Quantitative analysis of the generation of different striatal neuronal subtypes in the adult brain following excitotoxic injury

Experimental Neurology 195 (2005) 71 – 80 www.elsevier.com/locate/yexnr

Regular Article

Quantitative analysis of the generation of different striatal neuronal subtypes in the adult brain following excitotoxic injury Tove Collina,c, Andreas Arvidssona,c, Zaal Kokaiab,c, Olle Lindvalla,c,* a

Laboratory of Neurogenesis and Cell Therapy, Section of Restorative Neurology, Wallenberg Neuroscience Center, BMC A11, SE-221 84 Lund, Sweden b Laboratory of Neural Stem Cell Biology, Section of Restorative Neurology, Stem Cell Institute, BMC B10, SE-221 84 Lund, Sweden c Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund, Sweden Received 26 November 2004; revised 22 February 2005; accepted 30 March 2005 Available online 2 June 2005

Abstract Recent findings in adult rodents have provided evidence for the formation of new striatal neurons from subventricular zone (SVZ) precursors following stroke. Little is known about which factors determine the magnitude of striatal neurogenesis in the damaged brain. Here we studied striatal neurogenesis following an excitotoxic lesion to the adult rat striatum induced by intrastriatal quinolinic acid (QA) infusion. New cells were labeled with the thymidine-analogue 5-bromo-2V-deoxyuridine (BrdU) and their identity was determined immunocytochemically with various phenotypic markers. The unilateral lesion gave rise to increased cell proliferation mainly in the ipsilateral SVZ. At 2 weeks following the insult, there was a pronounced increase of the number of new neurons co-expressing BrdU and a marker of migrating neuroblasts, doublecortin, in the ipsilateral striatum, particularly its non-damaged medial parts. About 80% of the new neurons survived up to 6 weeks, when they expressed the mature neuronal marker NeuN and were preferentially located in the outer parts of the damaged area. Lesion-generated neurons expressed phenotypic markers of striatal medium spiny neurons (DARPP-32) and interneurons (parvalbumin or neuropeptide Y). The magnitude of neurogenesis correlated to the size of the striatal damage. Our data show for the first time that an excitotoxic lesion to the striatum can trigger the formation of new striatal neurons with phenotypes of both projection neurons and interneurons. D 2005 Elsevier Inc. All rights reserved. Keywords: Neurogenesis; Excitotoxic injury; Rat striatum

Introduction In the intact adult brain, the neural stem/precursor cells in the subventricular zone (SVZ), lining the lateral ventricle, Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; BrdU, 5-bromo-2V-deoxyuridine; ChAT, choline acetyltransferase; DARPP-32, dopamine- and adenosine 3V:5V-monophosphateregulated phosphoprotein with a molecular weight of 32 kDa; Dcx, doublecortin; GABA, g-aminobutyric acid; KPBS, potassium phosphatebuffered saline; MCAO, middle cerebral artery occlusion; NeuN, neuronspecific nuclear antigen; NPY, neuropeptide Y; PARV, parvalbumin; PBS, phosphate-buffered saline; PFA, paraformaldehyde; QA, quinolinic acid; SVZ, subventricular zone. * Corresponding author. Laboratory of Neurogenesis and Cell Therapy, Section of Restorative Neurology, Wallenberg Neuroscience Center, BMC A11, SE-221 84 Lund, Sweden. Fax: +46 46 222 0560. E-mail address: [email protected] (O. Lindvall). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.03.017

generate new interneurons which migrate to the olfactory bulb (Altman, 1969; Lois and Alvarez-Buylla, 1994). Recent findings in rodents have provided evidence that the SVZ precursors can be recruited following stroke induced by transient middle cerebral artery occlusion (MCAO), which causes extensive damage to striatum and overlying parietal cortex. Stroke was found to trigger increased neural proliferation in the SVZ (Jin et al., 2001; Zhang et al., 2001). The new immature neurons migrate into the damaged striatum, where a substantial portion of them express markers of striatal medium spiny projection neurons (Arvidsson et al., 2002; Jin et al., 2003; Parent et al., 2002). Thus, the new neurons seem to develop the phenotype of most neurons destroyed by the ischemic lesion. In addition, intraventricular infusion of epidermal growth factor after stroke in mice has been reported to generate cells expressing

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parvalbumin, i.e., a marker of a specific type of striatal interneuron (Teramoto et al., 2003). The findings that following a stroke, new neurons can be formed in damaged regions in which neurogenesis does not normally occur, e.g., striatum, have raised the possibility that such self-repair could become of therapeutic value (Abrahams et al., 2004). However, it is unclear whether the new neurons are functional and if they become integrated into existing neural and synaptic networks. Furthermore, the stroke-induced neurogenic response may be ineffective because the majority of the new neurons die during the first weeks after they have been formed (Arvidsson et al., 2002). In order to be able to promote self-repair in the brain, it is necessary to identify those mechanisms which regulate the formation of new neurons. Currently, very little is known about which factors determine the occurrence and magnitude of adult striatal neurogenesis. Here, we have studied striatal neurogenesis following an excitotoxic lesion to the adult rat striatum, produced by intrastriatal quinolinic acid (QA) infusion. The objectives were two-fold: First, to explore whether injury to the striatum caused by a nonischemic insult can induce the generation of new striatal neurons with the phenotypes of projection neurons and interneurons; second, to determine if the extent of injury influences the various steps of striatal neurogenesis, and what is the location of the new neuroblasts and mature neurons in relation to the damage in the striatum.

Materials and methods Animals Male Wistar rats (n = 23; body weight 530 –580 g) were obtained from Charles River (Germany). The animals were housed under 12:12-h light/dark conditions with ad libitum access to food and water. Experimental procedures were conducted according to the guidelines set by the Malmo¨Lund Ethical Committee for the use and care of laboratory animals. Quinolinic acid and BrdU administration Rats were anaesthetized with sodium pentobarbital (60 mg/kg ip) and infused with QA (2 Al (225 nmol dissolved in phosphate-buffered saline (PBS)) during 2 min; the Hamilton syringe was withdrawn after 5 min) into the right striatum at the following coordinates: 1.2 mm rostral and 3.2 mm lateral to bregma and 5.0 mm ventral from dura, tooth-bar at 0 mm (Paxinos and Watson, 1997). Rats subjected to sham surgery were infused with saline. Intraperitoneal injections of BrdU (5-bromo-2V-deoxyuridine, 50 mg/kg body weight, SigmaAldrich, St Louis; dissolved in PBS) were given twice daily during 2 weeks starting on the day after surgery. A group of intact rats, not subjected to any surgeries, were injected similarly. Animals were allowed to survive 1 day (six QA-

infused, six saline-infused and six intact animals) or 4 weeks (five QA-infused animals) after the last BrdU injection. Immunohistochemistry Animals were deeply anaesthetized with sodium pentobarbital and transcardially perfused with saline followed by ice-cold formaldehyde solution (4% paraformaldehyde (PFA) in 0.1 M PBS, pH 7.4). Brains were removed and postfixed in the same solution overnight. The PFA-solution was then replaced by 20% sucrose in 0.1 M PBS for at least 24 h for cryoprotection. The brains were sectioned on a freezing microtome at 30 Am thickness. Sections were stored at 20-C in cryo-protective solution. Free-floating sections were denatured by incubation in 1 M hydrochloric acid for 30 min at 65-C (except for staining including the anti-choline acetyltransferase (ChAT); 1 M hydrochloric acid for 10 min at 65-C followed by 20 min at room temperature). Following rinsing with potassium phosphate-buffered saline (KPBS), sections were preincubated for 1 h in 5% of appropriate sera (normal donkey serum for BrdU, normal horse serum for doublecortin (Dcx), neuron-specific nuclear antigen (NeuN), dopamine- and adenosine 3V:5Vmonophosphate-regulated phosphoprotein with a molecular weight of 32 kDa (DARPP-32), parvalbumin (PARV) or ChAT, and normal goat serum for neuropeptide Y (NPY)) in KPBS containing 0.25% Triton-X. Sections were then incubated with rat anti-BrdU (1:100, Oxford Biotechnology Ltd, Oxfordshire, UK) and either of the following primary antibodies against phenotypical markers; goat anti-Dcx (1:400, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-NeuN (1:100, Chemicon International, Temecula, CA), mouse anti-DARPP-32 (1:20000, provided by P. Greengard), mouse anti-PARV (1:2000, Sigma), rabbit anti-NPY (1:1500, Sigma), or mouse anti-ChAT (1:750, Chemicon International, Temecula, CA) in pre-incubation solution for 36 h at 4-C. Sections were then rinsed with KPBS containing 0.25% Triton-X, followed by 2 h incubation with Cy3-conjugated donkey anti-rat secondary antibody (1:200, Jackson ImmunoResearch Laboratories Inc, West Grove, PA) for visualization of BrdU together with either biotinylated horse antigoat (for Dcx), horse anti-mouse (for NeuN, DARPP-32, PARV, and ChAT), or goat anti-rabbit (for NPY) (1:200, Vector Laboratories, Burlingame, CA) at room temperature in darkness. Following rinsing, sections were finally incubated with Alexa 488-conjugated streptavidin (1:200, Molecular Probes Inc, Eugene, OR) in KPBS for 2 h, at room temperature in darkness. The sections were mounted onto glass slides. After drying, they were rinsed in distilled water and dried again before being cover-slipped with PVADABCO mounting medium. Microscopical analysis All analyses were conducted by observers blind to treatment conditions. BrdU+ cells in the SVZ were counted

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using 100 objective on an epifluorescence microscope in three coronal sections per animal at rostrocaudal levels +2.2, +1.3, +0.4 mm from bregma (Paxinos and Watson, 1997) using the fractionator method on stereological equipment. Counting of Dcx+, BrdU+/Dcx+, BrdU+/NeuN+, BrdU+/ DARPP-32+, BrdU+/PARV+, BrdU+/NPY+, and BrdU+/ ChAT+ cells in the striatum was performed using 40 objective on an epifluorescence microscope in three sections per animal from the same levels as above. For analysis of the relationship between number of new cells and size of striatal damage, we counted cells also in two additional sections ( 0.5 and 1.4 mm from bregma). The accuracy of counting double-labeled cells using epifluorescence microscopy was determined for BrdU/Dcx and BrdU/NeuN stainings by analysis of all cells in one random section, using a confocal laser scanning microscope (BioRad Microscience, Hemel Hempstead, UK). For the other stainings, where comparatively few cells were found, a subset of the counted cells were identified and validated similarly with confocal microscopy. Cells were considered double-labeled if co-labeling with relevant morphology was seen throughout the extent of the nucleus for nuclear markers, or if a cytoplasmic marker surrounded a nuclear marker, when viewed in x – y cross section, as well as in x – z and y –z cross sections produced by orthogonal reconstructions from z-series (z-step, 0.5 Am) taken with 63 objective. Since virtually all cells (>92%) were doublelabeled according to this confocal validation, no adjustments of the counted cell numbers were performed. For estimations of shrinkage and cell densities, we measured areas of total remaining striatal tissue and non-damaged striatal tissue, respectively, in BrdU/NeuN-stained sections at levels +2.2, +1.3, and +0.4 mm from bregma in QA-infused and intact animals using stereological equipment. Two additional sections (at 0.5 and 1.4 mm from bregma) were also analyzed for area of non-damaged striatal tissue when assessing the relation between size of damage and cell numbers.

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almost complete loss of cells in the lateral and rostral striatum, as revealed in sections stained for the neuronspecific marker NeuN (Fig. 1). The most medial parts of the striatum were spared. In 5 out of 6 rats in the 2-week group and 4 out of 5 rats in the 6-week group, the overlying parietal cortex was also damaged. There was a significant shrinkage of the ipsilateral striatum both at 2 and 6 weeks following the QA infusion (by 23.3 T 1.9 and 43.0 T 5.4%, respectively, as compared to mean striatal tissue volume in intact animals). The shrinkage of the striatum contralateral to the QA infusion was only minor (17.3 T 2.3% and 16.0 T 2.9% at 2 and 6 weeks, respectively). We first explored whether the excitotoxic striatal lesion influenced cell proliferation in the SVZ. Animals were injected with BrdU to label dividing cells twice daily for 2 weeks following the QA infusion and sacrificed 1 day thereafter. The sham operation did not induce any change of SVZ proliferation compared to that in intact animals (Fig. 2). In contrast, the QA lesion gave rise to elevated numbers of BrdU+ cells in the ipsilateral SVZ. Also on the contralateral side there was a significant increase of SVZ cell proliferation in the QA-treated rats as compared to intact animals. The increase of cell proliferation in the SVZ was significantly higher ipsilateral to the QA infusion as compared to contralaterally (56.7 and 23.3% higher number of BrdU+ cells as compared to sham, respectively; Fig. 2).

Statistical analysis All values are means T SEM. Comparisons were performed using Student’s paired t test, Student’s unpaired t test, or one-way analysis of variance (ANOVA) followed by post hoc Bonferroni/Dunn test. Differences were considered significant if P < 0.05. Pearson’s correlation was used to assess the relation between size of damage and cell numbers.

Results Excitotoxic striatal injury stimulates cell proliferation in SVZ The QA infusion caused a massive lesion of the striatum. Both at 2 and 6 weeks following the infusion, there was an

Fig. 1. Overview of the distribution of neuronal loss following the excitotoxic striatal injury. The extent of the lesion at 2 weeks following the QA infusion is illustrated at five coronal levels (+2.7 to 1.3 mm from bregma) in NeuN-immunostained sections. Asterisks indicate damaged area. Scale bar is 5 mm.

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Fig. 2. Excitotoxic striatal injury increases cell proliferation in SVZ. Number of BrdU+ cells in ipsilateral and contralateral SVZ in intact rats and at 2 weeks following unilateral intrastriatal QA or saline infusion with BrdU injections twice daily until perfusion. Data are means T SEM; *P < 0.05 compared to intact and sham; +P < 0.05 compared to intact; one-way ANOVA with Bonferroni/Dunn post hoc test; .P < 0.05 compared to contralateral side, paired t test. n = 6 for all groups.

Excitotoxic striatal injury triggers formation of neuroblasts and mature striatal neurons To determine if the QA-induced increase of cell proliferation in the SVZ was associated with induction of striatal neurogenesis, we counted the number of Dcx+ neurons in the striatum at 2 weeks following the QA infusion. Dcx is a marker of neuroblasts, which is transiently expressed (during about 2– 3 weeks) in neuronal progenitors and newly generated olfactory bulb neurons originating in the SVZ (Brown et al., 2003). All animals showed strong Dcx-immunoreactivity bilaterally in the SVZ, with the most pronounced staining ipsilateral to the QA infusion (Figs. 3B –C). We found only very few Dcx+ cells in the striatum of intact animals (Figs. 3A, D, and M) or after sham surgery (Fig. 3M). In contrast, in rats subjected to QA infusion (Figs. 3C, F, and M), there was a pronounced increase of the number of striatal Dcx+ cells,

distributed in a gradient from the SVZ laterally and ventrally, in the striatum ipsilateral to the lesion (10-fold vs. sham). On the contralateral side (Figs. 3B and E) the increase was only modest (2-fold), and the cells were located in close proximity to the SVZ. We also counted the number of Dcx+ cells which had been labeled with BrdU during the 2-week injection period. At 2 weeks after the QA infusion, the number of BrdU+/Dcx+ cells was increased in the striatum, in particular on the side ipsilateral to the lesion (19-fold increase as compared to sham), but to a minor extent also on the contralateral side (Fig. 3M). We found that 57.8 T 5.1 and 66.6 T 4.2% of the total number of Dcx+ cells were labeled with BrdU in the ipsi- and contralateral striatum, respectively. We then assessed whether the QA-induced lesion could also lead to the formation of striatal neurons with a mature phenotype by counting the number of BrdU+/NeuN+ cells at 6 weeks after the QA infusion and 4 weeks after the last BrdU injection (Fig. 4). It has been shown that at this time point after administration of BrdU, about 90% of BrdU+ olfactory bulb cells, generated in the SVZ, have matured and are now co-labeled with NeuN (Brown et al., 2003). We found a substantial number of BrdU+/NeuN+ cells in the QA-lesioned striatum at 6 weeks whereas only single cells were seen on the contralateral side (Figs. 4A – D). This finding indicated that the BrdU+/Dcx+ cells observed at 2 weeks had matured and were now BrdU+/NeuN+. In agreement, we found a small number of BrdU+/NeuN+ cells in the striatum already at 2 weeks (data not shown), which is the time when such cells are beginning to appear in the olfactory bulb (Brown et al., 2003). Furthermore, virtually no BrdU+/Dcx+ cells could be detected in the striatum at 6 weeks (3.6 T 0.9 cells). At 2 weeks after the QA infusion, many Dcx+ but BrdU cells (42.2 T 5.1% of the total number of Dcx+ cells) were observed in the damaged striatum. In contrast, very few such cells were found on the contralateral side or in sham-operated or intact striatum. Thus, the occurrence of these Dcx+ striatal cells had been triggered by the QA lesion. Also at 6 weeks after infusion, we found a substantial number of lesion-induced Dcx+/BrdU cells in the damaged striatum (145.8 T 62.8 cells). Most of these cells had an immature morphology resembling that observed for Dcx+ cells at 2 weeks.

Fig. 3. Excitotoxic striatal injury stimulates formation of neuroblasts in damaged striatum. (A – F) Overview of BrdU (red) and Dcx (green) immunoreactivity in the dorsomedial striatum of an intact rat (A) and contralateral (B) and ipsilateral (C) to an intrastriatal QA infusion performed 2 weeks earlier. In the striatum of intact animals, Dcx immunoreactivity is virtually restricted to the SVZ, as seen in higher magnification (D) of the boxed area in panel A. On the side contralateral to the QA infusion, the Dcx+ and BrdU+/Dcx+ cells are predominantly located in the SVZ, but some cells (arrows) are scattered in the adjacent striatum (as seen on higher magnification (E) of boxed area in panel B). In the striatum ipsilateral to the QA infusion, abundant Dcx+ and BrdU+/Dcx+ cells are present (C and F). Scale bar is 200 Am for A and B, 95 Am for C, 65 Am for D and E, and 30 Am for F. (G – L) Orthogonal reconstructions from confocal z-series of a migratory neuroblast (G – I) and a neuroblast with more mature morphology (J – L) double-labeled with BrdU (red; G and J) and Dcx (green; H and K) 2 weeks after QA infusion. I and L show the merged images. Scale bar in L is 20 Am for G – L. (M) Total number of Dcx+ cells (broken lines) and BrdU+/Dcx+ cells (solid lines) in the ipsilateral and contralateral striatum in intact rats and at 2 weeks following QA or saline infusion with BrdU injections twice daily until perfusion. Data are means T SEM; *P < 0.05 compared to intact and sham (Dcx+ cells and BrdU+/Dcx+ cells on the ipsilateral side and BrdU+/Dcx+ cells on the contralateral side); +P < 0.05 compared to intact (Dcx+ cells on the contralateral side); one-way ANOVA with Bonferroni/Dunn post hoc test; .P < 0.05 compared to contralateral side (both Dcx+ cells and BrdU+/Dcx+ cells), paired t test. n = 6 for all groups.

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Injury-induced neuroblasts and mature neurons have different locations and their number is related to extent of damage In order to explore whether there was a preferential location of the new cells at different time points after their

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formation, we determined the density of these cells in the damaged vs. non-damaged parts of the striatum at 2 and 6 weeks after QA infusion. At both time points, the majority of Dcx+ cells were found in the non-damaged parts of the striatum (Fig. 5A). The pattern was similar for the BrdU+/ Dcx+ and BrdU /Dcx+ cells. In contrast, the new cells

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was found for mature neurons (BrdU+/NeuN+) at 6 weeks after the QA infusion (Fig. 6B; r = 0.919; P = 0.0251). In contrast, we observed no correlation between the size of the lesion and the magnitude of cell proliferation in the ipsilateral SVZ at 2 weeks (r = 0.119; P = 0.8357). Excitotoxic striatal injury generates mature striatal neurons of different phenotypes

Fig. 4. Neuroblasts generated by striatal injury become mature neurons. (A – C) Orthogonal reconstructions from confocal z-series showing two cells double-labeled with BrdU (red; A) and NeuN (green; B) 6 weeks after QA infusion. Merged image in panel C. Scale bar in C is 10 Am. (D) Number of BrdU+/NeuN+ cells in the striatum ipsilateral and contralateral to an intrastriatal QA infusion performed 6 weeks earlier. BrdU injections were given twice daily during the first 2 weeks after QA infusion. Data are means T SEM; *P < 0.05 compared to contralateral side, paired t test. n = 5.

exhibiting a mature neuronal phenotype (BrdU+/NeuN+) were significantly more frequent within as compared to outside the lesioned area at both 2 and 6 weeks (Fig. 5B). To further delineate the preferential distribution of the new neurons, we plotted the location of the BrdU+/NeuN+ cells in the striatum at 6 weeks following the QA infusion and then measured the distance to the border of the damaged area. As illustrated in Fig. 5C, the majority of the new cells (72.1 T 8.3%) were found in the outer part of the damaged area, within 0 –0.75 mm from the border of the lesion. We then explored whether the magnitude of injuryinduced striatal neurogenesis was dependent on the size of the lesion caused by QA. At 2 weeks, the number of immature neurons (Dcx+) in the striatum correlated significantly to the size of the damage (Fig. 6A; Pearson’s correlation: r = 0.828; P = 0.0409), and a similar correlation

Finally, we wanted to determine whether the new neurons generated by the QA lesion differentiated into various phenotypes of striatal neurons. The vast majority of neurons in the intact striatum are GABAergic medium spiny projection neurons (Kawaguchi et al., 1995). In addition, four major classes of striatal interneurons can be distinguished, i.e., ChAT interneurons, GABAergic interneurons containing either PARVor calretinin, and finally interneurons containing somatostatin, NPY, and nitric oxide synthase, which probably also are GABAergic (Kawaguchi et al., 1995). We used DARPP-32 as marker for medium spiny neurons and PARV, NPY, and ChAT as markers for different striatal interneurons. The number of cells co-expressing BrdU and any of these markers at 6 weeks after the QA lesion were counted using epifluorescence microscopy. Doublelabeling was then validated in a confocal microscope. BrdU+ cells clearly expressing DARPP-32, PARV, or NPY were detected in the damaged striatum (Fig. 7), whereas we could not find any BrdU+/ChAT+ cells. The number of BrdU+/ DARPP-32+, BrdU+/PARV+ and BrdU+/NPY+ cells corresponded to 6.4 T 1.8%, 5.4 T 3.0%, and 1.0 T 0.5%, respectively, of the total number of BrdU+/NeuN+ cells.

Discussion This study shows that unilateral infusion of QA into the adult rat striatum, causing extensive neuronal loss in this structure, leads to increased cell proliferation in the SVZ, and migration of newly formed neuroblasts into the damaged area, where they start to express markers of several classes of mature striatal neurons. Striatal neurogenesis has previously been demonstrated to be triggered by ischemic damage to the striatum (Arvidsson et al., 2002; Parent et al., 2002). Three main mechanisms with overlapping and redundant features are believed to underlie cell death during ischemic brain injury: excitotoxicity and ionic imbalance, oxidative and nitrosative stress, and apoptoticlike processes (for a review, see Lo et al., 2003). The present findings indicate that striatal lesions caused by excitotoxicity using QA, an agonist of the N-methyl-d-aspartate subtype of glutamate receptors, induce striatal neurogenesis comparable to that we previously observed after stroke (Arvidsson et al., 2002). It is unlikely, therefore, that the formation of new neurons in the striatum caused by striatal injury is triggered by mechanisms specific for ischemic damage.

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Fig. 5. Neuroblasts and mature neurons generated by striatal injury have different location in damaged vs. non-damaged parts of striatum. (A – B) Density of Dcx+ and BrdU+/Dcx+ cells (A) and of BrdU+/NeuN+ cells (B) in the striatum ipsilateral to QA infusion after 2 and 6 weeks survival. BrdU injections were given twice daily during the first 2 weeks following QA infusion. Data are means T SEM; *P < 0.05 compared to damaged tissue, paired t test. (C) Distribution of BrdU+/NeuN+ cells in the striatum at 6 weeks after QA infusion, as plotted in segments of 0.25 mm, where the border of the damage is set at 0. Data are means T SEM; n = 6 for 2 week group, and n = 5 for 6 week group.

Several lines of experimental evidence indicate that the majority of the new striatal neurons generated by the excitotoxic lesion originated in the SVZ and migrated towards and into the damaged striatum. First, we observed that striatal neurogenesis was associated with a marked increase of cell proliferation in this neurogenic region. Similar to unilateral MCAO (Jin et al., 2001) and traumatic injury to cerebral cortex (Tzeng and Wu, 1999), we found that

the QA lesion stimulated SVZ proliferation bilaterally but mainly on the ipsilateral side. Consistent with our data, Tattersfield et al. (2004) recently reported that unilateral intrastriatal QA infusion gave rise to increased cell proliferation in the ipsilateral SVZ as compared to both the contralateral side and to sham-operated animals. Second, the location of the new neurons at the two time points analyzed here suggested that the cells gradually moved away

Fig. 6. Extent of striatal injury influences magnitude of neurogenesis in striatum. (A – B) Relationship between number of (A) Dcx+ cells at 2 weeks or (B) BrdU+/NeuN+ cells at 6 weeks after QA infusion and size of damage in the striatum ipsilateral to an intrastriatal QA infusion performed 2 and 6 weeks earlier, respectively. Each animal is plotted individually.

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Fig. 7. Excitotoxic injury generates different phenotypes of mature striatal neurons. (A – I) Orthogonal reconstructions from confocal z-series of cells doublelabeled with BrdU (red; A, D, G) and DARPP-32 (green; B; marker of striatal medium spiny projection neurons), PARV (green; E; marker of parvalbumincontaining interneurons), or NPY (green; H; marker of neuropeptide Y-containing interneurons) in the damaged striatum at 6 weeks after QA infusion. Panels C, F, and I show the merged images. Scale bar in I is 5 Am for A – C and 10 Am for D – I.

from the SVZ. Thus, at 6 weeks the new mature neurons (BrdU+/NeuN+) were localized preferentially within the lesion. In contrast, at 2 weeks the immature neurons (BrdU+/ Dcx+) were found mainly in the non-damaged striatum, closer to the SVZ. Third, Jin et al. (2003) previously observed migration of stroke-generated Dcx+ neuroblasts from the SVZ into striatum. Finally, Chmielnicki et al. (2004) have demonstrated that the new striatal neurons generated in the intact brain by adenoviral overexpression of BDNF and noggin in the ventricular wall do not arise from progenitors in the striatal parenchyma but their migration can be tracked from the SVZ into the striatum. At 2 weeks after the QA infusion, about 58% of the Dcx+ neuroblasts were also labeled with BrdU. We have estimated (Arvidsson, Kokaia, and Lindvall, unpublished), based on data obtained after suppression of cell proliferation with the antimitotic drug Ara-C, that the present BrdU injection paradigm labels about 80% of all new cells formed after stroke during the same time period. If this is the case also following QA, our data indicate that about 70% of the total number of Dcx+ cells had been generated by QA-triggered cell proliferation. However, similar to after

stroke (Arvidsson et al., 2002), a substantial number (about 30%) of the Dcx+ neuroblasts may derive from cells which had undergone division before the QA infusion and were then recruited to the damaged area. Interestingly, our data suggest that the striatal neurogenesis continues beyond the first 2 weeks after the induction of the excitotoxic lesion. Thus, at 6 weeks, the number of Dcx+ striatal cells in the damaged striatum was still substantially increased although very few of these cells were co-labeled with BrdU. It has been shown that the number of BrdU+ cells expressing Dcx in the olfactory bulb decreases to very low levels by 4 weeks after BrdU injection and remain virtually undetectable thereafter (Brown et al., 2003). Based on these observations, it seems likely that the Dcx+ cells observed at 6 weeks after QA had been generated after the 2-week BrdU injection period. The number of Dcx+ and BrdU+/NeuN+ cells was dramatically higher in the striatum on the side of the damage as compared to the contralateral side. The mechanisms underlying this preferential migration of the newly formed neuroblasts into the injured striatal parenchyma are unknown. It is conceivable that the adenoviral overexpres-

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sion of BDNF and noggin in the ventricular wall performed by Chmielnicki et al. (2004) not only induced increased formation of new neurons in the SVZ but also made the striatal parenchyma permissive to neuroblast migration. A similar mechanism may operate following striatal injury caused by stroke or QA. However, the migration of the new neurons away from the SVZ and towards the lesion, and their aggregation to the border area between the damaged and non-damaged striatum strongly indicate the presence of attracting molecule(s). Similarly, transplanted stem cells migrate to the site of an ischemic lesion (Veizovic et al., 2001) or tumor (Aboody et al., 2000). Identification of these molecules could lead to the development of tools to efficiently direct neuroblasts generated in the SVZ and intended for neuronal replacement to a damaged area. We observed that the size of the damage in the QAinfused striatum correlated significantly to both the number of Dcx+ neuroblasts at 2 weeks and of mature BrdU+/ NeuN+ neurons at 6 weeks after the insult. Thus, our data indicate that the extent of a lesion in the parenchyma of the striatum is a major factor in determining the magnitude of the resulting striatal neurogenesis. In support of this hypothesis, we have in parallel experiments observed that longer duration of MCAO, causing more extensive striatal damage, triggers more marked striatal neurogenesis as compared to brief ischemic insults (Thored et al., in preparation). In the present study, no correlation was found between the size of the striatal damage and the magnitude of cell proliferation in the ipsilateral SVZ. Taken together, our findings suggest therefore that the extent of injury regulates neurogenesis predominantly through mechanisms involved in the recruitment of neuroblasts into the striatal parenchyma and not by influencing SVZ cell proliferation. The present data also underscore the importance of exploring whether the observed effects are mediated indirectly through alterations of the extent of striatal damage when assessing the influences on neurogenesis of various experimental manipulations such as drug administration. We have estimated that following a stroke, about 80% of the new striatal BrdU+/Dcx+ cells detected at 2 weeks have died at 6 weeks after the insult, as evidenced by the number of BrdU+/NeuN+ cells (Arvidsson et al., 2002). When we made the same comparison between cell numbers following the QA-induced damage, the loss of the new neurons was found to be markedly lower (only about 20%). The cause of the major degeneration of newly generated striatal neurons after stroke is not known but probably reflects an unfavorable environment with lack of trophic support and inflammation (Ekdahl et al., 2003; Monje et al., 2003). Our findings suggest that the environment is less hostile to the new neurons after a QA lesion as compared to MCAO, although both insults are accompanied by inflammation (Duan et al., 1998; Stoll et al., 1998). Comparisons between the stroke and QA models should be useful to reveal those mechanisms which are important for compromising cell survival during insult-induced neurogenesis.

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At 6 weeks after the QA-lesion, a substantial number of BrdU+ cells were double-labeled with the mature neuronal marker NeuN. Similarly, Tattersfield et al. (2004) recently reported the presence of BrdU+/NeuN+ cells at 37 days following intrastriatal QA infusion. Here we have extended these findings by demonstrating, using confocal microscopy, cells clearly double-labeled with BrdU and either DARPP-32, PARV, or NPY. Our findings show for the first time that striatal injury can trigger the formation of several classes of new neurons, expressing phenotypic markers of striatal medium spiny neurons and interneurons, respectively. In our previous study (Arvidsson et al., 2002), we estimated that 42% of the stroke-generated new neurons were DARPP-32+. Similarly, Parent et al. (2002) reported that a majority of new neurons in the peri-infarct area after stroke were DARPP-32+. The present data indicated that the fraction of BrdU+/NeuN+ cells expressing DARPP-32 after the QA lesion was clearly lower (6.4 T 1.8%). We also observed a higher proportion of mature neurons (BrdU+/ NeuN+) not expressing any striatum-specific neuronal marker following QA infusion (87%) than after MCAO (58%). Our data suggest that the maturation of the new neurons into specific striatal phenotypes is regulated by signals in the tissue environment. The relative levels of such signals could differ between insults. In support of this idea, we observed that the QA-induced damage gave rise to a significant number of cells which were double-labeled with BrdU and PARV, and a minor fraction co-expressing BrdU and NPY. Such cells were completely absent following MCAO in rats (Parent et al., 2002). In contrast, Teramoto et al. (2003) observed no BrdU+/DARPP-32+ cells but a substantial number of BrdU+/PARV+ cells after stroke in mice. Interestingly, we found that the excitotoxic injury to the adult striatum triggered the formation of cells with the phenotype of both striatal projection neurons and interneurons. These neuron types, which are all damaged by QA infusion, have different origins during embryonic development, i.e., lateral and medial ganglionic eminence, respectively (Marin et al., 2000). Whether the neuronal phenotypes generated in the adult brain arise from the same precursor population in the SVZ or if projection neurons and interneurons maintain separate origins within the SVZ is currently unknown.

Acknowledgments This work was supported by the Swedish Research Council, EU grant for project LSHBCT-2003-503005 (EUROSTEMCELL), the So¨derberg Foundation, and the Kock, Crafoord, Elsa and Thorsten Segerfalk, and King Gustav V and Queen Victoria Foundations and. The Lund Stem Cell Center is supported by a Center of Excellence grant in life science from the Swedish Foundation for Strategic Research.

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References Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., Snyder, E.Y., 2000. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U. S. A. 97, 12846 – 12851. Abrahams, J.M., Gokhan, S., Flamm, E.S., Mehler, M.F., 2004. De novo neurogenesis and acute stroke: are exogenous stem cells really necessary? Neurosurgery 54, 150 – 156. Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis: IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433 – 457. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O., 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963 – 970. Brown, J.P., Couillard-Despres, S., Cooper-Kuhn, C.M., Winkler, J., Aigner, L., Kuhn, H.G., 2003. Transient expression of doublecortin during adult neurogenesis. J. Comp. Neurol. 467, 1 – 10. Chmielnicki, E., Benraiss, A., Economides, A.N., Goldman, S.A., 2004. Adenovirally expressed noggin and brain-derived neurotrophic factor cooperate to induce new medium spiny neurons from resident progenitor cells in the adult striatal ventricular zone. J. Neurosci. 24, 2133 – 2142. Duan, W.M., Widner, H., Cameron, R.M., Brundin, P., 1998. Quinolinic acid-induced inflammation in the striatum does not impair the survival of neural allografts in the rat. Eur. J. Neurosci. 10, 2595 – 2606. Ekdahl, C.T., Claasen, J.H., Bonde, S., Kokaia, Z., Lindvall, O., 2003. Inflammation is detrimental for neurogenesis in adult brain. Proc. Natl. Acad. Sci. U. S. A. 100, 13632 – 13637. Jin, K., Minami, M., Lan, J.Q., Mao, X.O., Batteur, S., Simon, R.P., Greenberg, D.A., 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc. Natl. Acad. Sci. U. S. A. 98, 4710 – 4715.

Jin, K., Sun, Y., Xie, L., Peel, A., Mao, X.O., Batteur, S., Greenberg, D.A., 2003. Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum. Mol. Cell. Neurosci. 24, 171 – 189. Kawaguchi, Y., Wilson, C.J., Augood, S.J., Emson, P.C., 1995. Striatal interneurones: chemical, physiological and morphological characterization. Trends. Neurosci. 18, 527 – 535. Lo, E.H., Dalkara, T., Moskowitz, M.A., 2003. Mechanisms, challenges and opportunities in stroke. Nat. Rev., Neurosci. 4, 399 – 415. Lois, C., Alvarez-Buylla, A., 1994. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145 – 1148. Marin, O., Anderson, S.A., Rubenstein, J.L., 2000. Origin and molecular specification of striatal interneurons. J. Neurosci. 20, 6063 – 6076. Monje, M.L., Toda, H., Palmer, T.D., 2003. Inflammatory blockade restores adult hippocampal neurogenesis. Science 302, 1760 – 1765. Parent, J.M., Vexler, Z.S., Gong, C., Derugin, N., Ferriero, D.M., 2002. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann. Neurol. 52, 802 – 813. Paxinos, G., Watson, C., 1997. The Rat Brain in Stereotaxic Coordinates, Compact Third edR Academic Press. Stoll, G., Jander, S., Schroeter, M., 1998. Inflammation and glial responses in ischemic brain lesions. Prog. Neurobiol. 56, 149 – 171. Tattersfield, A.S., Croon, R.J., Liu, Y.W., Kells, A.P., Faull, R.L., Connor, B., 2004. Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington’s disease. Neuroscience 127, 319 – 332. Teramoto, T., Qiu, J., Plumier, J.C., Moskowitz, M.A., 2003. EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia. J. Clin. Invest. 111, 1125 – 1132. Tzeng, S.F., Wu, J.P., 1999. Responses of microglia and neural progenitors to mechanical brain injury. NeuroReport 10, 2287 – 2292. Veizovic, T., Beech, J.S., Stroemer, R.P., Watson, W.P., Hodges, H., 2001. Resolution of stroke deficits following contralateral grafts of conditionally immortal neuroepithelial stem cells. Stroke 32, 1012 – 1019. Zhang, R.L., Zhang, Z.G., Zhang, L., Chopp, M., 2001. Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia. Neuroscience 105, 33 – 41.