Abbocath, Hospira) over a period of 2 min.In the PTS group, dye injection was accompanied by a 20-min illumination period using a cold light source (KL 1500, Schott AG).To avoid calefaction damage, air cooling was performed using a custommade ventilation system.To standardize lesion size, a 4-mm-diameter stencil was placed above the forelimb area of the M1 (2 mm anterior and 2 mm lateral, relative to the bregma).Animals in the sham stroke group received identical surgical procedures, but no illumination was performed, so the injection of Rose Bengal dye did not lead to cortical vascular occlusion.

| Retrograde tracing
After stroke, changes in terms of gene expression (Carmichael, 2006), axonal sprouting (Carmichael, 2003), and dendritic remodeling (Brown et al., 2010) occur within the first 3-4 weeks.Moreover, exo-focal degeneration processes occur within a timeframe of at least 2 weeks after stroke onset (Hosp et al., 2020).To capture a stable state in which both PIC remodeling and degeneration of dopaminergic neurons are largely completed, we have chosen Day 30 after PTS/sham surgery for the injection of retrograde tracer that was performed as described earlier (Hosp et al., 2015).In brief, a craniotomy over the right hemisphere was performed (coordinates with respect to the bregma: 3 mm posterior, 6 mm anterior, 5 mm lateral, 0.5 mm medial).After craniotomy, the dura was gently removed, and tracer injections were performed under visual control through a stereotactic operating microscope using a digitally controlled microliter injection pump (Nanoject III and matching glass capillaries with an inner diameter of 0.53 mm and an outer diameter of 1.14 mm; Drummond Scientific Company).For the PTS group, 50 nL of the retrograde fluorescent tracer Micro Ruby (MR; 3.000 MW dextran; Cat. No. D7162, Thermo Fisher Scientific, 10% suspension in aqua dest.; injection speed: 1 nL/s) were injected into six adjacent positions within the medial and lateral PIC at a depth of 900 μm.The PIC constitutes the 1-mm zone surrounding the ischemic lesion.After each injection, the capillary was left in place for 2 min before being slowly retracted.Sham-treated animals were 1 : 1 matched to PTS rats, and MR injections were performed at coordinates corresponding to the PTS animal.Thus, each PTS-sham pair received MR-injections at identical coordinates.After tracer injection, the skull was reconstructed using the previously explanted bone flap and dental cement.

| Assessment of post-stroke deficits
Post-stroke deficits were assessed using the modified neurological severity score (mNSS) 1 day before and after PTS or sham stroke induction as well as retrograde tracing surgery (Figure 1c).The mNSS is a widely used and valid tool to evaluate neurological functional deficits in rodents after unilateral brain injury (Xiong et al., 2013).
Neurological function is based on motor, sensory, reflex, and balance tests and graded on a scale of 0-18 (normal score 0; maximum deficit score 18).As the absence of a neurological deficit (mNSS = 0) would question the presence of a relevant ischemic lesion, an mNSS of 0 was an exclusion criterion for our study.However, no animal had to be excluded because of this reason.mNSS assessments were documented by video footage (HERO3+, GoPro) and analyzed offline.

| Euthanasia, tissue preparation, histochemistry, and immunohistochemistry
Rats were deeply sedated (ketamine 80 mg/kg i.p., Medistar, Cat.No. 13690.00.00; xylazine 12 mg/kg i.p., Bayer, Cat.No. 6293841.00.00) and perfused transcardially with 4% paraformaldehyde (PFA, Sigma-Aldrich, Cat.No. 158127).Brains were quickly removed and kept in 4% PFA for 24 h before being transferred to a 30% sucrose solution (Sigma-Aldrich, Cat.No. S9378).Coronal sections were prepared using a sliding microtome with a freezing stage (Leica Microsystems GmbH).For analysis of the stroke, tracer placement, and PIC tissue, brain slices of 40 μm thickness were prepared from +4.0 to −2.5 mm with respect to the bregma and alternately collected in seven sampling wells.Thus, a sample contains every seventh section with a fixed distance of 280 μm between subsequent sections.For the analysis of dopaminergic midbrain nuclei, brain slices of 40 μm thickness were prepared from −5 to −7 mm with respect to the bregma and alternately collected in five sampling wells.Thus, a sample contains every fifth section with a fixed distance of 200 μm between subsequent sections; six to eight midbrain slices were analyzed per animal.Brain slices were stored in antifreeze solution (NaH 2 PO 4 at 13 mmol/L, Na 2 HPO 4 at 36.48 mmol/L, glycerin at 4.12 mol/L, and ethylene glycol at 5.36 mol/L) until further experimental assessment.All coordinates are based on Paxinos and Watson (Paxinos & Watson, 2013).

| Fluoro-Jade C (FJ-C) staining
Brain sections were washed in 0.05 M Tris-buffered saline (TBS, Sigma-Aldrich, Cat.No. T6664) for 5 min, mounted on glass slides coated with 0.3% gelatin, and then dried for 2 h in an incubator at 50°C.Slides were rinsed in 1% sodium hydroxide (Roth, Germany, Cat.No. P031.2) diluted in 80% ethanol for 5 min.They were then dehydrated in a graded series (ethanol 70% for 2 min, 0.45 M sodium chloride solution for 2 min) and subsequently incubated in 0.006% potassium permanganate (Sigma-Aldrich, Cat.No. 223468) for 10 min.The slides were washed with distilled water for 1 min and then placed in a 0.0001% F-J C solution (Chemicon, Cat.No. AG325) for 10 min.Lastly, the slides were washed three times in distilled water for 1 min, dried in an incubator at 40°C for 1 h, and cleared in xylol for 1 min.Then slides were cover-slipped with Depex mounting medium (Electron Microscopy Sciences).

| Histological analysis
For analysis of midbrain sections and stroke volumetry, brain sections were digitized using a fluorescent microscope (AxioImager, Zeiss AG; equipped with a motorized x-y stage; 10×/0.5 EC Plan-Neofluar objective).For analysis of TH-positive boutons within the PIC, confocal images were acquired using a Leica SP8 laser-scanning microscope equipped with a 40× oil immersion (NA 1.30; Leica) objective.Image stacks were acquired in tile scanning mode with the automated stitching function of the LasX software package (RRID:SCR_013673).
For analysis of anti-D1-receptor, anti-D2-receptor, and anti-DARPP-32-immunoreactivity, images were taken using a Leica TCS SP8 laser confocal microscope with 63× magnification (Resolution 1024 × 1024, laser speed 600).Unless noted otherwise, images were analyzed using Fiji software (RRID:SCR_002285; Schindelin et al., 2012).Quantitative assessment of anti-TH-and MR-positive cell counts as well as anti-TH-positive boutons was based on the presence of fluorescence signal and independent of fluorescence intensity.Image brightness and contrast were equally adjusted for all groups.

| Assessment of stroke volume and tracer placement
Lesioned tissue was identified by the FJ-C signal, indicating degenerating cells, and deposits of retrograde tracer were identified by MR fluorescence (Figure 1b).Lesion volume was computed using the frustum-formula V = (h•π/3)•((r1) 2 + r1•r2 + (r2) 2 ) based on the measured lesion area and the distance between subsequent sections (280 μm).Extensions of ischemic lesions were compared to the Paxinos and Watson atlas (Paxinos & Watson, 2013) to assure congruence with motor cortical topography.Even though the largest part of the lesion was confined to M1, tails invading the secondary motor cortex (M2) existed at the rostral pole of the stroke.Every photothrombotic lesion was restricted to the cortex, and all tracer depots were within the 1-mm zone of the PIC.No animal had to be excluded because of misplacement of the photothrombotic stroke/tracer.

| Analysis of dopaminergic midbrain nuclei
dopaminergic midbrain nuclei were identified based on TH-positivity, as TH was confirmed to be the most reliable marker for dopaminergic neurons in the rodents' midbrain (Margolis et al., 2010).The nomenclature of dopaminergic structures was adopted from Dahlström and Fuxe (Dahlström & Fuxe, 1964): nucleus A8 contains the retrorubral field (RRF), and nucleus A9 contains the substantia nigra pars compacta (SNC).Nucleus A10 contains the ventral tegmental area (VTA: including nucleus paranigralis, parabrachial pigmented nucleus, rostral linear nucleus raphe, the central linear nucleus (CLi), and the interfascicular nucleus (IF)).Anti-TH+ and anti-TH + MR+ double-labeled cells were counted within the entire section using Zeiss Zen Lite (RRID:SCR_023747), and their quantities were extrapolated for the whole midbrain structure.The position of anti-TH and MR double-labeled cells in midbrain slices was marked on a reconstructed representation of midbrain anatomy.

| Quantification of TH-positive boutons within PIC
For this analysis, three sections per animal were taken into account: the section in the middle of the ischemic lesion and the sections 1400 μm anterior and posterior to this mark.Single images were recorded medially and laterally at a distance of 750 μm from the scar and at depths of 300 and 800 μm from the brain surface.Regarding sham animals, sample sites were chosen at identical coordinates as the matched counterpart from the stroke group.For each image, the number of TH-positive boutons was related to associated cells that were identified based on DAPI signal.The dopamine-and cAMP-regulated phosphoprotein (DARPP-32) is expressed in cells receiving dopaminergic input but not in dopaminergic neurons themselves (Ouimet et al., 1984) and is an indicator of a functioning D1-receptor-mediated signaling cascade (Svenningsson et al., 2004)

| Statistics
Statistical analyses and graph presentations were performed using Prism version 8 (GraphPad Software, RRID:SCR_002798).For all tests, the normal distribution was checked using the Shapiro-Wilk test.Paired t-tests were used for within-group comparisons (e.g., lesioned vs. non-lesioned hemisphere).For analysis of anti-TH-positive cells, an ipsi/contra-ratio was calculated.For between-group comparisons, unpaired t-tests or the non-parametric Mann-Whitney-test were used.The evolution of mNSS was assessed via repeated measures ANOVA or the Friedman test whenever the data did not show a normal distribution.The sphericity assumption was tested using the Mauchly criterion, and the Greenhouse-Geisser correction was used where appropriate.Post-hoc analyses were performed using Tukey's multiple comparisons test.Numerical results were expressed as mean and standard deviation (SD)/standard error of the mean (SEM).

| RE SULTS
Application of a PTS induced an average lesion size of 9.56 ± 6.9 mm 3 whereas sham intervention did not lead to any brain lesions.Over the course of the experiment, motor performance was observed using the mNSS (Figure 1c).Motor performance differed significantly between

| PTS induces secondary degeneration of midbrain dopaminergic neurons
Following the PTS or sham procedure, animals in the PTS group demonstrated significant side effects in the affected brain regions.In total, 24.8 ± 7.9% fewer TH-positive neurons could be detected in the ipsilesional ventral midbrain compared to the contralesional side (Figure 2a).Across mesencephalic subfields, dopaminergic degeneration was most marked in the VTA and the SNC but not in the RRF.Sham animals did not exhibit significant side differences in TH-positive neurons in the ventral midbrain (Table 1).
For between-group comparisons, ipsilesional TH-positive cell

| Dopaminergic cell degeneration does not reduce the number of mesencephalic dopaminergic neurons projecting to the PIC
To analyze dopaminergic projections from the ventral midbrain to the PIC, cells positive for both TH and MR (TH+/ MR + -double-labeled cells) in the ventral midbrain were quantified (Figure 3a).In total, TH+/MR + -double-labeled cells in the ventral midbrain did not differ following PTS or sham procedure (PTS group: 130.0 ± 16.7 cells; sham group: 140.8 ± 27.5 cells, t = 0.83, DF = 10, p = 0.419, Figure 3b).
Regarding mesencephalic subfields, neither PTS nor sham showed significant differences across ipsi-and contralesional midbrain nuclei (Figure 3b; Table 2).The location of PIC-projecting dopaminergic neurons identified by their anti-TH-and MR-positivity was marked on reconstructed midbrain slices for PTS (n = 6, magenta dots) and corresponding sham-treated animals (n = 6, blue dots) that had received MR-injections into homologous sites in M1 (Figure 4).
The localization of dopaminergic PIC projections did not differ between PTS and sham.

| Density of dopaminergic boutons in the PIC is higher after PTS
In order to evaluate the density of dopaminergic nerve terminals in the PIC, the number of dopaminergic boutons (identified by their TH-positivity) per associated soma (identified based on DAPI signal) was assessed in the PIC and compared to the corresponding cortical localizations in the sham group (Figure 5a).Following PTS, the density of dopaminergic boutons was significantly higher compared to sham (Figure 5b, PTS: 3.6 ± 0.5 boutons/cell, n = 6, sham: 2.9 ± 0.3 boutons/cell, n = 6 per group, Mann-Whitney U = 1, p = 0.004).

| Dopamine receptors show a specific expression pattern and strong activation within the PIC
In an exploratory approach to display the expression patterns of dopamine receptors within the PIC, double-labeling of anti-D1-and D2-receptor with anti-NeuN-and anti-GFAP was performed.D1receptor expression was present in neurons (NeuN+, Figure 6a), but no colocalization could be found in astrocytes (GFAP+; Figure 6b).
Interestingly, D1-receptor expression in neurons within the PIC was more pronounced compared to the homotopic contralesional cortex (Figure 6a).Regarding D2-receptors, no expression could be detected in NeuN+ neurons, whereas sparse signal was present TA B L E 1 Anti-TH-positive cell counts ipsi-and contralesionally in the PTS and sham groups.in ramifications of GFAP+ astrocytes (Figure 6c,d).Interestingly, D2-signal could be detected in axons, possibly indicating presynaptic D2-autoreceptors in dopaminergic fibers (Figure 6c).To illustrate an intact dopamine receptor signaling cascade, we performed doublelabeling of anti-DARPP-32-immunoreactivity with NeuN and GFAP.

| DISCUSS ION
Despite a 25% reduction of ipsilesional dopaminergic neurons, the number of PIC-projecting TH-positive cells in PTS animals was not different to homotopic TH-positive projecting neurons in the sham group 1 month after stroke.Moreover, the density of dopaminergic innervation in the PIC was even significantly higher compared to homotopic cortical areas in controls.Thus, the sprouting of dopaminergic terminals into the PIC is the most plausible explanation for this finding.Such sprouting into perilesional tissue has been investigated in a mouse model of traumatic striatal injury (Batchelor et al., 2002) and was dependent on neurotrophins, such as the brain-and the glia-cell line-derived neurotrophic factors [i.e., BDNF and GDNF; (Skaper, 2012)].GDNF, for example, promotes the survival and differentiation of cultured dopaminergic neurons (Lin et al., 1993) and has even been discussed as a therapeutic agent against Parkinson's disease (Lang et al., 2006).After striatal brain injury, GDNF release from activated macrophages near the wound margin attracted axonal outgrowth and sprouting of dopaminergic nigro-striatal fibers (Batchelor et al., 2002).Within the PIC, D1-and D2-dopaminergic receptors were expressed by activated microglia cells, and D1receptor activation stimulated GDNF synthesis 1 week after stroke

PTS sham
induction (Kuric et al., 2013).Adopting the model of a GDNFmediated chemotaxis to meso-cortical dopaminergic fibers within the margin of an ischemic lesion, the view of a positive feedback circuit emerges: GDNF released from activated microglia guides axonal sprouting of dopaminergic fibers into the PIC, whereas dopamine released from these terminals in turn increases GDNF synthesis.In line with these thoughts and our findings, TH-content and density of TH-positive fibers within the PIC were increased compared to homotopic cortical areas of controls 14 days after transient MCAO in mice (Talhada et al., 2021).
With respect to the postsynaptic side, our study revealed expression of D1-receptors in neurons within the PIC, whereas D2receptors were present in astrocytes and axons.This pattern clearly differs from previous reports showing D1-and D2-receptor expression in astrocytes (Kuric et al., 2013;Ruscher et al., 2012).Also, with respect to the expression of DARPP-32, a marker of functional intracellular signal transduction and also a measure of dopamine receptor activation (Svenningsson et al., 2004), our study revealed a neuronal pattern, whereas an astrocytic expression was reported by Ruscher and colleagues (Ruscher et al., 2012).Apart from the different stroke models (MCAO in mice vs. PTS in rats), different expression patterns can be likely explained by different time points that were assessed: as dopaminergic signaling within the PIC predominantly affected astrocytes 7 days after stroke (Kuric et al., 2013;Ruscher et al., 2012), a neuronal activation pattern prevailed 34 days after stroke induction, at which time plastic changes within the PIC are largely completed (Carmichael, 2006).These observations argue for long-term reorganization and maturation processes in the dopaminergic innervation of the PIC.Moreover, our observation of increased expression of D1-receptors and DARPP-32 within the PIC compared to the contralesional homotopic cortex further highlights the functional relevance of dopaminergic signaling for this particular region.
As a sequel of suspended blood supply, the production of reactive oxygen species leads to the induction of proinflammatory genes, activation of inflammatory cells, and production of cytokines and chemokines, which are associated with secondary tissue damage and progression of ischemic lesions (Batchelor et al., 2002;Macrez et al., 2011).Interestingly, dopaminergic receptors are expressed on various cell types involved in post-stroke inflammation, such as microglia, regulatory-and effector-T-cells and dendritic cells (Talhada et al., 2018).In a rat model of MCAO, levodopa treatment during the first days after stroke reduced the number of cytotoxic T cells in the ischemic hemisphere, decreased the concentration of the Tcell-associated cytokine interleukin 5, and reduced the expression of the intercellular adhesion molecule 1 that mediates CNS-invasion of leucocytes (Kuric & Ruscher, 2014a).Furthermore, expression of the major histocompatibility complex class II protein in microglia within the infarct core becomes reduced (Kuric & Ruscher, 2014b).Thus, dopamine may attenuate the post-ischemic inflammatory cascade, thereby exerting a neuroprotective effect.
After MCAO in rats, increased ipsi-and contralesional expression of the neurite growth inhibitory factor NOGO-A could be observed within various cortical (including peri-infarct) regions during the later (i.e., 2-4 weeks) post-ischemic stage (Cheatwood et al., 2008).
Inter alia, NOGO-A prevents axonal sprouting and synapse formation, thereby limiting repair of various types of neural injury (Schwab & Strittmatter, 2014).Levodopa treatment reduced NOGO A-levels, the expression of its receptor NgR1, and the number of NOGO-Apositive oligodendrocytes within the PIC after PTS and MCAO in mice (Talhada et al., 2021), likely via stimulation of dopamine 2 and 3 receptors that are expressed on mature oligodendrocytes (Rosin et al., 2005).As neutralization of NOGO-A by intrathecally administered antibodies supports motor recovery after stroke in rats (Wahl et al., 2020), dopamine-associated down-regulation of the NOGO-A pathway within the PIC could support functionally relevant reorganization processes.
Apart from neuroprotection and providing a plasticity-promoting environment, dopamine could directly support neuroplasticity within the PIC.For M1, these modulating effects are well investigated and may also be relevant for the peri-infarct zone (Hosp & Luft, 2013).
As motor recovery is thought to rely on similar mechanisms that are required for motor learning (Krishnan, 2006), insights from learning-dependent changes in healthy animals could improve the understanding of neuroplasticity relevant for repair.Basically, the M1 is a prerequisite for successful motor learning (Hosp et al., 2011).
Within M1, dopamine inversely regulates spine formation and elimination via D1-and D2-receptor-dependent processes and is critically involved in the formation of long-term plasticity (Guo et al., 2015).
Dopamine depletion in turn compromised motor learning-induced spine dynamics, selective stabilization of spines that are functionally relevant, consolidation of learning-dependent changes in synaptic weights, and the formation of task-specific correlation structures within layer V motor neuron populations (Guo et al., 2015;Li et al., 2017).Regarding M1 physiology, dopamine increases cortical excitability and strengthens cortical limb representations (Hosp et al., 2009).Thus, dopaminergic signaling within the PIC may facilitate network plasticity, which forms the basis for motor recovery and successful rehabilitation.In line with this hypothesis, blocking D1-and D2-receptors in the ischemic region impaired the recovery of motor skills on Days 7-12 after PTS (Vitrac et al., 2022).In turn, preventing post-stroke degeneration of dopaminergic midbrain

F
I G U R E 1 (a) Schematic depiction of the experimental timeline.Animals received either a PTS (PTS, n = 6) in the right sensorimotor cortex or a sham stroke procedure (n = 6).Motor function was assessed with the modified neurological severity score (N/mNSS) on the days before and after surgical interventions.30 days after PTS induction, the retrograde tracer Micro Ruby (MR) was injected into the medial and lateral peri-infarct cortex (PIC).Injection site coordinates were recorded for each PTS animal, allowing homotopic injection of MR into the cortex of sham stroke animals that were matched with a PTS animal each.Transcardial perfusion was performed 4 days after MR injection, and tissue samples were prepared for histological and immunohistochemical analysis.(b) A series of representative microscopy images (10× objective) depicting a PTS in the primary motor cortex (M1) visualized via Fluoro-Jade C staining and MR deposits in the medial and lateral PIC.Dashed white lines indicate the border of the ischemic stroke.Scale bar: 1000 μm.(c) mNSS in PTS-and sham-treated animals.The PTS group exhibited a motor deficit shortly after stroke induction, which improved significantly over time.MR injection into the PIC did not affect performance in the mNSS.D, day; MR, Micro Ruby; N/mNSS, modified neurological severity score; PTS, photothrombotic stroke.Data are presented as mean ± SEM, *p < 0.05, **p < 0.01.
. To address specific expression patterns in the PIC, double-labeling against the neuronal marker NeuN and the astrocyte marker GFAP were performed.Leica X Files were converted to the IMARIS format (version 10, Oxford Instruments).Maximum Intensity Projection (MIP) mode was employed to assess staining quality and determine the maximum (MAX) and minimum (MIN) values for surface reconstruction.Consistent MAX values of 250-255 were used, while the MIN value varied between 0 and 100 depending on the background.Once the appropriate MIN values were determined, the surface mode was utilized to reconstruct spots or cells.As a result of variations in intensity across sections, the initial threshold for surface reconstruction was set independently for each image.For qualitative assessment of anti-D1-receptor-, anti-D2-receptor-, and anti-DARP-32-immunoreactivity, regions of interest measuring x = 320 pixel, y = 250 pixel, and z = 32 pixel were taken into account.The spots mode was used to detect a pointed signal of at least 0.575 μm in diameter.In order to exclusively detect spots colocalizing with NeuN/GFAP-surfaces, a further filter was applied (distance to surface 1 < 0 μm).

F I G U R E 3
Despite dopaminergic cell loss in ventral midbrain nuclei, the numbers of PIC-projecting dopaminergic cells were not significantly reduced in the PTS group when compared to sham-treated animals.(a) Representative microscopy images (30× objective) of anti-TH and MR-positive cells.Top: Arrowheads indicate a MR-positive but TH-negative cell in the ipsilesional SNC.Bottom: arrowheads indicate a MR-and anti-TH-positive (i.e., double-labeled) cell in the contralesional SNC, both top and bottom images from PTS animals.Scale bar: 30 μm.(b) Quantitative analysis of MR-and anti-TH-positive cell counts in the midbrain revealed no differences in the distribution of PIC-projecting dopaminergic neurons across mesencephalic subfields.Extra: extranuclear area of the ventral midbrain, MR, Micro Ruby; RRF, retrorubral field; SNC, substantia nigra pars compacta; TH, tyrosine hydroxylase; VTA, ventral tegmental area.Data presented as mean ± SEM. /MR+ cell counts in the ipsi-and contralesional midbrain of PTS and sham group animals.

F
Retrograde tracing identifies somata of PIC-projecting dopaminergic neurons within the mesencephalon.The location of PIC-projecting dopaminergic neurons identified by their anti-TH and MR-positivity was marked on reconstructed midbrain slices in PTS (n = 6, magenta dots) and corresponding sham-treated animals (n = 6, blue dots) that had received MR-injections into homologous sites in M1.The ipsilesional hemisphere is indicated by black arrows, and the coronal distance from the bregma is indicated above each image.

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After photothrombotic stroke, dopaminergic innervation of the peri-infarct cortex was increased compared to sham.(a) Representative confocal microscopy images (40× objective) of cortical cell somata with associated dopaminergic boutons identified by anti-TH-positivity.Scale bar 10 μm.(b) Quantitative assessment of anti-TH-positive boutons per cell soma in the PIC in PTS animals compared to homologous cortical area in sham-treated animals.Data presented as mean ± SEM. **p < 0.01.DAPI, 4′,6-diamidino-2-phenylindole; TH, tyrosine hydroxylase.projections from the ventral midbrain to

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I G U R E 6 D1 but not D2-receptor immunoreactivity colocalizes with NeuN+ cells in the PIC.(a) Representative microscopy images (63× objective) depicting colocalization of D1-receptor immunoreactivity (green) and anti-NeuN+ cells (magenta) in the PIC and, to a lesser extent, in the contralesional cortex.Lower panel: 3D IMARIS reconstruction of selected anti-NeuN+ cells and their anti-D1-receptor immunoreactivity.(b) Representative images (63× objective) of anti-GFAP+ cells (magenta) and anti-D1-receptor immunoreactivity (green) in the PIC illustrate that D1-receptor immunoreactivity did not colocalize with anti-GFAP+ astrocytes.(c) Representative images (63× objective) of anti-NeuN+ cells (magenta) and anti-D2-receptor immunoreactivity (green) in the PIC demonstrating no colocalization.The right panel illustrates a representative D2-immunoreactivity signal that resembles an axonal distribution but no D2-immunoreactivity in neuronal nuclei.(d) representative images of anti-GFAP+ astrocytes (magenta) and anti-D2-receptor immunoreactivity (green) in the PIC showing anti-D2-immunoreactivity in an axonal pattern that did not colocalize with the anti-GFAP+ cells with the exception of sparse anti-D2-immunoreactivity in astrocyte ramifications (demonstrated in a selected astrocyte in the lower panel).GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclear protein.