TY - JOUR
AU - Kiss, Jozsef, Zoltan
AB - Abstract Transplantation of appropriate neuronal precursors after injury is a promising strategy to reconstruct cortical circuits, but the efficiency of these approaches remains limited. Here, we applied targeted apoptosis to selectively ablate layer II/III pyramidal neurons in the rat juvenile cerebral cortex and attempted to replace lost neurons with their appropriate embryonic precursors by transplantation. We demonstrate that grafted precursors do not migrate to replace lost neurons but form vascularized clusters establishing reciprocal synaptic contacts with host networks and show functional integration. These heterotopic neuronal clusters significantly enhance the activity of the host circuits without causing epileptic seizures and attenuate the apoptotic injury-induced functional deficits in electrophysiological and behavioral tests. Chemogenetic activation of grafted neurons further improved functional recovery, and the persistence of the graft was necessary for maintaining restored functions in adult animals. Thus, implanting neuronal precursors capable to form synaptically integrated neuronal clusters combined with activation-based approaches represents a useful strategy for helping long-term functional recovery following brain injury. cerebral cortex, embryonic neural precursors, neuronal apoptosis, neuronal circuit, transplantation Introduction Restoring neuronal circuits after damaging insults or degeneration is a major challenge of regenerative medicine. Since the intrinsic neurogenic capacity of the postnatal mammalian brain is very limited, cell-replacement approaches utilizing transplanted progenitor/precursor cells derived from embryos and newborns have developed into a key strategy for neural tissue repair (see review in Grealish et al. 2016; Grade and Gotz 2017; Barker et al. 2018). Over the last 15 years, clinical studies and experiments performed in animal models of diverse pathologies, including neurodegenerative disorders and stroke, have provided proof of concept that transplanted stem/progenitor cells can survive, differentiate into neurons, and successfully integrate into the appropriate functional circuits from both homotopic and heterotopic sites (Lindvall et al. 1989, 1990; Bachoud-Levi et al. 2000, 2006; Olanow et al. 2003; Shin et al. 2012; Feldman et al. 2014; Grealish et al. 2015; Mazzini et al. 2015; Falkner et al. 2016), and see review in Bjorklund and Lindvall (2000). Transplanted neurons display remarkably high specificity when integrating to the pre-existing network (Fricker-Gates et al. 2002; Espuny-Camacho et al. 2013; Michelsen et al. 2015; Wuttke et al. 2018) and could be activated by appropriate stimuli (Tornero et al. 2013; Falkner et al. 2016). In addition, immature grafted neurons possess their own intrinsic activity (Mire et al. 2012; Tolner et al. 2012; Kirkby et al. 2013; Kiss et al. 2014); however, the relevance of this activity for the host networks, and in general the impact of the graft on host networks, remains to be determined. Similarly, the influence of the injured environment on the differentiation and network integration of the graft remains poorly investigated. Recently, we demonstrated that diphtheria toxin (DT)-induced cell death is a valuable, clinically relevant injury model, offering an opportunity to explore cellular responses in the environment of dying neurons (Petrenko et al. 2015, 2018). Our investigations revealed that deficiency in glutamatergic neurons after induced apoptotic cell death in the immature rat somatosensory cortex is not followed by de novo neurogenesis (Petrenko et al. 2015). Here, we tested the hypothesis that transplantation of embryonic precursors of the lost neurons in the vicinity of lesion could result in neuronal replacement and improved functions. We found that grafted embryonic precursors remain in cluster vascularized by the host and do not invade apoptotic lesion sites. Transplanted cells in this configuration survive long term (up to 360 days) and show functional integration into the recipient cortex, improving functional recovery as measured with sensory-evoked activity at the EEG level and with behavioral tests. The specific chemogenetic activation of transplanted neurons further improved their integration and significantly ameliorated the behavioral deficit after cortical lesion. These experiments demonstrate high capacity of embryonic neuronal grafts for synaptic integration into the pre-existing host neuronal circuits and the direct impact of their activation on functional recovery. Material and Methods Animals and Animal Procedures Principles of laboratory animal care have been followed in accordance with the Swiss law, and all procedures have been previously approved by the Geneva Cantonal Veterinary authority. Wistar rats and β-actin-GFP (green fluorescent protein) transgenic rats, ubiquitously expressing enhanced GFP under the control of the cytomegalovirus-enhancer and the chicken β-actin promoter (Ito et al. 2001), are used for the experiments. In total, 240 rats were used. All animals were maintained at a constant temperature (22–24 °C) and humidity (40–60%) in a 12 h light–dark cycle with free access to food and water. During the surgical procedures, body temperature was maintained at 36–37 °C by an electrical heating blanket. Tear-gel was applied to keep the open eye lubricated. Postoperative analgesia was provided by acetaminophen in the concentration 250 mg/kg dissolved in the drinking water. All animals were examined two times daily for signs of infection, severe neurological injury, or discomfort. After transplantation of the cell suspension, no immunosuppression was applied. Rat Model Based on In Utero Electroporation In Utero electroporation was performed in Wistar rats on embryonic day 18 (E18) as previously described (Petrenko et al. 2015). Briefly, timed E18 pregnant Wistar rats were anesthetized with 2% isoflurane in a mixture of 30% O2 and 70% of air. A 4-cm midline laparotomy was performed and the uterus was taken out. About 1 μL of 5 μg/μL DNA solution of plasmid in 10 M Tris-HCl, pH 8.0 mixed with Fast Green colorant, 1:1000 (Sigma-Aldrich), was injected through the uterus wall into the lateral ventricle of the fetus using the mouth-controlled aspirator tube with calibrated microcapillary pipette (Sigma-Aldrich). Electric field was generated by square wave electroporator (NepaGene, CUY21SC), and the fetus was exposed between platinum forceps electrodes (d = 0.5 cm, NepaGene, CUY611P3–1) in the way to label part of the fetal SVZ, which gives origin to the layer II/III neurons in somatosensory cortex (positive electrode was posed on the injected hemisphere, axis between two electrodes made 45–60° angle with the sagittal axis of the head). The following settings of electroporator have been used: voltage, 50 V; exposure time, 50 ms; time between pulses, 950 ms; and number of pulses, 5. Then, the horns of the uterus were repositioned into the abdominal cavity; the abdominal wall and skin were sewed up using a suture Prolene 5-0 (Ethicon). Preparation of Embryonic Cell Suspension for the Transplantation Following the rules of asepsis, the uterine horns containing E18–E19 donor embryos were removed from timed pregnant Wistar rats anesthetized with 2% isoflurane (Baxter) in a mixture of 30% O2 and 70% of air, and the rats were cervically dislocated. Uterine horns were immediately placed in ice-cold phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (Sigma-Aldrich), and embryos were removed. Brains of embryos were isolated and placed into the ice-cold neurobasal medium (Gibco; Invitrogen) supplemented with 1 mM sodium pyruvate (Sigma-Aldrich) and 2-mM N-acetylcysteine (Sigma-Aldrich). Rostral and caudal poles of both hemispheres were removed, and meninges were peeled away. The cortical plate was dissected and placed in the ice-cold neurobasal medium. The obtained tissue was transferred into a 15-mL centrifuge tube containing neurobasal medium and mechanically triturated with a series of 9-inch fire-polished Pasteur pipettes to form a single-cell suspension and then filtered through a 40-μm nylon cell strainer (BD Falcon; BD Biosciences Discovery Labware). A suspension of embryonic cells was centrifuged for 7 min at 1100 rpm and resuspended in 10 mL of neurobasal medium. Similar centrifugation was repeated twice. Embryonic cells were counted, and cell suspension with the density of 3 × 104 cells/μL was prepared for transplantation. Cell preparation was carried out in less than 1 h. Cell viability was tested using Trypan blue 0.4% (Gibco; Invitrogen) cell exclusion. Only cell suspensions with a viability of 75% or greater were used. During the transplantation, cell suspension was kept on ice and gently triturated before each animal injection. To reduce damage to embryonic cells during repeated trituration, final cell suspension was divided into three equal aliquots. Preparation of Neuronal Cell Culture E18–E19 embryonic cells were isolated as described above. After the third centrifugation, cells were counted and plated on 2-cm dishes with the glass coverslips previously coated with 0.24 mg/cm2 of Matrigel (BD Biosciences), with a concentration of 1 × 106 cells per dish. Cells were cultured in neurobasal medium supplemented with 2% B27 (Gibco; Invitrogen), 1% GlutaMax (Gibco; Life Technologies), 1 mM sodium pyruvate (Sigma-Aldrich), 2 mM N-acetylcysteine (Sigma-Aldrich), and 0.5% penicillin/streptomycin (Gibco; Life Technologies). About 1 of 2 mL of supplemented neurobasal medium was changed every second day. Cell cultures were fixed with 4% paraformaldehyde (PFA) at days 2 and 7 in vitro (DIV2, DIV7) followed by immunocytochemistry. Transplantation Technique For transplantation experiments, Wistar or β-actin-GFP rats at postnatal day 20 (P20) were anesthetized with 2% isoflurane (Baxter, Switzerland) in a mixture of 30% O2 and 70% of air. The head of the rat was shaved and stabilized in a stereotaxic frame. The area of the surgery was disinfected with 2% chlorhexidine (B. Braun Medical). A small skin midline incision was performed at the surface of the skull. Three burr holes were drilled on the left hemisphere with the coordinates from bregma: 1, 2.5, and 4 mm caudally and 3 mm laterally. Cell suspension was stereotaxically injected into the cortex through burred holes with a WPI Nanoliter 2000 (World Precision Instruments, Inc.), where the capillary made a 45° angle with the sagittal axis of the brain. Injection was accomplished into the left hemisphere in the lateral direction, 2 mm ventrally. In total, three angled injections of 1 × 104 donor cells were performed. The capillary was left for 1 min before slowly retracting it. Prior to every single-cell injection, the capillary was filled with a new portion of cells. In the part of animals, similar transplantation technique was performed into the right hemisphere. After transplantation of the cell suspension, the skin of the skull was closed with a suture Prolene 6-0 (Ethicon), and the animal was returned to the mother when completely awake after anesthesia. The surgery for every animal was completed within 15 min. Rats exposed to the surgery were compared with nonsurgical animals from the same litter for weight gain and nursing. All animals showed normal weight gain. No death after transplantation was detected. Rats that received neuronal cell injection were sacrificed at days post-transplantation (DPT) 7, 14, 30, 60, 90, 180, and 360. Plasmids Lesion model To introduce a high-affinity receptor to DT (DTR), the plasmids pCLX–UBI–DTR–ires2–tRFP (further indicated as DTR–tRFP) and pCWX–UBI–DTR–PGK–GFP (further indicated as DTR–GFP) were used in the lesion group recipient animals. These plasmids express DTR together with turbo red fluorescent protein (tRFP) (Evrogen) or GFP, allowing labeling and tracking of electroporated cells. Labeling of layer II/III donor progenitor cells The following plasmids were used for labeling of donor cells: pCLX–UBI–GFP to mark with GFP (further—ubi-GFP) for transplantation into Wistar rat hosts or pCLX–UBI–tRFP to label the cells with turbo red fluorescent protein (further—ubi-tRFP) for transplantation into β-actin-GFP hosts. Specific chemogenetic activation of grafted cells To introduce the gene of modified muscarinic receptor hM3Dq into transplanted layer II/III cells, pCWX–UBI–hM3Dq–PGK–GFP plasmid (further indicated as hM3Dq) was used. Detailed information on these plasmids and the procedures that have been used for their preparation can be obtained at http://lentilab.unige.ch/. Layer-Specific Apoptotic Lesion To introduce a high-affinity DTR into the layer II/III neurons, DTR–tRFP plasmid was electroporated to E18 recipient Wistar rats. Death of DTR electroporated cells was induced on postnatal day 16 (P16) by a single subcutaneous injection of DT at the dose of 50 μg/kg. The extent of the lesion was examined postmortem by immunohistochemical analysis for GFAP marker. Labeling of Donor Cells For in vivo experiments, we used E18–E19 primary embryonic cell culture with different types of cell labeling, depending on the purpose of the experiment (Fig. S2A): 1. β-actin-GFP-positive donors (morphology of the transplanted cluster, detection of the separated grafted cells in the host tissue). 2. Specific labeling of layer II/III neuronal population of somatosensory cortex by in utero electroporation of E18 donors with: ubi-GFP plasmid (transplantation into Wistar host, single donor neuron, and dendritic protrusion reconstructions) and ubi-tRFP plasmid (transplantation into β-actin-GFP-positive hosts to visualize ingrowing host elements, transplantation into previously electroporated with ubi-GFP plasmid layer IV host to discern ingrowing host layer IV axonal fibers). In utero electroporation of E18 Wistar rat embryos was performed as described above. One day after electroporation, the donor cell suspension was prepared and transplanted into the host. Specific Stimulation of Transplanted Neurons The gene of modified muscarinic receptor hM3Dq was introduced into transplanted layer II/III cells by electroporation of hM3Dq plasmid into donor brains at E18. Specific activation of transplanted electroporated neurons was achieved by intraperitoneal injections to the host rats of clozapine-N-oxide (CNO) (Sigma-Aldrich), a specific stimulator of modified hM3Dq receptor, daily in the concentration 1 mg/kg, starting from the third day after transplantation to the next 14 days. Animals have been sacrificed at DPT 7, 14, and 30. In Vivo Lentivector Injections P30 Wistar rats that have previously undergone transplantation were anesthetized with 2% isoflurane in a mixture of 30% O2 and 70% of air and placed in a stereotaxic frame. Incision of skin up to 5-mm length was carried out above the level of bregma. An accurate skull hole was drilled to allow access with the needle using the following coordinates: 3.2 mm caudally, 3.2 mm laterally, and 6.0 mm ventrally from the bregma level. About 1 μL of concentrated lentivector suspension was injected using a Hamilton syringe with a 28-gauge needle (a 5 μL Hamilton, Reno, NV). pXL-DTR-ires2-Cherry (pCS2-DTR-iresCherry) lentivector was used in these experiments (Addgene reference 14 883) with titers ranging from 108 to 109 transducing units [TU]/ μL (see (Giry-Laterriere et al. 2011) for details on lentivector production and titration). The needle was left for 1 min before slowly retracting it. Further, the skin of the skull was closed with a suture Prolene 6-0 (Ethicon). The surgery for every animal was completed within 20 min. All procedures using lentivectors have been performed in a level 2 animal facility. Animals have been sacrificed at P60. Behavior Tests Female and male Wistar rats were used for the behavioral experiments. Animals were kept in cages with food and water ad libitum. Experimental animals were caged at least two per cage. All rats were weaned at P21 and separated by gender at P28. During habituation, animals were handled for seven consecutive days before the test. During the last 3 days, animals were placed for 5 min per day in the testing device. All behavioral tests were performed during the first 6 h of the light phase of the 12 h light–dark cycle. Whisker-sensing abilities and sensorimotor coordination were examined by the gap-crossing test. Rats were placed on a 40 × 44 cm platform, elevated from the floor by 24 cm, with a central 7- or 9-cm-wide gap (for P35 or P49 animals, respectively, this is a size, requiring animals to extend their heads and to use their whiskers to estimate the width of the gap) separating the two sides of the chamber: one brightly illuminated and the other one obscured and containing nesting material from the home cage. Animals placed in the bright side were let to explore or to cross freely while measuring the time to cross and the number of whisking trials before each cross. All animals were tested twice and each trial was compared between the groups. If one animal failed a trial (i.e., fell), it was removed from this trial analysis. To assess somatosensory touch sensation and complex sensorimotor functions, the adhesive patch removal task was used. A 6-mm diameter circular adhesive patch was placed on the plantar surface of one hind paw, after which the rats were released in the testing arena and observed for a maximum of 240 s. The latency to detect the patch (first snout contacts with the patch) and the time taken to remove the patch were measured. If an animal did not remove the patch before the maximal time of 240 s, the test was considered as failed, and only the time for the first touch was considered. Rats underwent three consecutive trials (contralesional paw–ipsilesional paw–contralesional paw), and the values of each trial were compared between the groups. Physiological Activation of Barrel Cortex by Enriched Environment Experiments with physiological activation of barrel cortex were performed in special cages with enriched environment in the dark phase of 12 h light–dark cycle for 1.5 h right before sacrificing the animals. Neuronal activity was evaluated by colocalization of neuronal marker NeuN with an acute marker of activity c-Fos. Donor Cell Transduction with DTR Lentivirus, Following Transplantation and Cell Cluster Elimination A single-cell suspension from Wistar rat’s donor embryos was prepared as described before. Further, donor cells were incubated for 6 h with tRFP-DTR lentiviruses with the ratio 1:2 in neurobasal medium (Gibco; Invitrogen). After 6 h, the suspension of the embryonic cells was centrifuged for 7 min at 1100 rpm and resuspended in 10 mL of neurobasal medium. The same centrifugation was repeated twice. Embryonic cells were counted, and cell suspension with the density of 3 × 104 cells/μL was prepared for transplantation. Cell viability was tested using Trypan blue 0.4% (Gibco; Invitrogen) cell exclusion. Only cell suspensions with a viability of 75% or greater were used. During the transplantation, cell suspension was kept on ice and gently triturated before each animal injection. To reduce damage to embryonic cells during repeated trituration, final cell suspension was divided into three equal aliquots. Transplantation of donor cells was performed, as previously described. Electrophysiological Examination EEG recordings were performed as described previously (Megevand et al. 2008; Quairiaux et al. 2010). Differential potentials were recorded under isoflurane anesthesia with a custom-made amplifier (gain 5000x) and digitally converted (A/D converter DT3004, Data Translation) using custom-made scripts in VEE Pro 6 (Agilent). Epicranial sensory-evoked potentials (SEPs) were recorded from the same animal at DPT 14 and 30 using 16 stainless steel electrodes dipped in EEG paste (EC2, Grass Technologies). Electrode coordinates from bregma for the right and left hemispheres were (rostrocaudal/mediolateral; see Fig. 5A) −7.5 mm/4 mm (e1, e15), −4.75/5 (e2, e14), −3.5/2.25 (e3, e13), −1.5/5 (e4, e12), −0.75/2.25 (e5, e11), 1.25/4 (e6, e10), 3.25/2.25 (e7, e9), 0/0 (e8), and −6.25/0 (reference) and ground electrode at 6/0. Unilateral stimuli were delivered simultaneously to all large whiskers on the one side of the snout through a solenoid-based stimulator device controlled by the acquisition software. All analyses were performed using Cartool software by Denis Brunet (http://brainmap- ping.unige.ch/cartool) and Matlab toolboxes (MathWorks). SEP was calculated off-line by averaging responses, 100 ms prestimulus to 500 ms poststimulus. Grand averages were obtained by averaging individual SEPs for each group of animals. The maximum absolute amplitude of the SEPs was registered in S1 that first receives whisker-evoked activity through thalamocortical fibers (electrodes 12 (right stimulation) and 4 (left stimulation); Fig. 5A). For each animal, we compared SEPs in lesioned and intact hemispheres. Paired two-tailed Student’s t-test was used to test response difference between hemispheres. Tissue Processing and Immunohistochemistry Under the deep pentobarbital anesthesia, animals were perfused intracardially with 0.9% saline followed by 4% PFA. Then, the brains were postfixed in 4% PFA overnight. The 50-μm-thick coronal slices cut with vibratome (Leica) were used for subsequent immunostaining using the free-floating section technique. Immunostaining was performed as described previously (Petrenko et al. 2015). Briefly, sections were incubated with a primary antibody diluted in PBS/0.5% bovine serum albumin/0.3% Triton X-100 and then incubated with the appropriate secondary antibodies. The following primary antibodies have been used: rabbit anti-c-Fos (1:5000; Santa Cruz), mouse anti-GAD67 (1:5000; Millipore), rabbit anti-GFAP (1:2500; DakoCytomation), mouse anti-GFAP (1:2500; Millipore), goat anti-GFP (1:2500; Novus Biologicals), rabbit anti-GFP (1:500; Millipore), rabbit anti-Iba1 (1:2500; Wako), mouse anti-NeuN (1:500; Millipore), mouse anti-RECA-1 (1:250; AbD Serotec), mouse anti-Rip (1:10 000; Chemicon), rabbit anti-S100β (1:1000; Abcam), mouse anti-SATB2 (1:250; Abcam), mouse anti-Tuj1 (1:5000; Covance), rabbit anti-TurboRed (1:1000; Evrogen), and rabbit anti-V-Glut1 (1:1000; SYnaptic SYstems). To recognize the signal, the following secondary antibodies were used: donkey anti-rabbit Alexa-488, Alexa-568, and Alexa-647; donkey anti-mouse Alexa-488, Alexa-568, and Alexa-647; and donkey anti-goat Alexa-488, Alexa-568, and Alexa-647 (1:1000, Molecular Probes by Invitrogen), diluted in PBS/0.5% bovine serum albumin. Hoechst (Invitrogen, Molecular probes) staining was performed at the final step of the procedure to visualize the nuclei. Sections were mounted on microscope glass slides. Electron Microscopy and Immunoelectron Microscopy For electron microscopy (EM) studies, brains were perfused with 2% PFA containing 0.2% glutaraldehyde in 0.1 M PBS and then incubated for 2 h at 4 °C in the same fixative. Right after the brain fixation, 150-μm vibratome slices were obtained with Leica vibratome (Switzerland) and postfixed in the fixative solution for 1 h at 4 °C. Tissue was cryoprotected in 20% dimethyl sulfoxide and 2% glycerol in PBS for 20 min and frozen in liquid nitrogen followed by thawing in cryoprotectant solution. After rinsing in PBS, slices were pretreated with 0.3% peroxide in PBS, then rinsed in PBS, and preincubated in PBS with 0.5% BSA. Incubation with primary antibodies anti-GFP goat (1:2500; Novus Biologicals) was performed overnight at 4 °C. Then, slices were washed in PBS-BSA 0.1% and incubated with biotinylated rabbit anti-goat secondary antibodies (1:200, DAKO A/S) for 2.5 h at room temperature. After washing in TRIS-buffer saline (TBS) (pH 7.6), slices were incubated in avidin–biotin–peroxidase complex (VECTASTAIN ABC kit, Vector labs) in TBS for 2 h at room temperature. Then, slices were rinsed in TBS and incubated with a solution of 3.3′-diaminobenzidine (DAB) tetrachloride (Sigma, USA) (0.04% DAB and 0.015% peroxide in TBS) for 18 min. After washing in TBS and PBS, the tissue was postfixed in 1% OsO4 in 0.1 M PBS for 1 h at 20 °C in the dark. After postfixation in OsO4, further rinsing and dehydration through ascending series of ethanol concentrations and absolute acetone (three changes for 5 min), the samples were infiltrated through graded acetone/Epon mixtures (1:1, 1:3, 2 h each) and immersed overnight in Epon, followed by the flat embedding in Epon. Ultrathin sections (40–50 nm thick) were cut from the region of DAB-positive cells with an LKB 8800 ultratome (Sweden), stained with uranyl acetate and lead citrate, and examined with a Tecnai Transmission Electron Microscope (FEI). Single Neuron Dendrite Reconstruction Slices have been examined with a Nikon Eclipse 80I microscope (Nikon Corporation) using Nikon objectives and photographed with a digital camera MBF Bioscience CX9000 under the control of PictureFrame software (version 2.3). Dendrites of the neurons labeled by electroporation were reconstructed using a computer-based Neurolucida system (Microbrighfield) on 50-μm-thick coronal sections with ×100 objective. Only pyramidal cells of layer II/III with noncut dendrites were chosen for reconstructions. A morphometric analysis of total dendritic length and dendritic complexity including the number of primary dendrites and terminal tips (endings) was performed with Neurolucida Explorer software (Microbrighfield). Distribution of the dendritic arbors from the cell body was expressed on the polar histograms, where 0° corresponds to the cortical surface and 180°—to the corpus callosum (external capsule). At least 25 layer II/III neurons from four animals were reconstructed at 7, 14, and 30 days posttransplantation. Imaging and Data Analysis For post hoc analyses, immunostained slices were examined with a Nikon Eclipse TE2000-U microscope using Nikon objectives and photographed with a digital camera (Retiga EX; Qimaging) controlled by the Openlab software (version 3.1.2; Improvision). For the analysis high-power magnification confocal pictures were done with Zeiss LSM 700 confocal microscopes (Carl Zeiss) using Plan-Neofluar ×20, ×40, ×63/1.3 oil objectives. Image processing was performed with the software LSM Image Browser version 4.2.0.121. Quantification of fluorescent cells was done using ImageJ software (version 1.45, ImageJ Inc.) or LSM Image Browser version 4.2.0.121. Except when indicated, images have been taken from at least three adjacent areas within grafted cluster from a minimum three independent experiments. Layer II/III was recognized by the position and typical morphology of the pyramidal cells; layer II/III lesion zone was recognized by the reactive astrogliosis, marked with GFAP immunostaining. Levels of glutamic acid decarboxylase 67 (GAD67)-positive elements for inhibitory innervation inside the cluster and in the somatosensory cortex were quantified as the surface density (SD) of fluorescence using MetaMorph outline software (Molecular device). The amount of layer IV GFP-positive fibers inside the cluster was quantified as the total length of fluorescent elements using MetaMorph outline software. For quantification of dendritic protrusions, confocal images were obtained on Zeiss LSM 700 confocal microscope with Plan-Neofluar ×63/1.3 oil objective, ×3 zoom, Z-stack for at least 10 electroporated layer II/III neurons inside the cluster from transplanted animals or 10 electroporated random neurons of layer II/III in the case of the control nontransplanted animals, for each time point. The following sampling criteria were utilized: complete or near complete dendritic arbors, unobscured by surrounding cells, presence of the second- and third-order dendritic branches. The spine density on a pyramidal neuron was calculated by dividing the total number of spines on a neuron by the total length of its dendrites and was expressed as the number of spines/mm. Except when indicated, the results are expressed as mean ± standard error of the mean (s.e.m.) or presented as a percentage, as marked in the figure legend, where n = number of analyzed brains. Statistical significance was measured by ordinary one-way or two-way ANOVA test with Bonferroni’s multiple comparison test and defined at *P < 0.05; **P < 0.01; or ***P < 0.001. Results In order to produce local and selective deficiency of layer II/III pyramidal neurons in host juvenile animals, we applied a noninvasive cell-specific ablation using the DTR/DT system as described (Petrenko et al. 2015) with some modifications. At embryonic day 18 (E18), rat embryos were electroporated with plasmid carrying DTR and turbo red fluorescent reporter protein (tRFP) (Fig. 1A), leading to DTR expression in 35.18 ± 3.7% (n = 5) of layer II/III pyramidal neurons in the somatosensory barrel cortex (Fig. 1B1). To induce ablation of electroporated neurons, animals were injected subcutaneously with DT at postnatal day 16 (P16) (Fig. 1A). As previously reported (Petrenko et al. 2015), DT-triggered apoptosis is a slowly progressing process, lasting approximately for 5–7 days (Fig. 1B2). Microglia activation and proliferation of undifferentiated progenitor cells in adjacent cortical layers represented the earliest cellular response from day 3 after apoptotic death initiation, followed by reactive astrogliosis (Petrenko et al. 2015), (Fig. 1B3). To replace lost cells, we microdissected somatosensory cortex cells expressing either GFP or tRFP from E18 rats, a time point when layer II/III pyramidal precursors have already been generated. Cells were dissociated and grafted in the close vicinity of the injured area 4 days after triggering apoptosis (Fig. 1C), the timing that corresponds to the period of the prominent neuronal apoptosis (Petrenko et al. 2015). In vitro characterization of the cellular composition of the graft revealed that more than 70% of donor cells were postmitotic pyramidal neuronal precursors, expressing V-Glut1 (Fig. S1). Figure 1 Open in new tabDownload slide Embryonic cortical cells transplanted into the adolescent cerebral cortex remain in long-term surviving clusters. (A) Timeline of the experimental procedures. (B) Confocal images showing phases of DT-mediated ablation of DTR electroporated layer II/III cortical neurons. (1) DTR–tRFP electroporated layer II/III neurons and their projections in LV of somatosensory cortex (high-power confocal images of layers II/III and V of electroporated cortex are shown on adjacent panels (1a) and (1b), respectively, scale bar = 50 μm). (2) Dead layer II/III DTR-tRFP electroporated neurons 7 days after DT-triggered neuronal death initiation (DPI, days postinjection). (3) Reactive astrogliosis after layer II/III neuronal death, which is displayed by immunostaining for GFAP. Scale bar = 200 μm. (C) Example of the cluster. Scale bar = 200 μm. (D) (1) Schematic illustration and epifluorescent photo of transplanted cells cluster in the vicinity of apoptotically lesioned layer II/III host cortex. Scale bar = 100 μm. Graph, representing populations of migrated neuronal progenitors toward the lesion and toward the external capsule (opposite to the lesion) at DPT 7. Difference between two groups in the graph examined by paired two-tailed Student’s t-test; n = 5. Data expressed as a mean ± s.e.m., ns: difference is not significant. (2–3) Representative confocal images showing separated from the GFP-positive donor cluster glial cells. (2) High-power confocal image showing colocalization of GFP and Rip, a marker of oligodendrocytes (arrows), in the same cells. (3) High-power confocal image sowing colocalization of GFP and GFAP, a marker of astrocytes (arrows), in the same cells. 2–3 scale bars = 20 μm. (E) Confocal images of GFP-positive ingrowing host elements into transplanted ubi-tRFP electroporated donor cell cluster (experimental approach is shown on the adjacent schema). (1) Confocal image of ingrowing GFP-positive host elements (arrows) into the donor cell cluster, where single-layer II/III neurons are labeled with tRFP plasmid by E18 in utero electroporation. Scale bar = 50 μm. (2) High-power confocal image of GFP-positive host vessels in the cluster colocalizing with RECA-1 endothelial marker (arrows). Scale bar = 25 μm. (3) High-power confocal image of GFP-positive host microglial cell in the cluster colocalizing with Iba1 marker (arrow). Scale bar = 10 μm. Figure 1 Open in new tabDownload slide Embryonic cortical cells transplanted into the adolescent cerebral cortex remain in long-term surviving clusters. (A) Timeline of the experimental procedures. (B) Confocal images showing phases of DT-mediated ablation of DTR electroporated layer II/III cortical neurons. (1) DTR–tRFP electroporated layer II/III neurons and their projections in LV of somatosensory cortex (high-power confocal images of layers II/III and V of electroporated cortex are shown on adjacent panels (1a) and (1b), respectively, scale bar = 50 μm). (2) Dead layer II/III DTR-tRFP electroporated neurons 7 days after DT-triggered neuronal death initiation (DPI, days postinjection). (3) Reactive astrogliosis after layer II/III neuronal death, which is displayed by immunostaining for GFAP. Scale bar = 200 μm. (C) Example of the cluster. Scale bar = 200 μm. (D) (1) Schematic illustration and epifluorescent photo of transplanted cells cluster in the vicinity of apoptotically lesioned layer II/III host cortex. Scale bar = 100 μm. Graph, representing populations of migrated neuronal progenitors toward the lesion and toward the external capsule (opposite to the lesion) at DPT 7. Difference between two groups in the graph examined by paired two-tailed Student’s t-test; n = 5. Data expressed as a mean ± s.e.m., ns: difference is not significant. (2–3) Representative confocal images showing separated from the GFP-positive donor cluster glial cells. (2) High-power confocal image showing colocalization of GFP and Rip, a marker of oligodendrocytes (arrows), in the same cells. (3) High-power confocal image sowing colocalization of GFP and GFAP, a marker of astrocytes (arrows), in the same cells. 2–3 scale bars = 20 μm. (E) Confocal images of GFP-positive ingrowing host elements into transplanted ubi-tRFP electroporated donor cell cluster (experimental approach is shown on the adjacent schema). (1) Confocal image of ingrowing GFP-positive host elements (arrows) into the donor cell cluster, where single-layer II/III neurons are labeled with tRFP plasmid by E18 in utero electroporation. Scale bar = 50 μm. (2) High-power confocal image of GFP-positive host vessels in the cluster colocalizing with RECA-1 endothelial marker (arrows). Scale bar = 25 μm. (3) High-power confocal image of GFP-positive host microglial cell in the cluster colocalizing with Iba1 marker (arrow). Scale bar = 10 μm. Grafted Embryonic Precursors Remain in Vascularized Cluster and Do Not Invade Apoptotic Lesion Sites Transplantation of cell suspension (approximately 3 × 104 cells per brain) from the β-actin-GFP rat allowed us to visualize all grafted elements in the host cortex, representing both neuronal and glial cells. To evaluate the integration of grafted cells into the intact or apoptotically injured juvenile cortex, we histologically analyzed transplanted brains at different time points after transplantation. Grafts, surviving up to 360 days in the lesion or intact environment, were typically located oblique to the pial surface in the somatosensory cortex throughout layers I–V (Figs 1C and S2A). The elliptic dense conglomerate of transplanted cells did not display any obvious cytoarchitecture, and the transplanted cell population remained in cluster even after longer survival time (Fig. S2B). We did not detect any significant dispersion of cells in the tangential plane, and only few, sparsely distributed cells were separated from the main cluster in the radial plane (Fig. 1D1). These single migrating cells displayed glial phenotypic markers (GFAP- and S100β-positive astrocytes and Rip-positive oligodendrocytes; Figs 1D2,3 and S3). The presence or absence of apoptotic lesion did not influence the migratory behavior of cells, and we found no evidence for the attraction of grafted cells toward the lesion site (Fig. 1D1). In order to visualize host-derived structural elements ingrowing into the cluster, we transplanted E18 layer II/III neurons that express ubi-tRFP red fluorescent protein after E18 electroporation, into the β-actin-GFP host animal brain (Fig. 1E). Starting from 7 days after transplantation, we detected host blood vessels coexpressing RECA-1 and GFP growing into the graft (Fig. 1E2). In addition, we observed migration of microglial Iba1-positive cells from the host tissue into the transplanted cluster (Fig. 1E3). Inside the graft we also observed a considerable number of GFAP-positive, GFP-negative reactive astrocytes, most likely originating from the donor tissue (Fig. S4). Host astrocytes were located mainly on the border of the cluster, with merely a few processes found inside the cluster. Together, these observations demonstrate that transplanted embryonic pyramidal precursors do not invade the injured cerebral cortex but survive in compact clusters receiving cellular elements and vascularization from the host. The Apoptotic Environment Influences the Structural Maturation of Transplanted Neuronal Precursor Cells Next, we explored the differentiation and maturation of embryonic neuronal precursors in grafted cell clusters. Already at 7 DPT, we found a larger number of NeuN-positive neurons in grafts implanted in lesioned hemispheres compared with grafts implanted in intact hemispheres (Fig. 2A). We also detected that a significant number of neurons expressed markers of supragranular pyramidal cells such as CUX1 and SATB2 (Fig. S5). We analyzed the density of the neuronal marker NeuN in relation to the nuclear marker Hoechst inside the donor cluster at DPT 7, 14, and 30 in the intact and lesioned cortex. In both cases, we observed decreasing density of the cell nuclei (Hoechst positive) in the cluster with time, most likely due to the development of neuropil and increase of the extracellular matrix volume (Fig. 2A). At DPT 14, we observed a significantly faster decrease of nuclear density in the clusters placed in an apoptotic environment, raising the possibility of a faster maturation of the cluster in the apoptotic lesion environment. At the same time, the percentage of NeuN-positive cells was significantly higher within grafts in the lesioned hemisphere than in grafts in an intact cortex at all analyzed time points (on average 53.5% NeuN-positive cells in the intact vs. 68.5% in the lesioned hemisphere). Remarkably, when the transplantation was postponed by 2 weeks, significantly lowered percentage of NeuN-positive cells were observed in the cluster (Fig. S6A,B), similar to nonlesioned cortex. The NeuN/Hoechst ratio remained stable at different time points after transplantation in intact as well as apoptotic hemispheres (Fig. 2A). These findings raise the possibility that the apoptotic environment may promote neuronal survival as well as accelerate the maturation of grafted embryonic precursors. Figure 2 Open in new tabDownload slide Apoptotic lesion environment influences the survival and maturation of transplanted neuronal precursors. (A) Representative confocal images showing NeuN/Hoechst costaining inside the transplanted cluster in the intact or injured hemisphere at the indicated time points. Scale bar = 20 μm. Graphs representing common cell density (Hoechst) and neuronal cell density (NeuN/Hoechst) changes inside the donor cluster in intact or lesioned host cortex at marked time points; n > 4. (B) Neurolucida single-cell reconstructions representing typical morphology of the layer II/III neurons in the host and after transplantation into the intact and lesioned cortex at mentioned time points. Scale bar = 50 μm. Graphs displaying main characteristics of dendrites (number of primary dendrites, average number of terminal branches, and total length of dendrites) at indicated time points. Polar histogram representing average distribution of the dendritic length based on the Neurolucida reconstruction, where the polar axis represents the angle from cortical surface and y-axis represents the length of dendrites in micrometers. n > 25 cells from at least four animals per group. (C) High-magnification confocal images showing representative dendritic branches of nontransplanted layer II/III neurons or transplanted into intact or lesioned cortex at indicated time points and corresponding quantification of protrusion density (middle panel). The difference in width of mushroom spines between neurons transplanted in intact and lesioned cortex at DPT 30 is shown on the right panel (n = 3 animals per group). At least four animals per group were used for analysis per time point; three dendrites per cell in at least 10 cells were quantified per animal. The difference between groups is examined by ordinary two-way ANOVA with Bonferroni’s multiple comparison test. Data expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P <0.0001. Figure 2 Open in new tabDownload slide Apoptotic lesion environment influences the survival and maturation of transplanted neuronal precursors. (A) Representative confocal images showing NeuN/Hoechst costaining inside the transplanted cluster in the intact or injured hemisphere at the indicated time points. Scale bar = 20 μm. Graphs representing common cell density (Hoechst) and neuronal cell density (NeuN/Hoechst) changes inside the donor cluster in intact or lesioned host cortex at marked time points; n > 4. (B) Neurolucida single-cell reconstructions representing typical morphology of the layer II/III neurons in the host and after transplantation into the intact and lesioned cortex at mentioned time points. Scale bar = 50 μm. Graphs displaying main characteristics of dendrites (number of primary dendrites, average number of terminal branches, and total length of dendrites) at indicated time points. Polar histogram representing average distribution of the dendritic length based on the Neurolucida reconstruction, where the polar axis represents the angle from cortical surface and y-axis represents the length of dendrites in micrometers. n > 25 cells from at least four animals per group. (C) High-magnification confocal images showing representative dendritic branches of nontransplanted layer II/III neurons or transplanted into intact or lesioned cortex at indicated time points and corresponding quantification of protrusion density (middle panel). The difference in width of mushroom spines between neurons transplanted in intact and lesioned cortex at DPT 30 is shown on the right panel (n = 3 animals per group). At least four animals per group were used for analysis per time point; three dendrites per cell in at least 10 cells were quantified per animal. The difference between groups is examined by ordinary two-way ANOVA with Bonferroni’s multiple comparison test. Data expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P <0.0001. Figure 3 Open in new tabDownload slide Graft-host neuronal connections. (A) Schematic drawing of the pattern of axonal projections at DPT 30 in the lesioned and intact hemispheres, where transplanted cluster is indicated with red arrows and representative microscopic photos of GFP-positive axonal projections in the host cortex. (1) Epifluorescent microphotograph of GFP-labeled donor cluster with projecting fibers. Scale bar = 100 μm, insertion bar = 10 μm. (2) High-magnification confocal images of GFP-positive donor axonal fibers detected within ipsilateral cortex: MCx, motocortex; SSCx, somatosensory cortex; and InsCx, insular cortex. Scale bar = 10 μm. (B) Timeline of the experimental procedures and confocal images showing the presence of the host cortical axons inside the transplanted cluster. (1) Low-magnification confocal image of E18 ubi-tRFP electroporated donor cell cluster in layer IV ubi-GFP electroporated host cortex. Scale bar = 250 μm. (2) Confocal image of ubi-GFP labeled host layer IV axons inside of ubi-tRFP labeled donor cluster. Scale bar = 20 μm. (3) Confocal images showing progressive increase of the host ubi-GFP-labeled axons inside the donor cell cluster from DPT 7 to 30. Scale bar = 10 μm. (3a) Graph representing total length in μm of GFP-positive axons inside the grafted cluster. (4) High-power confocal image of the contacts between donor tRFP-labeled dendritic spines and host GFP-positive layer IV axons inside the transplanted cluster. Scale bar = 2 μm. Data are shown for at least five animals per group; ordinary one-way ANOVA with Bonferroni’s multiple comparison test was used to examine the differences between time points. (C) Schematic drawing and representing confocal images of thalamocortical mCherry-labeled fibers inside the transplanted GFP-positive cluster. Scale bar = 100 μm, insertion bar = 20 μm. (D) Confocal images showing distribution of inhibitory neuronal fibers in the cluster immunostained for glutamic acid decarboxylase 67 (GAD67) at the indicated time points in intact and lesioned hemispheres. Graph represents ratio between SD covered by GAD67-positive elements inside and outside of the cluster at corresponding time points. Scale bar = 50 μm. Difference between groups examined for at least six animals per group, using ordinary two-way ANOVA with Bonferroni’s multiple comparison test. (E) Electron micrographs illustrating synaptic contacts between host and DAB-labeled transplanted cells. (1) Contacts between host presynaptic axonal terminals with synaptic vesicles (blue) with postsynaptic dendritic spines of transplanted cells (in yellow). (2) Contacts between presynaptic axonal boutons of transplanted neurons (in yellow) and host cortical neuron spines (in blue). Scale bars = 0.5 μm. Data in all graphs expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; and ***P < 0.001. Figure 3 Open in new tabDownload slide Graft-host neuronal connections. (A) Schematic drawing of the pattern of axonal projections at DPT 30 in the lesioned and intact hemispheres, where transplanted cluster is indicated with red arrows and representative microscopic photos of GFP-positive axonal projections in the host cortex. (1) Epifluorescent microphotograph of GFP-labeled donor cluster with projecting fibers. Scale bar = 100 μm, insertion bar = 10 μm. (2) High-magnification confocal images of GFP-positive donor axonal fibers detected within ipsilateral cortex: MCx, motocortex; SSCx, somatosensory cortex; and InsCx, insular cortex. Scale bar = 10 μm. (B) Timeline of the experimental procedures and confocal images showing the presence of the host cortical axons inside the transplanted cluster. (1) Low-magnification confocal image of E18 ubi-tRFP electroporated donor cell cluster in layer IV ubi-GFP electroporated host cortex. Scale bar = 250 μm. (2) Confocal image of ubi-GFP labeled host layer IV axons inside of ubi-tRFP labeled donor cluster. Scale bar = 20 μm. (3) Confocal images showing progressive increase of the host ubi-GFP-labeled axons inside the donor cell cluster from DPT 7 to 30. Scale bar = 10 μm. (3a) Graph representing total length in μm of GFP-positive axons inside the grafted cluster. (4) High-power confocal image of the contacts between donor tRFP-labeled dendritic spines and host GFP-positive layer IV axons inside the transplanted cluster. Scale bar = 2 μm. Data are shown for at least five animals per group; ordinary one-way ANOVA with Bonferroni’s multiple comparison test was used to examine the differences between time points. (C) Schematic drawing and representing confocal images of thalamocortical mCherry-labeled fibers inside the transplanted GFP-positive cluster. Scale bar = 100 μm, insertion bar = 20 μm. (D) Confocal images showing distribution of inhibitory neuronal fibers in the cluster immunostained for glutamic acid decarboxylase 67 (GAD67) at the indicated time points in intact and lesioned hemispheres. Graph represents ratio between SD covered by GAD67-positive elements inside and outside of the cluster at corresponding time points. Scale bar = 50 μm. Difference between groups examined for at least six animals per group, using ordinary two-way ANOVA with Bonferroni’s multiple comparison test. (E) Electron micrographs illustrating synaptic contacts between host and DAB-labeled transplanted cells. (1) Contacts between host presynaptic axonal terminals with synaptic vesicles (blue) with postsynaptic dendritic spines of transplanted cells (in yellow). (2) Contacts between presynaptic axonal boutons of transplanted neurons (in yellow) and host cortical neuron spines (in blue). Scale bars = 0.5 μm. Data in all graphs expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; and ***P < 0.001. To further test this hypothesis, we carried out single-cell reconstruction of transplanted neurons. We electroporated E18 rat embryos with ubi-GFP plasmid to specifically label layer II/III neurons and the next day transplanted freshly isolated donor cell suspension. To determine the influence of transplantation by itself and transplantation into the apoptotic environment on the development of the dendrites of donor neurons, we performed single-cell reconstruction in grafts within the intact or the apoptotic hemispheres at DPT 7, 14, and 30. We compared these data with those from control (nontransplanted) layer II/III pyramidal cells electroporated with ubi-GFP plasmid at E18. Grafted neurons in both intact and apoptotic hemispheres displayed morphologies with complex dendritic arborization without clear polarization typical for layer II/III pyramidal cells. We detected a progressively increasing number of dendritic terminal branches and common lengths after transplantation into the intact hemisphere that was further enhanced if transplanted neurons have been placed in the apoptotic lesion environment (Fig. 2B). We observed a similar phenomenon for primary dendrites at DPT 7. Thus, grafted neurons appear to develop complex dendrites in the host, and the apoptotic environment significantly enhances this process. At the same time, we did not find the typical polarization with apical dendrite oriented to the pial surface, as in control layer II/III neurons (Fig. 2B). We observed larger surface coverage by the dendrites of transplanted cells in comparison with control layer II/III neurons that was confirmed by the polar histogram depicting average dendritic lengths within equal angle domains around the perikaryon (Fig. 2B, green and red lines). In addition, this analysis revealed avoidance of dendritic spread in the direction of adjacent apoptotic region in the lesion group. Next, we performed comparative analysis for the density of dendritic protrusions at the abovementioned time points in the three groups of layer II/III neurons. Grafted cells, similar to the control host neurons, exhibited progressively increasing density of the protrusions (Fig. 2C). The density of dendritic protrusions in donor layer II/III cells was higher in the intact cortex at DPT 14 and 30 as compared with control nontransplanted neurons. Transplantation into the injured cortex promoted an even higher number of protrusions at DPT 14 and results in greater width of mushroom spines. Interestingly, when transplantation was performed at 2 weeks after neuronal ablation, this stimulatory effect of the lesion on spine formation disappeared (Fig. S6C). These data demonstrate that transplanted donor layer II/III neurons develop a significant number of dendritic protrusions, and this process is stimulated by the acute apoptotic lesion environment. Taken together, these observations raise the possibility that transplantation by itself, as well as the apoptotic environment, promotes a faster dendritic development of transplanted neurons. Grafted Embryonic Neuronal Precursors Project over Long Distances and Receive Appropriate Functional Innervation from the Host Cortex To further examine axonal outgrowth from grafted cells in the lesioned hemisphere, we used donor cells strongly labeled by E18 electroporation with ubi-GFP plasmid (Figs 3A and S2A). After mapping the distribution of labeled axons at DPT 30, we found a significant number of GFP-positive axons in the ipsilateral, but not in the contralateral, hemisphere (Fig. 3A). We routinely observed widely distributed fibers locally in the somatosensory cortex, but donor efferent projections also extended longer distances, reaching the prefrontal, motor, and insular cortices. These regions appear to be the appropriate in vivo targets of layer II/III pyramidal cells of the somatosensory cortex, according to Allen Mouse Brain Connectivity Atlas. In order to explore the penetration of host-derived axons into the grafts, E16 wild-type recipients were electroporated with ubi-GFP plasmid, to label layer IV neurons. At P20, we transplanted ubi-tRFP electroporated E19 donor cells. Using this bicolor labeling technique, we detected green-labeled host axons inside the cluster of red-labeled layer II/III pyramidal neurons (Fig. 3B1,2). By measuring the total length of the GFP-positive fibers inside the transplanted cluster at DPT 7, 14, and 30, we detected an increase of layer IV axons in the graft by time (Fig. 3B3). Higher magnification at DPT 14 and 30 revealed numerous adjacent contacts between red-labeled donor layer II/III dendritic spines and green-labeled host layer IV axonal buttons, reliably proving the presence of appropriate connections between host and donor neurons (Fig. 3B4). We also investigated the presence of thalamocortical input inside the graft. It is known that around 40% of thalamocortical fibers are directly in contact with layer II/III neurons (Viaene et al. 2011). To visualize thalamocortical fibers within the transplanted cluster, we stereotactically labeled ventral posterior nucleus of the thalamus with the mCherry-lentivirus at DPT 30. Seven days after lentivirus injection, we detected mCherry-labeled thalamocortical fibers inside the transplanted cluster, suggesting connection between donor neurons and appropriate afferents from the thalamus (Fig. 3C). Since the large majority of the donor cells were excitatory glutamatergic neurons (Fig. S1), we hypothesized that inhibitory GABAergic innervation, at least in part, is provided by the host cortical network. To characterize the development of the GABAergic system inside the grafted cluster, we performed immunohistochemical analysis of GAD67-positive element density at DPT 7, 14, and 30 in the intact and lesioned hemisphere. At DPT 7, we observed a few GABAergic fibers inside the cluster, but at later time points, the quantity of inhibitory fibers increased in both groups (Fig. 3D). Moreover, comparison of GABAergic fiber density in apoptotic and intact environment revealed significant increase of inhibitory innervation in the cluster located in the vicinity of the lesion at DPT 14. In addition, expression of K+/Cl– cotransporter KCC2 was weaker in the graft than in surrounding host cortex in 7 days after transplantation, and it was markedly increased after 30 days, correlating with development of GABAergic elements inside the cluster (Fig. S7A). It is noteworthy that the density of GAD67-positive neurons inside the cluster was comparable between time points with no significant difference between intact and lesion hemispheres (Fig. S7B). These results demonstrate a progressively increasing host inhibitory GABAergic input to the transplanted cluster and acceleration of this process by the apoptotic environment. Next, we took advantage of the pre-embedding immunoperoxidase technique to reveal host-derived GFP labeling with DAB using EM. We were able to find well-developed synaptic contacts, labeled both pre- and post-synaptically, between host and donor neurons (Fig. 3E). Postsynaptic DAB labeling, colocalized with postsynaptic density (Fig. 3E1, in yellow), was found in the proximity of the cluster. In addition, we detected DAB-labeled axonal terminals with multiple synaptic vesicles (Fig. 3E2, in yellow), indicating presynaptic contacts established on the transplanted neurons. To further confirm the functional integration of the grafted neurons into the host lesioned cortex, we examined if these neurons respond specifically to the somatosensory stimuli. For this purpose, we used c-Fos immunoreactivity as neuronal activity reporter (Herdegen and Leah 1998; Filipkowski et al. 2000; Vanelzakker et al. 2011). We compared c-Fos expression in the clusters at DPT 40 in injured animals without sensory stimulation and after 1-h stimulation in an enriched environment (Fig. 4A). While only a few transplanted neurons expressed c-Fos at the basal state, a significant 2-fold increase in c-Fos-labeled NeuN-positive neurons was observed in the transplanted clusters after sensory stimulation in the enriched environment (Fig. 4A). Moreover, in the experiments in which we transplanted electroporation-labeled layer II/III donor neurons, grafted cells displayed c-Fos immunoreactivity after sensory stimulation (Fig. 4B). Figure 4 Open in new tabDownload slide Functional integration of the donor neurons. (A) Confocal images showing activated donor neurons inside the grafted GFP-positive cell cluster with or without enriched environment condition (“enriched env+” and “enriched env-,” respectively). Scale bar = 100 μm. Graph representing density of activated neurons (NeuN/c-Fos double-positive cells) inside the GFP-positive cluster. Data presented for at least four animals per group and analyzed by unpaired Student’s t-test. Data in graph expressed as a mean ± s.e.m.; ***P < 0.001. (B) Confocal image of grafted cell cluster, where donor GFP-positive layer II/III neurons were activated (NeuN/c-Fos/GFP-positive cells, indicated by arrows) by enriched environment conditions. Scale bar = 100 μm, insertion scale bar = 20 μm. Figure 4 Open in new tabDownload slide Functional integration of the donor neurons. (A) Confocal images showing activated donor neurons inside the grafted GFP-positive cell cluster with or without enriched environment condition (“enriched env+” and “enriched env-,” respectively). Scale bar = 100 μm. Graph representing density of activated neurons (NeuN/c-Fos double-positive cells) inside the GFP-positive cluster. Data presented for at least four animals per group and analyzed by unpaired Student’s t-test. Data in graph expressed as a mean ± s.e.m.; ***P < 0.001. (B) Confocal image of grafted cell cluster, where donor GFP-positive layer II/III neurons were activated (NeuN/c-Fos/GFP-positive cells, indicated by arrows) by enriched environment conditions. Scale bar = 100 μm, insertion scale bar = 20 μm. These observations demonstrate the efficient structural and functional integration of the transplanted neuron clusters into the host circuit. Transplantation of Embryonic Neuronal Progenitors Improves Recovery from Lesion-Induced Functional Deficits Next, we tested the hypothesis that grafting embryonic neuronal precursors could promote functional recovery after apoptotic injury. We first addressed this question by measuring SEPs by EEG recordings in response to the stimulation of the left or the right whiskers (Fig. 5A). We compared the amplitude of the evoked potentials in the injured and noninjured S1 regions in response to contralateral whisker stimulations. In the S1 region engaged in targeted neuronal ablation in layer II/III, the average amplitude of the first SEP positive peak was significantly weaker by 37% (in 20 days after DT injection) and 20% (in 35 days after DT injection) as compared with the response in the noninjured hemisphere (Fig. 5B,D). In animals that received early transplantation of neuronal progenitors, the amplitude of SEPs was not different anymore between the injured and noninjured hemisphere at DPT 14 and 30, suggesting that the graft improved functional recovery of the SEP (Fig. 5C,E). Noteworthy, we failed to detect epileptiform cortical activity after transplantation. Figure 5 Open in new tabDownload slide Partial recovery of the lesion-induced functional deficit. (A) Under isoflurane anesthesia, 16 stainless steel electrodes were placed in contact with the skull. SEPs are recorded in response to upward deflections of all whiskers on one side of the face. (B, D) Left panels: grand average traces of the SEPs recorded above the left and right S1 cortices in response to respective contralateral stimulations in rats with apoptotic lesion in layer II/III at P34 and P50 (18 and 34 days after induction of neuronal ablation, equal to 14 and 30 days after transplantation in the experimental group). Right panels: corresponding histograms showing the mean ± s.e.m. amplitude quantifications for the negative and positive peaks of the SEP. Note, lower amplitude of the response in lesioned hemisphere (right stimulation) at 18 and 34 days after DT injection (average ± s.e.m. of 50 traces). (C, E) Similarly, grand average traces and mean SEP peaks in lesion groups of rats with grafts of embryonic neuronal progenitors at P34 and P50, 14 and 30 days post-transplantation. The reduced SEP peaks in the lesion group were restored in 2 weeks following embryonic neuronal progenitor cell transplantation. Data presented for 7 animals (B, D) or 10 animals (C, E) and analyzed by paired Student’s t-test. Data in graph expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01. (F) Graph representing results of gap-crossing test at DPT 14 and 30 for the control group (white column, n = 15 rats); layer II/III apoptotic lesion group (red column, n = 15 rats); layer II/III apoptotic lesion with the following transplantation of donor cells (dark green column, n = 19 rats). (G) Graph representing results of adhesive patch removal test at DPT 14 and 30 for the control group (black line, n = 15 rats); layer II/III apoptotic lesion group (red line, n = 15 rats); and layer II/III apoptotic lesion with the following transplantation of donor cells (dark green line, n = 19 rats). Difference between groups in F and G examined by two-way ANOVA with Bonferroni’s multiple comparison test. Data expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; and ****P < 0.0001; and ns, not significant. Figure 5 Open in new tabDownload slide Partial recovery of the lesion-induced functional deficit. (A) Under isoflurane anesthesia, 16 stainless steel electrodes were placed in contact with the skull. SEPs are recorded in response to upward deflections of all whiskers on one side of the face. (B, D) Left panels: grand average traces of the SEPs recorded above the left and right S1 cortices in response to respective contralateral stimulations in rats with apoptotic lesion in layer II/III at P34 and P50 (18 and 34 days after induction of neuronal ablation, equal to 14 and 30 days after transplantation in the experimental group). Right panels: corresponding histograms showing the mean ± s.e.m. amplitude quantifications for the negative and positive peaks of the SEP. Note, lower amplitude of the response in lesioned hemisphere (right stimulation) at 18 and 34 days after DT injection (average ± s.e.m. of 50 traces). (C, E) Similarly, grand average traces and mean SEP peaks in lesion groups of rats with grafts of embryonic neuronal progenitors at P34 and P50, 14 and 30 days post-transplantation. The reduced SEP peaks in the lesion group were restored in 2 weeks following embryonic neuronal progenitor cell transplantation. Data presented for 7 animals (B, D) or 10 animals (C, E) and analyzed by paired Student’s t-test. Data in graph expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01. (F) Graph representing results of gap-crossing test at DPT 14 and 30 for the control group (white column, n = 15 rats); layer II/III apoptotic lesion group (red column, n = 15 rats); layer II/III apoptotic lesion with the following transplantation of donor cells (dark green column, n = 19 rats). (G) Graph representing results of adhesive patch removal test at DPT 14 and 30 for the control group (black line, n = 15 rats); layer II/III apoptotic lesion group (red line, n = 15 rats); and layer II/III apoptotic lesion with the following transplantation of donor cells (dark green line, n = 19 rats). Difference between groups in F and G examined by two-way ANOVA with Bonferroni’s multiple comparison test. Data expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; and ****P < 0.0001; and ns, not significant. In order to assess behavioral changes following layer II/III apoptotic lesion with and without transplantation, we tested animals in commonly used behavior tests of the somatosensory system (Bocchi et al. 2017). The gap-crossing task requires active use of the whiskers and the somatosensory cortex by the animal (Learoyd and Lifshitz 2012), while the adhesive patch removal test is used to evaluate long-term sensorimotor effects (paw-mouth sensitivity and dexterity) (Bouet et al. 2009). We observed that DT-induced depletion of layer II/III neurons results in long-lasting whisker-sensory deficit, measured as a total number of trials to cross the gap 2 and 4 weeks after the lesion. This deficit was completely recovered in grafted group at 14 days after transplantation and persisted up to DPT 30 for the lesioned group (Fig. 5F). In contrast, the adhesive patch removal test failed to detect any significant postlesion recovery after transplantation even at DPT 30 (Fig. 5G). Thus, successful functional integration of grafted embryonic cells into the host could improve large-scale network activity as well as whisker-related behavior deficit. On the other hand, complex sensorimotor behavior that requires precise paw–mouth coordination and correct dexterity could not be completely recovered. In order to determine whether the long-term graft survival was required to maintain the functional recovery, we transplanted neurons transduced with DTR-encoding lentiviral construct to the apoptotic environment and eliminated the cluster by DT injection at 30 days post-transplantation after functional improvement (Fig. 6A). Given a relatively short half-life of DT in the blood (Wrobel et al. 1990), we expected this time being sufficient to eliminate the DT from the first injection. While in the control group DTR-tRFP expressing clusters were present in the cortex, these were not visible in animals that received a second DT injection. Instead, the place of the ablated cluster was marked by an important astrogliosis (Fig. 6B). Longitudinal behavioral tests (paired t-test) indicated that animals needed significantly more trials to cross the gap after ablation of the graft (Fig. 6C). This phenomenon was not observed with animals with an intact graft (Fig. 6C). Moreover, we observed further improvement of patch removal time between DPT 30 and 55 in animals with intact grafts, but not those after graft ablation (Fig. 6D). These results demonstrate that the presence of graft is needed for maintaining functional improvements after transplantation. Figure 6 Open in new tabDownload slide Long-term functional improvement relies on the persistence of the cluster in the injured cortex. (A) Timeline of the experimental procedures. (B) Confocal images showing phases of DT-mediated ablation of DTR-transduced donor neurons. (1) DTR–tRFP transduced donor cluster before DT injection, moderate GFAP-stained astrogliosis. (2) Reactive astrogliosis (GFAP) on the place of elimination by DT injection donor cluster. Scale bars = 200 μm. (C) Graphs representing the number of trials to cross the gap in gap-crossing test at DPT 30 and 55 for the groups with remained (red line, n = 11 rats) or eliminated (blue line, n = 12 rats) donor cluster. (D) Graphs representing results of adhesive patch removal test at DPT 30 and 55 for the groups with remained (red line, n = 11 rats) or eliminated (blue line, n = 12 rats) donor cluster. The difference between groups in C and D was assessed by paired Student’s t-test. Each line on the graph represents individual animal; *P < 0.05; and ns, not significant. Figure 6 Open in new tabDownload slide Long-term functional improvement relies on the persistence of the cluster in the injured cortex. (A) Timeline of the experimental procedures. (B) Confocal images showing phases of DT-mediated ablation of DTR-transduced donor neurons. (1) DTR–tRFP transduced donor cluster before DT injection, moderate GFAP-stained astrogliosis. (2) Reactive astrogliosis (GFAP) on the place of elimination by DT injection donor cluster. Scale bars = 200 μm. (C) Graphs representing the number of trials to cross the gap in gap-crossing test at DPT 30 and 55 for the groups with remained (red line, n = 11 rats) or eliminated (blue line, n = 12 rats) donor cluster. (D) Graphs representing results of adhesive patch removal test at DPT 30 and 55 for the groups with remained (red line, n = 11 rats) or eliminated (blue line, n = 12 rats) donor cluster. The difference between groups in C and D was assessed by paired Student’s t-test. Each line on the graph represents individual animal; *P < 0.05; and ns, not significant. Chemogenetic Activation of Transplanted Neurons Leads to Enhanced Dendritic Protrusion Density and Further Improves Functional Recovery Since neuronal activity drives functional network formation during neurodevelopment, we hypothesized that neuronal ablation may result in reduced neuronal activity in the host cortex and the graft may have a positive impact by activation of preserved host circuits. To evaluate the activity level of neurons, animals were placed in an enriched environment for 1 h and then immediately sacrificed. As described above, we used immunostaining for c-Fos as neuronal activity reporter and determined the percentage of NeuN-positive cells that also contained c-Fos immunoreactivity. We observed that after DT-induced neuronal lesion in layer II/III, the number of c-Fos-expressing neurons in the apoptotic neighborhood was significantly lower than in noninjured littermates (Fig. 7A). We then examined whether grafted neurons may have an impact on the activity of host neurons. We found that transplantation leads to a 2-fold increase in the number of NeuN/c-Fos double-positive host layer II/III neurons under basal conditions (Fig. 7A). To test the idea that the activity level of grafted neurons is important for this effect, we took advantage of graft-specific chemogenetic activation using designer receptor exclusively activated by designer drug. We transplanted E19 cortical precursors that were electroporated at E18 with the GFP-hM3Dq construct (Fig. 7B). Starting from the third day after transplantation, animals received intraperitoneal injection of CNO or vehicle solution (PBS) daily for 14 days. We first tested if the activity of the graft influences the activity of host circuits by quantifying the number of NeuN/c-Fos double-positive neurons in lesioned host LII/III at DPT 40 that experienced enriched environment. In addition, we compared obtained data with animals transplanted with GFP electroporated cells. We observed that the number of c-Fos expressing neurons in the host was significantly higher in animals that were stimulated by CNO than in vehicle-injected control rats or in animals transplanted with GFP-expressing progenitors (Figs 7C and S8). Note that the density of NeuN-positive neurons within clusters with and without activation was comparable (Fig. 7D). Figure 7 Open in new tabDownload slide Specific chemogenetic activation of transplanted neurons improves their integration and the general behavioral performance. (A) Representative confocal images showing activated layer II/III neurons in the brain of control, lesioned and lesioned-transplanted animals in basal condition. Scale bar = 20 μm. Graph, representing density of activated layer II/III neurons (NeuN/c-Fos double-positive cells) in the indicated group’s host brains. Data presented for at least four animals per group and examined by ordinary one-way ANOVA with Bonferroni’s multiple comparison test. (B) Timeline of the experimental procedures. (C) Graph, representing density of activated layer II/III neurons (NeuN/c-Fos double-positive cells) in the indicated group’s host brains at 40 DPT. Data are presented for at least five animals per group, and the difference is examined by ordinary one-way ANOVA with Bonferroni’s multiple comparison test. (D) Graphs representing the neuronal cell density (NeuN/Hoechst) and the common cell density (Hoechst) inside the donor cluster at marked time points in animals following the transplantation of neurons electroporated with ubi-GFP (control group) or with hM3Dq later injected with PBS (control group) or CNO (experimental group); n > 3. (E) High-magnification confocal images showing representative dendritic branches of transplanted layer II/III donor neurons and corresponding quantifications (right panel) in three groups of animals: transplanted with control ubi-GFP electroporated neurons or hM3Dq electroporated neurons and later injected with PBS or CNO—n > 3 animals per time point. Scale bar = 5 μm. (F) Graph representing results of gap-crossing test at DPT 14 and 30 for the group with simple GFP-labeled donor cells (dark green column, n = 19 rats); the group with grafted hM3Dq cells and treated with PBS (light green column, n = 12 rats); and for the CNO-treated hM3Dq group (purple column, n = 12 rats). (G) Graphs representing results of adhesive patch removal test at DPT 14 and 30 for the group with simple GFP-labeled donor cells (dark green line, n = 19 rats); the group with grafted hM3Dq cells and treated with PBS (light green line, n = 12 rats); and for the CNO-treated hM3Dq group (purple line, n = 12 rats). Difference between groups in A, C, D, and E examined by ordinary two-way ANOVA with Bonferroni’s multiple comparison test. Data expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Figure 7 Open in new tabDownload slide Specific chemogenetic activation of transplanted neurons improves their integration and the general behavioral performance. (A) Representative confocal images showing activated layer II/III neurons in the brain of control, lesioned and lesioned-transplanted animals in basal condition. Scale bar = 20 μm. Graph, representing density of activated layer II/III neurons (NeuN/c-Fos double-positive cells) in the indicated group’s host brains. Data presented for at least four animals per group and examined by ordinary one-way ANOVA with Bonferroni’s multiple comparison test. (B) Timeline of the experimental procedures. (C) Graph, representing density of activated layer II/III neurons (NeuN/c-Fos double-positive cells) in the indicated group’s host brains at 40 DPT. Data are presented for at least five animals per group, and the difference is examined by ordinary one-way ANOVA with Bonferroni’s multiple comparison test. (D) Graphs representing the neuronal cell density (NeuN/Hoechst) and the common cell density (Hoechst) inside the donor cluster at marked time points in animals following the transplantation of neurons electroporated with ubi-GFP (control group) or with hM3Dq later injected with PBS (control group) or CNO (experimental group); n > 3. (E) High-magnification confocal images showing representative dendritic branches of transplanted layer II/III donor neurons and corresponding quantifications (right panel) in three groups of animals: transplanted with control ubi-GFP electroporated neurons or hM3Dq electroporated neurons and later injected with PBS or CNO—n > 3 animals per time point. Scale bar = 5 μm. (F) Graph representing results of gap-crossing test at DPT 14 and 30 for the group with simple GFP-labeled donor cells (dark green column, n = 19 rats); the group with grafted hM3Dq cells and treated with PBS (light green column, n = 12 rats); and for the CNO-treated hM3Dq group (purple column, n = 12 rats). (G) Graphs representing results of adhesive patch removal test at DPT 14 and 30 for the group with simple GFP-labeled donor cells (dark green line, n = 19 rats); the group with grafted hM3Dq cells and treated with PBS (light green line, n = 12 rats); and for the CNO-treated hM3Dq group (purple line, n = 12 rats). Difference between groups in A, C, D, and E examined by ordinary two-way ANOVA with Bonferroni’s multiple comparison test. Data expressed as a mean ± s.e.m.; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. Post hoc analysis at DPT 7, 14, and 30 showed that dendritic protrusion density in the grafted layer II/III neurons was significantly increased at DPT 14, when donor hM3Dq-expressing neurons were activated with CNO, suggesting their enhanced structural integration (Fig. 7E). This phenomenon was stably present even 12 days after the last CNO injection, at DPT 30. The increased numbers of dendritic spines on activated grafted neurons raise the possibility that such neurons may be better integrated into host circuits. We then tested whether grafted neuron stimulation could improve the functional outcome in the behavior tests. Although we did not observe any further improvement in the gap-crossing test after activation of transplanted cells compared with the transplanted group without activation (Fig. 7F), we found that enhanced activation of transplanted neurons with CNO resulted in significant increase in the number of rats that succeeded to remove the adhesive patch when compared with both transplanted control groups at DPT 14 and 30 (Fig. 7G). Based on these observations, we concluded that transplanted cells increase the activity of host circuits and that enhancing the activity of the graft is an efficient way to further improve this effect, leading to better functional recovery. Discussion A critical issue for transplantation-based neuronal replacement strategies is the capacity of grafted cells to integrate into host circuitry and to compensate for functional deficit (see in Dayer et al. 2007; Wuttke et al. 2018). In this study, we induced apoptotic death of layer II/III pyramidal neurons in the juvenile cerebral cortex and tested how grafted embryonic precursors are integrated in an apoptotic microenvironment. We found that grafted excitatory neuronal precursors do not migrate to positionally replace lost neurons but rather remain in compact clusters. We demonstrate that despite the lack of repopulation of lesion sites, the cluster as a whole is structurally as well as functionally integrated into the host tissue, and the apoptotic microenvironment accelerates this process. Most importantly, grafted neurons establish reciprocal synaptic contact with the host cerebral cortex, enhance the activity of host circuits, and significantly improve functional recovery after the apoptotic death of layer II/III pyramidal cells. Targeted chemogenetic activation of the graft further improves functional recovery. Finally, we found that the presence of the graft is necessary for maintaining improved functional activity in adult animals. We conclude that grafted aggregates of neurons are not isolated “heterotopias” but functionally relevant for the host and could compensate for activity loss of preserved cortical circuits after injury, thereby promoting functional recovery. We hypothesized that grafted neuronal precursors in the vicinity of apoptotic death environment will migrate to replace lost neurons in the supragranular layer. Our results demonstrate that this is not the case. A frequently reported observation in transplantation studies is that grafted cells form discrete neuronal aggregates and only rare cells migrate into the surrounding host tissue (Gaspard et al. 2008; Ladewig et al. 2014; Falkner et al. 2016). This has been related to the large number of transplanted cells and the reciprocal attractions between grafted cells preventing cells from migrating into the host tissue (Ladewig et al. 2014). On the other hand, microtransplantation of very small numbers of neurons resulted in cell dispersion and cellular level integration into the host (Wuttke et al. 2018). It has been proposed that migration of grafted cells into the recipient tissue provides a more appropriate, individual integration of cells with the host circuitry (Dayer et al. 2007; Wuttke et al. 2018). Here, we provide several lines of evidence for a functionally relevant integration of grafted compact aggregates. First, using distinct fluorescent labeling of grafts and donors, we demonstrate that vascular elements grow into grafted neuronal aggregates from the host, thus providing access to nutritional support as well as oxygen supply. This agrees with a previous study showing that the vascularization of grafted embryonic neurons could be derived from the host as well as from the graft (Peron et al. 2017). We also show here that microglia of host origin invade the donor cluster. The host-derived microglia had a nonactivated morphology inside the cluster suggesting that the recipient accepts the cluster immunologically as its own structure and that host microglial cells likely serve the graft by providing their physiological functions. Second, we found that the complexity of dendritic arbors within the grafted cluster progressively increased during 30 days posttransplantation, which correlated with the normal dendritic development of layer II/III cells. In parallel, dendritic spine density gradually increased between 14 and 30 days posttransplantation, and the presence of the synapses and well-developed neuropil was confirmed by EM. Similar results were obtained in the visual cortex with two-photon live imaging of transplanted to the lesion site layer II/III neurons, in which growth of apical and basal dendrite, appearance, and turnover of dendritic spines resemble the normal developmental program (Falkner et al. 2016). However, we noticed that transplanted neurons establish primary dendrites earlier than age-matched control cells in the layer II/III, followed by increased complexity and expansion of dendrites at later stages. In addition, we observed higher spine density in the grafted neurons. Third, we demonstrate that grafted cells grow both short-distance and long-distance axonal projections to a number of cortical areas. We showed that axons from transplanted layer II/III cells extend locally to the ipsilateral somatosensory cortex and distantly to the ipsilateral insular and motor cortex, reflecting physiological targets of layer II/III neurons in S1 (Fabri and Burton 1991; Zakiewicz et al. 2014). Importantly, we demonstrated that labeled donor axons formed synapses with the host dendrites. Fourth, in agreement with previous published studies (Tornero et al. 2017), we show that grafted neurons in cell clusters receive synaptic input from the host brain. These may include GABAergic inhibitory innervation, input from neighboring layer 4, as well as thalamic inputs as we show in the present study. Consistent with these observations, we found that whisker stimulations elicit the activation of grafted neuronal clusters as shown by c-Fos immunostaining. Together, the presented results give strong support to previous observations that the host brain can influence the activity of neurons in intracortical implants (Bragin et al. 1988; Girman and Golovina 1990; Grabowski et al. 1993; Michelsen et al. 2015). Furthermore, our findings indicate that neurons in grafted cell clusters structurally as well as functionally integrate into the recipient through bidirectional connections. Following DTR-targeted lesion in layers II/III of somatosensory cortex, rats were impaired in their ability to remove the adhesive patch. Future studies should explore whether this inability is related to the sensory feedback alterations or a motor coordination following the lesion. We found that animals with apoptotic lesion of layer II/III pyramidal neurons of the somatosensory cortex displayed an altered performance in behavior tests. We demonstrate that functional deficits could be significantly improved after grafting embryonic neurons and that long-term recovery requires the persistence of the graft. Transplantation of embryonic neurons has improved somatosensory functions in the gap-crossing task, but the adhesive patch removal test evaluating long-term sensorimotor integration (Bouet et al. 2007; Balkaya et al. 2013; Duricki et al. 2019) failed to detect any significant recovery. The adhesive patch-removal task is considered as one of the most sensitive behavior tests for the focal cortical lesions (Sughrue et al. 2006; Komotar et al. 2007; Bouet et al. 2009). It assesses two parameters: the latency to contact the patch on the impaired paw is used to assess sensory impairment, while the latency to remove is influenced by both sensory and motor systems (Balkaya et al. 2013). Following DTR-targeted lesion in layers II/III of somatosensory cortex, rats were impaired in their ability to remove the adhesive patch. Future studies should explore whether this inability is related to the sensory feedback alterations or a motor coordination following the lesion. While the precise mechanism by which grafted cells may improve functional recovery remains to be determined, our results give support to the hypothesis that transplanted neurons increase the activity of host circuits. Activity plays a crucial role in development and functioning of neuronal circuits (reviewed in (Kiss et al. 2014)). Reduction of cortical activity was previously detected after hypoxic–ischemic injury in neonatal human and rodent brains, resulting in decreased activity-dependent protein expression, impaired dendritogenesis, and plasticity in the barrel cortex (Ranasinghe et al. 2015). Moreover, depressed EEG-defined cortical activity is an important predictive factor for poor neurodevelopmental outcome (Wikstrom et al. 2012; Natalucci et al. 2013; Benders et al. 2015). We found a significantly decreased activity of host neurons after the lesion that was enhanced by transplanting embryonic pyramidal precursors. Consistent with these findings, chemogenetic activation of grafted neurons further increases the activity level of host circuit and promotes functional recovery. We showed that early chemogenetic activation of the grafted layer II/III neurons with CNO significantly improved functional recovery assessed by the adhesive patch removal test. The underlying mechanism of this effect remains to be determined. It is likely that while intrinsic activity plays an important role in the initial steps of the cluster integration and its appropriate connection to the host network, a specific experience-dependent activity could promote further connectivity and functioning of the grafted neurons, recapitulating the normal developmental program (Cheetham et al. 2007; Zhao et al. 2009; Kiss et al. 2014; Crocker-Buque et al. 2015). On the other hand, the activation of the cluster may influence plasticity in the host cortical networks. In agreement with previous works on the activity-driven structural plasticity (Holtmaat et al. 2006; Hamilton et al. 2012; Ping Yu et al. 2019), we showed that chemogenetic stimulation of the grafted neurons significantly increases the number of dendritic spines in a cell-autonomous manner. Such enhanced plasticity in the layer II/III constitutes a substrate for learning and memory based on tactile information (Guic-Robles et al. 1992), thus explaining improved performances in the gap-crossing and adhesive patch removal tests after cortical lesion in our experiments. In conclusion, we propose that grafts from the embryonic cortex rescue decreased neuronal activity level in injured networks and improve functional recovery after neuronal loss. This effect may involve synaptic connections between host and graft. Exogenous activation of the graft further improves functional recovery of the recipient. Finally, the presence of the graft is necessary for maintaining adequate functional activity in the adult animals. Together, our results suggest new possibilities for combined neuronal transplantation/activation-based approaches for treating brain injury-related functional deficits. Funding This work was supported by the Swiss National Foundation (grant number 31003A_140940/1). Notes We thank E. Husi and C. Saadi for technical assistance. Conflict of Interest: None declared. References Bachoud-Levi AC , Gaura V, Brugieres P, Lefaucheur JP, Boisse MF, Maison P, Baudic S, Ribeiro MJ, Bourdet C, Remy P, et al. 2006 . Effect of fetal neural transplants in patients with Huntington's disease 6 years after surgery: a long-term follow-up study . Lancet Neurol. 5 : 303 – 309 . Google Scholar Crossref Search ADS PubMed WorldCat Bachoud-Levi AC , Remy P, Nguyen JP, Brugieres P, Lefaucheur JP, Bourdet C, Baudic S, Gaura V, Maison P, Haddad B, et al. 2000 . 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TI - Transplanted Embryonic Neurons Improve Functional Recovery by Increasing Activity in Injured Cortical Circuits
JF - Cerebral Cortex
DO - 10.1093/cercor/bhaa075
DA - 2020-06-30
UR - https://www.deepdyve.com/lp/oxford-university-press/transplanted-embryonic-neurons-improve-functional-recovery-by-1GSa0JH6J5
SP - 4708
EP - 4725
VL - 30
IS - 8
DP - DeepDyve
ER -