Midbrain Dopamine Neurons Associated with Reward Processing Innervate the Neurogenic Subventricular Zone

13078 • The Journal of Neuroscience, September 14, 2011 • 31(37):13078–13087

Development/Plasticity/Repair Midbrain Dopamine Neurons Associated with Reward Processing Innervate the Neurogenic Subventricular Zone

Jessica B. Lennington,1,2 Sara Pope,1,2 Anna E. Goodheart,1 Linda Drozdowicz,1 Stephen B. Daniels,1 John D. Salamone,3 and Joanne C. Conover1,2 1Department of Physiology and Neurobiology, 2Center for Regenerative Biology, and 3Department of Psychology, University of Connecticut, Storrs, Connecticut 06269

Coordinated regulation of the adult neurogenic subventricular zone (SVZ) is accomplished by a myriad of intrinsic and extrinsic factors. The neurotransmitter dopamine is one regulatory molecule implicated in SVZ function. Nigrostriatal and ventral tegmental area (VTA) midbrain dopamine neurons innervate regions adjacent to the SVZ, and dopamine synapses are found on SVZ cells. Cell division within the SVZ is decreased in humans with Parkinson’s disease and in animal models of Parkinson’s disease following exposure to toxins that selectively remove nigrostriatal neurons, suggesting that dopamine is critical for SVZ function and nigrostriatal neurons are the main suppliers of SVZ dopamine. However, when we examined the aphakia mouse, which is deficient in nigrostriatal neurons, we found no detrimental effect to SVZ proliferation or organization. Instead, dopamine innervation of the SVZ tracked to neurons at the ventrolateral boundary of the VTA. This same dopaminergic neuron population also innervated the SVZ of control mice. Characterization of these neurons revealed expression of proteins indicative of VTA neurons. Furthermore, exposure to the neurotoxin MPTP depleted neurons in the ventrolateral VTA and resulted in decreased SVZ proliferation. Together, these results reveal that dopamine signaling in the SVZ originates from a population of midbrain neurons more typically associated with motivational and reward processing.

Introduction borders the dorsolateral SVZ. These neurons mediate sensorimo- Neurogenesis in the adult brain occurs primarily in two stem cell tor function (DeLong et al., 1983). The ventral SVZ is bordered niches, the dentate gyrus of the hippocampus and the subventricular by the nucleus accumbens, which is innervated by A10 dopamine zone (SVZ) along the lateral wall of the lateral ventricle (Altman and neurons originating from the ventral tegmental area (VTA). Das, 1966; Hinds, 1968; Cameron and Gould, 1994; Eriksson et al., These neurons mediate affect, reward, and motivational learning 1998; Gould et al., 1999). Here we focus on the SVZ, which through- (Montague et al., 1996; Hollerman and Schultz, 1998). Injection out adulthood generates immature neurons that migrate to the of neuronal tracers into the midbrain of nonhuman primates olfactory bulb, integrate into the existing neuronal network, and revealed organized, topographical projections of midbrain dopa- function in odor perception discrimination (Lois and Alvarez- mine neurons to the SVZ (Freundlieb et al., 2006). Additionally, Buylla, 1993; Doetsch et al., 1999; Be´dard and Parent, 2004; Curtis et it has been shown that SVZ cells possess dopamine receptors and al., 2007). Several molecules released by cells within, and adjacent to, dopaminergic synapses (Ho¨glinger et al., 2004). Based on studies the SVZ have regulatory roles in SVZ neurogenesis, including showing declines in SVZ proliferation in Parkinson’s disease pa- growth factors such as FGF, EGF, and VEGF, and several neu- tients and following A9-selective lesioning in rodents, the origin rotransmitters, notably GABA, glutamate, and 5-HT (for review, see of midbrain inputs to the SVZ was thought to be SNpc neurons Lennington et al., 2003; Pathania et al., 2010; Conover and Shook, (Baker et al., 2004; Ho¨glinger et al., 2004). However, others have 2011). Another neurotransmitter, dopamine, has been reported to proposed that A10 VTA afferents, in addition to innervating the stimulate proliferation within the SVZ (Ho¨glinger et al., 2004), but neighboring nucleus accumbens, extend into the ventromedial the origin and role of dopamine afferents in the SVZ are still unclear. striatum immediately adjacent to the SVZ (Bjo¨rklund and Dun- A9 nigrostriatal dopamine neurons originating from the sub- nett, 2007). Innervation by the VTA would implicate the motiva- stantia nigra pars compacta (SNpc) innervate the striatum, which tional reward system in olfactory neurogenesis, versus the sensorimotor system as currently reported. To date, the molecular signature and projection targets of midbrain dopaminergic Received March 8, 2011; revised June 21, 2011; accepted July 22, 2011. neuron populations and their contribution to SVZ neurogenesis Author contributions: J.B.L., S.P., J.D.S., and J.C.C. designed research; J.B.L., S.P., A.E.G., L.D., and S.B.D. performed research; J.B.L., S.P., and J.C.C. analyzed data; J.B.L., J.D.S., and J.C.C. wrote the paper. have not been fully investigated. J.C.C. was supported by NINDS Grant NS05033. J.B.L. was supported in part by the Parkinson’s Disease Founda- To examine the origin and role of dopamine signaling in the tion. We thank Drs. K. S. Kim and D. Y. Hwang (McLean Hospital, Harvard Medical School) for the ak mice. SVZ, we evaluated the aphakia (ak) mouse, a genetic model for The authors declare no competing financial interests. dopaminergic neuron loss and an alternative to standard chemi- Correspondence should be addressed to Joanne C. Conover, Department of Physiology and Neurobiology, Uni- versity of Connecticut, 75 North Eagleville Road, Unit 3156, Storrs, CT 06269. E-mail: [email protected]. cal methods that produce acute lesions of dopaminergic neurons. DOI:10.1523/JNEUROSCI.1197-11.2011 During ak development, midbrain dopamine neurons are born, Copyright © 2011 the authors 0270-6474/11/3113078-10$15.00/0 but up to 90% of SNpc neurons are lost by birth, resulting in Lennington et al. • VTA Innervates the SVZ J. Neurosci., September 14, 2011 • 31(37):13078–13087 • 13079

by the Institutional Animal Care and Use Committee of the University of Connecticut and conformed to National Institutes of Health guidelines. Immunohistochemistry. Mice were perfused transcardially with 0.9% saline followed by 3% paraformaldehyde in PBS. Brains were fixed overnight in 3% paraformaldehyde at 4°C and then washed in PBS before cutting 50 ␮m sections with a vibratome (VT-1000S, Leica). Free-floating sections were washed in 0.1% Triton X-100 (Sigma) in PBS three times for 10 min, blocked in 10% donkey serum (Sigma) in PBS/0.1% Triton X-100 for 1 h, and incubated with the following primary antibodies: anti- bromodeoxyuridine (BrdU, 5 ␮g/ml, cat. #OBT0030, Accurate Chemical and Scientific Corporation), anti-Ki-67 (1:500, MAB377, Millipore), anti-doublecortin (DCX, 1 ␮g/ml, cat. #SC-8066, Santa Cruz Biotechnology), anti-dopamine transporter (DAT, 1:400, cat. #MAB369, Millipore Bioscience Research Re- agents; Triton X-100 exposure in blocking phase only); anti-Caspase3 (6 ␮g/ml, cat. #AF355, R&D Systems), anti-GABA (44 ␮g/ ml, #55545, Sigma), anti-tyrosine hydroxylase (1:500, cat. #ab113, AbCam), anti-Otx2 (1:400, cat. #AB9566, Millipore Bioscience Research Reagents), anti-Calbindin (1:500, cat. #C9848, Sigma), and anti-GIRK2 (1:500, cat. #APC- 006, Alomone Labs). Sections were incubated with appropriate Alexa Fluor dye-conjugated secondary antibodies (Invitrogen) for1hand washed three times for 10 min in PBS. Second- ary antibodies alone were used as a control. Mounted sections were washed for 5 min in PBS and coverslipped using Aqua-Poly/Mount (Polysciences). Bromodeoxyuridine immunohistochemistry. Mice were injected with 300 mg of BrdU/kg 2 h before perfusion for 2 h pulse studies. This concentration was previously determined to Figure1. Dopamineafferentsareseverelydepletedinthedorsalstriatum,butretainedwithintheSVZofakmice.a,Schematic label the maximal number of S-phase cells ϩ ϩ indicating area evaluated. Boxes indicate position of dorsal and ventral regions of the SVZ used in d–g. b,TH /DAT afferents (Cameron and McKay, 2001). BrdU immuno- ϩ ϩ (purple) are observed in the dorsal and ventral striatum and along the SVZ in C57BL/6 control mice. c,TH /DAT afferents are staining of 50 ␮m sections was conducted as ϩ severelyreducedinthedorsalstriatum,butretainedintheregionofthenucleusaccumbensinakmousebrain.BrdU cells(green) described previously (Luo et al., 2006) and an- define the SVZ, which is located along the lateral wall of the lateral ventricle. d–g, High-magnification images of the SVZ (left of alyzed by quantifying BrdU ϩ cells in 18 con- ϩ dottedline)fromcontrolandakmicerevealedthatTH afferents(purple,arrows)arepresentinthedorsal(d,e)andventral(f, g) secutive anterior forebrain sections from SVZ.h,AverageintensityofTHissignificantlyreducedinakmiceinthestriatum(C57BL/6n ϭ 4,aknϭ 4,p ϭ 0.000232), coordinates 0.5–1.4 mm anterior, relative to but not in the dorsal SVZ or ventral SVZ. lv, Lateral ventricle; Str, striatum; NAc, nucleus accumbens. Scale bars: b, c, 250 bregma. ␮m; d–g,50␮m. Intensity analysis. Average intensities of tyrosine hydroxylase (TH) were calculated using severe depletion of dopaminergic innervation of the striatum. ImageJ by sampling a 28 ϫ 28 pixel area, in the VTA dopaminergic neuron projections to the nucleus accum- dorsal and ventral SVZ, as well as in the corpus callosum and striatum, in ϫ bens are only modestly affected (Hwang et al., 2003; Nunes et al., 40 images taken from four consecutive sections from coordinates 2003; van den Munckhof et al., 2003). Since ak mice have normal 1.10–1.25 mm anterior, relative to bregma. Values are reported as average intensity above background (corpus callosum) Ϯ SD. lifespans, we could examine whether loss of SNpc neuron- Whole mounts. The entire lateral wall of the lateral ventricle was pre- generated dopamine affected SVZ organization and function. pared as previously described and immunostained for doublecortin Here, we report that a subpopulation of dopaminergic neurons (DCX, Santa Cruz Biotechnology) (Luo et al., 2006). Whole mounts were with characteristics more typically associated with VTA neurons trimmed, placed onto glass slides, coverslipped with Aqua-Poly/Mount is the primary supplier of dopamine to the SVZ in ak as well as (Polysciences), and imaged by epifluorescence microscopy. control mice. Neuronal tracing. Mice were anesthetized with 1.5% isoflurane in 2

L/min O2 flow while placed in a stereotaxic frame. Mice received a ste- Materials and Methods reotaxic injection of 1 ␮l of 2 mg/ml wheat germ agglutinin conjugated to Animals. Male ak mice were provided by Drs. Kwang-Soo Kim and FITC (WGA-FITC; Sigma L-4895) and transport was analyzed after 5–7 Dong-Youn Hwang (McLean Hospital, Harvard Medical School, Bel- d. For anterograde transport analysis, WGA-FITC was injected into the mont, MA). Male C57BL/6 mice were purchased from The Jackson Lab- ventrolateral VTA region of the midbrain (Ϫ3.08 mm caudal, 0.4 mm oratory. Animal procedures were performed under protocols approved lateral, 4.6 mm ventral, relative to bregma). For retrograde analysis, 13080 • J. Neurosci., September 14, 2011 • 31(37):13078–13087 Lennington et al. • VTA Innervates the SVZ

WGA-FITC was injected into the lateral ventricle (0.0 mm caudal, 0.9 mm lateral, 2.3 mm ventral, relative to bregma). We confirmed that 5–7 d was sufficient for retrograde transport of FITC via TH ϩ afferents to the midbrain. In addition, our observation of FITC ϩ cells in the raphe´nucleus after 5–7 d, via retrograde transport by serotonergic neurons known to innervate the region of the SVZ (Lorez and Richards, 1982), served as a positive control. The extent of WGA-FITC diffusion into brain tissue following injection into the lateral ventricle was analyzed at 6 and 24 h after injection in the coronal sections containing the lateral and third ventricles. Needle insertion into the cortex (0.0 mm caudal, 0.9 mm lateral, 1.0 mm ventral, relative to bregma) revealed that 5–7 d later no WGA-FITC tracked to TH ϩ cells in the VTA. In addition to imaging for WGA-FITC, 50 ␮m sections were immunostained with antibodies for tyrosine hydroxylase, Otx2, Calbindin, and GIRK2. In the midbrain, FITC ϩ cells in six sections from Ϫ2.85 to Ϫ3.56 mm, relative to bregma, were counted and evaluated for coexpression of TH with Otx2, Calbindin, or GIRK2. Results are reported as percentage of the total FITC ϩ cells. MPTP lesioning. Mice received four intraperitoneal injections of 10 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Sigma) or 0.9% saline at 2 h intervals followed by eight intraperitoneal injections of 50 mg/kg BrdU, at 6 h intervals, as previously described (Ho¨glinger et Figure 2. SNpc A9 neuron deficiency does not affect proliferation in the SVZ of ak mice. Cell al., 2004). Animals were analyzed 3 d after the first MPTP injection. proliferation was quantified in the SVZ followinga2hpulse BrdU injection. a–c, Based on Midbrain analysis. SNpc and VTA midbrain neurons were character- incorporation of BrdU (green nuclei), levels of cell proliferation were similar in control and ak ized by TH, Otx2, Calbindin, and GIRK2 expression, and counted using mice in both the dorsal and ventral SVZ. Ac, Anterior commissure; lv, lateral ventricle; Str, stereological methods. SNpc and VTA regions in every third section from striatum; NAc, nucleus accumbens. Scale bars: a, b, 200 ␮m. Ϫ2.85 to Ϫ3.56 mm relative to bregma were traced and cells were counted using MicroBrightField stereological algorithms and settings neuron afferents were significantly reduced in the striatum of ak as follows: counting frames were 50 ϫ 50 in a 150 ϫ 150 grid. Fifty- mice, compared to C57BL/6 control mice (Fig. 1a–c). However, micrometer sections (cut thickness) were used and safety zones were set to exclude edge areas such that 30 ␮m in the center of the section were high-powered magnification revealed that afferents containing counted. Results are reported based on mean measured thickness. TH, the rate-limiting enzyme in the production of dopamine, Microscopy and statistical analysis. Sections were imaged on an Axios- were still found in the region of the SVZ in ak mice (Fig. 1d–h). ϩ kop 2 mot plus microscope (Carl Zeiss MicroImaging), using a Retiga Specifically, we found that TH neuronal afferents remained in 1300 EX digital camera (Q-Imaging), on a TCS SP2 confocal laser both the dorsal and ventral regions of the SVZ in ak mice. scan microscope (Leica), or on an Axio Imager M2 with Apotome (Carl Zeiss MicroImaging) using a Orca R2 (Hamamatsu). Statistical SNpc dopamine neuron loss does not affect SVZ cell Ϯ results are reported as mean SD. Differences in means were assessed proliferation or niche organization using a two-tailed unpaired Student’s t test. The level of significance We next examined whether the severe reduction in A9 nigrostri- was set at p Ͻ 0.05. atal dopamine afferents affected SVZ proliferation in the ak mouse. Based on incorporation of the thymidine analog BrdU, Results we found that the number of dividing cells in the SVZ was not Dopaminergic afferents are retained in the SVZ following loss significantly different in 3-month-old ak compared to C57BL/6 of SNpc neurons control mice (C57BL/6 n ϭ 10; ak n ϭ 16; p ϭ 0.34) (Fig. 2a–c). Several studies, including investigations of patients with Parkin- Similarly, no difference in proliferation levels was observed at son’s disease, have correlated SNpc A9 neuron loss with de- either P7 or 6 months of age in ak compared to control mice (data creased SVZ proliferative function (Baker et al., 2004; Ho¨glinger not shown). As BrdU incorporation can also occur in dying cells, et al., 2004; O’Keeffe et al., 2009). However, VTA A10 dopami- we quantified expression of caspase-3 and found that cell death nergic neurons are also thought to innervate regions adjacent to levels were not significantly different in the SVZ of ak mice com- the SVZ (Bjo¨rklund and Dunnett, 2007). To determine the re- pared with controls (C57BL/6 31.83 Ϯ 13.50, n ϭ 6; ak 21.17 Ϯ spective contributions of A9 and A10 midbrain dopaminergic 10.91, n ϭ 6; p ϭ 0.163). neuron populations to the innervation and regulation of the SVZ, We also evaluated whether the absence of nigrostriatal A9 we evaluated the ak mouse strain, which is selectively deficient in dopaminergic neurons affected either SVZ organization or the A9 midbrain neurons (Hwang et al., 2003; Nunes et al., 2003). migratory capacity of newly generated neuroblasts. Transmission The ak mouse has greatly reduced expression of Pitx3, a homeo- electron microscopy (TEM) was used to examine the cytoarchi- box transcription factor that facilitates the survival and differen- tectural organization of the SVZ in ak and control mice. Micro- tiation of A9 dopamine neurons (Semina et al., 1997; Hwang et graph montages were prepared from 3-month-old ak and control al., 2003; Nunes et al., 2003; van den Munckhof et al., 2003, 2006; mice, and cell types in the SVZ were identified and counted (Fig. Baker et al., 2004; Maxwell et al., 2005; Amano et al., 2009; Papa- 3a–c) (Luo et al., 2006). The SVZ contains four main cell types: nikolaou et al., 2009). Analysis of the midbrain of ak mice shows astrocytes, a subset of which are stem cells; transit-amplifying early degeneration of midbrain dopamine neurons from E14.5 progenitor (TAP) cells; neuroblasts, the highly migratory im- through postnatal day 0 (Smidt et al., 2004). Both A9 and A10 mature neurons; and ependymal cells that line the ventricles and dopaminergic neurons are born in the ak mouse embryo; how- form the apical border of the SVZ (Luskin, 1993; Doetsch et al., ever, nearly all A9 dopamine neurons die during late embryonic 1997). The numbers of neuroblasts, astrocytes, ependymal cells, development, while A10 neurons are relatively spared (Hwang et and transit-amplifying cells in the young adult ak SVZ were not al., 2003, 2009; Nunes et al., 2003). Similar to the work of others significantly different from C57BL/6 control mice (C57BL/6 n ϭ (Beeler et al., 2009), we found that TH ϩ and DAT ϩ dopamine 3; ak n ϭ 3; neuroblasts p ϭ 0.15, astrocytes p ϭ 0.18, ependymal Lennington et al. • VTA Innervates the SVZ J. Neurosci., September 14, 2011 • 31(37):13078–13087 • 13081

SNpc A9 neuron deficiency, ak mice re- tain full SVZ organization and function.

Dopaminergic neurons from the ventrolateral VTA innervate the SVZ Since previous studies implicated SNpc A9 neurons in providing dopamine critical for normal SVZ regulation (Baker et al., 2004; Ho¨glinger et al., 2004), we won- dered how the ak mouse, which is deficient in A9 dopamine neurons, was able to maintain and regulate SVZ functions. We determined that dopamine neuron afferents were still present in the SVZ of ak mice (Fig. 1), yet the precise identity of these innervating neurons was unclear. To determine their origin, WGA conjugated to FITC was injected unilaterally into the lateral ventricle, using stereotaxic coordinates, of both C57BL/6 control and ak mice (Fig. 4). Retrograde tracking of labeled neurons was performed 5–7 d later. In control C57BL/6 mice, WGA was transported via SVZ afferents to cell bod- ies found in a discrete midbrain region at the ventrolateral border of the VTA, adjacent to the SNpc (Fig. 4a,c). In A9- deficient ak mice, retrograde tracking from the SVZ revealed similar tracking of WGA-FITC to midbrain cells located at the ventrolateral border of the VTA, immediately adjacent to where the SNpc would typically be found (Fig. 4b,d). Based on transcriptome comparisons, several markers have been identified that Figure 3. SNpc A9 neuron deficiency does not affect the SVZ of adult ak mice. a, b, Representative electron micrograph are differentially expressed between the montages of the SVZ reveal the cellular composition and distribution of the four major cell types within the SVZ of C57BL/6 control A9 and A10 dopaminergic neuron popu- and ak mice. SVZ cells are pseudo-colored to indicate cell type. c, Quantification of each of the major cell types of the SVZ revealed lations. A10 neurons that populate the no significant difference between control and ak mice. d, Schematic indicating region of whole-mount preparation of lateral wall VTA express significantly higher levels of of the lateral ventricle (boxed area in gray region). e, f, Whole-mount preparations of doublecortin (DCX) immunolabeled neuro- ortho-denticle-related homeobox 2 blastchainsindicatenosignificantdifferenceinneuroblastchainorganizationincontrolversusakmice.g,Wholebrainsincluding (Otx2) and Calbindin, while A9 neurons olfactory bulbs from 3-month-old control and ak mice indicate no gross anomaly in ak brain morphology. h–k, Immunolabeling that populate the SNpc express higher with GABA and TH indicate no deficits in olfactory bulb organization in ak mice. Gl, Glomerular region; mi, mitral cell region; gr, levels of G-protein coupled inwardly rec- ␮ ␮ ␮ ␮ granule cell region; lv, lateral ventricle; Str, striatum. Scale bars: a, b,10 m; e, f, 200 m; h, i, 200 m; j, k, 100 m. tifying potassium channel 2 (GIRK2), aldehyde dehydrogenase 2 (AHD2), and glycosylated dopamine transporter cells p ϭ 0.11, transit-amplifying cells p ϭ 0.06). Similarly, no signif- (glyco-Dat) (C. Y. Chung et al., 2005, 2010; S. Chung et al., 2005; icant differences in SVZ organization were observed in ak versus Di Salvio et al., 2010). To define the neurons in the ventrolateral control mice at P7 or 6 months of age (data not shown). region of the VTA, we combined retrograde tracking with immu- Further analysis of SVZ functions included examination of the nohistochemical characterization in both C57BL/6 and ak mice organization of neuroblasts into chains. Chain migration is the (see Fig. 5a,b, schematic representation of midbrain region). Fol- mechanism by which neuroblasts migrate across the lateral ven- lowing 5–7 d of retrograde transport, quantification of FITC ϩ tricle wall, along the RMS, and into the olfactory bulb (Lois et al., cells in C57BL/6 control mice revealed that 96% of the FITC ϩ 1996; Wichterle et al., 1997). Neuroblasts were immunolabeled cells were TH ϩ. Of those FITC ϩ cells expressing TH, 46% coex- with doublecortin in whole-mount preparations of the lateral wall. pressed Otx2 (Fig. 5c–e, Table 1) and 22% coexpressed Calbindin The organization and number of chains of doublecortin ϩ neuro- (Fig. 5i–k, Table 1), while 41% coexpressed GIRK2 (Fig. 5o–q, blasts transiting the lateral ventricle wall in ak mice did not differ Table 1). In the ak mouse, quantification of the FITC ϩ cells fol- from those found in control mice (Fig. 3d–f). Morphological lowing 5–7 d of retrograde transport showed that 99% were observation revealed that the brains and, in particular, the olfac- TH ϩ, with 63% coexpressing Otx2 (Fig. 5f–h, Table 1), 31% tory bulbs of adult ak mice were comparable in size and morphol- coexpressing Calbindin (Fig. 5l–n, Table 1), and 39% coexpress- ogy to control mice (Fig. 3g). In addition, no gross abnormalities ing GIRK2 (Fig. 5r–t, Table 1). In the A9-deficient ak mouse, were observed in adult olfactory bulb organization or composi- these results indicate that the midbrain neurons that innervate tion (Fig. 3h–k). Together, these data demonstrate that despite an the SVZ include a mixed population of Otx2 ϩ, Calbindin ϩ, and 13082 • J. Neurosci., September 14, 2011 • 31(37):13078–13087 Lennington et al. • VTA Innervates the SVZ

GIRK2 ϩ neurons that reside along the ventrolateral border of the VTA. Our studies also emphasize that the classification of midbrain dopamine neurons into A9 and A10 populations based on expression of markers such as Calbindin or GIRK2 alone is overly simplified. In the SNpc-deficient ak mouse, we observed GIRK2 expression in the VTA consistent with previous studies that have found low levels of GIRK2 expression in A10 populations and low levels of Calbindin expression in A9 populations (C. Y. Chung et al., 2005). While relative levels of proteins such as GIRK2 and Calbindin are pres- ently used to identify A9 and A10 populations, there is not yet a distinct biomarker for either population (C. Y. Chung et al., 2005). To assess the extent to which WGA- FITC diffuses into regions neighboring the SVZ, we examined coronal sections of the anterior forebrain at the level of the lateral ventricle at 6 and 24 h after injection. At 6 h, WGA-FITC was largely restricted to the ependyma and the subependyma of the lateral ventricle (Fig. 6a,b). We also determined that WGA- FITC is restricted to the ependyma in sections including the third ventricle (Fig. 6c). By 24 h, WGA-FITC intensity decreased significantly along the walls of both the lateral and third ventricles due to ventricular clearance. Only very faint staining was evident beyond the SVZ (Fig. Figure4. TheSVZisinnervatedbydopaminergicneuronsoriginatingfromtheventrolateralregionoftheVTA.WGAconjugated 6d–f). This faint staining may represent to FITC was unilaterally injected into the lateral ventricle, and after 5–7 d, the midbrain was analyzed for WGA-FITC retrograde the initial transport of WGA-FITC by tracking.a,Incontrolmice,FITC-labeledneuronswerefoundintheventrolateralregionoftheVTAborderingtheSNpc,withsome SVZ afferents. These studies confirm that FITC ϩ cells in regions of the SNpc. b, Retrograde tracking of WGA-FITC from the SVZ of ak mice also revealed FITC ϩ cells in the WGA-FITC diffusion into tissue neigh- ventrolateral region of the VTA. (Note the absence of TH ϩ neurons in the area of the SNpc in ak mice.) c, d, Schematic of midbrain boring the SVZ is very limited. dopaminergic neuron populations that innervate the striatum and nucleus accumbens, modified from Bjo¨rklund and Dunnett (2007). Here we represent midbrain dopamine neuron populations that innervate the striatum, nucleus accumbens, and SVZ in Midbrain WGA-FITC injection results C57BL/6controlmice(c)andinakmice(d).Inakmice,SNpcinnervationofthestriatumisdepleted,whilemidbraindopaminergic in anterograde transport to the neuron innervation of the NAc and SVZ remains. ac, Anterior commissure; cc, corpus callosum; lv, lateral ventricle. Scale bars: a, b, ␮ subventricular zone 500 m. Anterograde transport experiments al- lowed us to track midbrain dopaminergic neuron projections to Reduction in SVZ proliferation following injection of MPTP has the SVZ, providing further support of our retrograde tracing been attributed to the removal of A9 nigrostriatal midbrain neu- studies. WGA-FITC was stereotaxically injected into the region rons (Ho¨glinger et al., 2004). Above, our results indicate that dopa- of the ventrolateral VTA of ak and control mice, and anterior minergic neuron tracts originating from a region at the ventrolateral forebrain sections were examined after 5–7 d. In C57BL/6 control border of the VTA innervate the SVZ. We sought to determine mice, analysis of WGA-FITC in anterior forebrain sections revealed whether, in addition to depleting SNpc cells, MPTP exposure also anterograde transport to both the dorsal and ventral SVZ (Fig. 6k,l). reduces neurons in this ventrolateral VTA region. We administered Similarly, in the SNpc-deficient ak mouse, anterograde transport of MPTP to ak and control mice in a series of intraperitoneal injections. WGA-FITC resulted in labeled afferents in the dorsal and ventral BrdU was injected immediately after the final MPTP injection (four SVZ (Fig. 6m,n). Together, these data reveal that a population of injections at 6 h intervals) to label dividing cells, and the SVZ and dopaminergic neurons, present in the SNpc-deficient ak mouse, in- midbrain were evaluated 3 d after the first MPTP injection nervates the SVZ. (Ho¨glinger et al., 2004). Quantification of the number of BrdU incorporating cells revealed that MPTP injection, compared to injec- Chemical lesioning by MPTP affects dopamine neurons in the tion of vehicle alone, caused a significant decrease in the number of ventrolateral region of the VTA proliferating cells in the SVZ of both ak and C57BL/6 mice (Fig. ϩ The compound 1-methyl-4-phenylpyridinium (MPPϩ), the toxic 7a–d). In C57BL/6 mice, proliferation was reduced by 36% [BrdU metabolite of MPTP, is a neurotoxin that rapidly and selectively SVZ cells: saline (n ϭ 4) 272.2 Ϯ 40.64; MPTP (n ϭ 6) 174.28 Ϯ targets nigrostriatal dopaminergic neurons (Langston et al., 1983). 52.11; p ϭ 0.0135], similar to the levels reported previously Lennington et al. • VTA Innervates the SVZ J. Neurosci., September 14, 2011 • 31(37):13078–13087 • 13083

Figure 5. Characterization of midbrain dopaminergic neurons that innervate the SVZ. a, b, Schematic representation of dopaminergic neurons in the midbrain. Inset boxes indicate area imaged inc–t.Incontrolandakmice,FITC ϩ neuronswereidentifiedfollowingretrogradetransportofWGA-FITC.ImmunostainingresultedincellsthatwereTH ϩ/Otx2 ϩ (arrowheads)andTH ϩ/Otx2 Ϫ (arrows) (c–h); TH ϩ/Calbindin ϩ (arrowheads) and TH ϩ/Calbindin Ϫ (arrows) (i–n); and TH ϩ/GIRK2 ϩ (arrowheads), as well as TH ϩ/GIRK2 Ϫ (arrows) (o–t). In the ak mouse midbrain, the italicized SNpc denotes the area typically populated by SNpc A9 neurons. Scale bars: c, f, i, l, o, r, 100 ␮m; others, 25 ␮m.

Table 1. Classification of FITC ؉ cells in the midbrain following retrograde (Ho¨glinger et al., 2004), and we detected a 38% reduction in prolif- transport in C57Bl/6 and ak mice erating BrdU ϩ cells in ak mice [BrdU ϩ SVZ cells: saline (n ϭ 4) ϭ ϭ C57BL/6 (n 3) ak (n 3) 276.78 ϩ 35.29; MPTP (n ϭ 5) 170.70 Ϯ 38.29; p ϭ 0.00368]. Fur- THϩ 96.89 Ϯ 3.73 99.15 Ϯ 1.48 ther analysis of the effects of MPTP exposure revealed a significant ϩ ϩ TH /Otx2 46.15 Ϯ 6.61 63.17 Ϯ 10.42 decline in SVZ proliferation in the dorsal SVZ compared to saline Ϫ TH 3.11 Ϯ 3.73 0.85 Ϯ 1.48 vehicle in both control and ak mice [dorsal SVZ % decline (MPTP- THϩ 88.88 Ϯ 6.03 97.58 Ϯ 2.04 treated vs saline-treated): control 40.125%, p ϭ 0.0105; ak 44.344, THϩ/Calbindinϩ 21.52 Ϯ 7.03 31.27 Ϯ 7.24 p ϭ 0.00950]; whereas no significant decline was observed in the Ϫ TH 11.12 Ϯ 6.03 2.42 Ϯ 2.04 ventral SVZ of MPTP-treated versus saline vehicle-treated control THϩ 94.31 Ϯ 3.06 90.67 Ϯ 4.82 and ak mice [ventral SVZ % decline (MPTP-treated vs saline-treat- THϩ/GIRK2 ϩ 41.12 Ϯ 7.30 39.17 Ϯ 5.48 ed): control 28.909%, p ϭ 0.067; ak 26.706, p ϭ 0.110]. Ϫ TH 5.69 Ϯ 3.06 9.33 Ϯ 4.82 Analysis of the midbrain of MPTP-treated C57BL/6 mice re- Data are recorded as percentage of FITC ϩ cells Ϯ SD, n ϭ 3 per experimental grouping. vealed a loss of both SNpc and VTA neurons, including neurons 13084 • J. Neurosci., September 14, 2011 • 31(37):13078–13087 Lennington et al. • VTA Innervates the SVZ

in the ventrolateral region of the VTA (Fig. 7e,f). Specifically, in C57BL/6 mice, the neurons in the ventrolateral region of the VTA were reduced by 56%, compared to saline-injected controls (p ϭ 0.0134) (Fig. 7i). In ak mice, neurons at the ventrolateral border of the VTA were reduced by 39% (p ϭ 0.0462) (Fig. 7g,h,j). These results indicate that, in addition to depleting SNpc neurons, MPTP also targets neurons located in the ventrolateral VTA—precisely the population of neurons that innervates the SVZ. Discussion Examination of the SNpc-deficient ak mouse revealed no deficien- cies in proliferation or organization in the SVZ, suggesting that dopamine neurons from another source supply sufficient dopamine to regulate the SVZ. Indeed, we report that a population of midbrain dopamine neurons in the ventrolateral VTA innervates the SVZ in both C57BL/6 control and ak mice and likely provides the SVZ with adequate access to the neurotransmitter dopamine. Although the traditional view of dopamine neuron anatomy is often expressed in terms of a clear distinction between the nigrostriatal system, which originates in the SNpc (A9) and terminates in the neostriatum, and the mesocorticolimbic system, which originates in the VTA (A10) and terminates in the ventral striatum and cortex, the present findings are consistent with the more complex view that the distinction between A9 and A10 projection zones is not so clear and that the VTA has a wide projection area that includes various cortical, limbic, and striatal regions (Beckstead et al., 1979; Fallon and Loughlin, 1995), as well as the SVZ. Likewise, the traditional view of dopamine function has been that the mesolimbic DA system mediates “reward” processes, while the nigrostriatal system regulates motor functions; this dogma also has been challenged, because the mesolimbic dopamine system is involved in multiple functions that span several realms, including motor control and learning, behavioral activation, and aversive as well as appetitive aspects of motivation (Salamone et al., 2007; Salamone, 2010). Our finding that the SVZ is innervated by midbrain dopamine neurons more generally associated with the VTA system indicates that mesolimbic dopamine signaling regulates SVZ neurogenesis. Thus, it is possible that the appetitive, aversive, or intense conditioned and unconditioned sen- sory stimuli that are generally thought to activate VTA dopamine neurons may also participate in the regulation of olfactory neurogenesis (Anstrom and Woodward, 2005; Salamone et al., 2007; Brischoux et al., 2009; Schultz, 2010). In view of the extended time- scale for the process of SVZ neurogenesis and migration to the olfac-

The initial diffusion of WGA-FITC was analyzed at 6 and 24 h following injection into the lateral ventricle. a–c, Six hours after injection of WGA-FITC into the lateral ventricle revealed trace in theependymalandsubependymalregions.Similarly,WGA-FITCwasfoundprimarilyassociated withtheependymainthethirdventricle.d–f,At24hafterinjection,theintensityofWGA-FITC labeling was reduced, but restricted to the ependyma and subependyma. Some FITC ϩ afferents were detected beyond the SVZ (arrows). Monochrome images are shown below fluores- cent images. g–i, Insertion of the needle into the cortex resulted in minimal WGA-FITC label in thecortexandnoWGA-FITClabelingofTH ϩ neuronsintheVTA.ArrowindicatesWGA-FITC ϩ/ TH Ϫ cell. j–n, Anterograde transport was analyzed following injections into the ventrolateral VTA.j,SchematicdepictinganterogradeinjectionintoventrolateralVTA.k,l,InC57BL/6control mice injection of WGA-FITC into the ventrolateral region of the VTA resulted in transport to afferents visible in the SVZ (arrows) in the dorsal and ventral SVZ. Transport of WGA-FITC to targets in both the medial striatum and nucleus accumbens can also be seen, as would be predicted by diffusion at the injection site and arborization of neuronal projections. m, n, Sim- ilarly, in the SNpc-deficient ak mouse, there was transport to both the dorsal and ventral SVZ (arrows). Transport of WGA-FITC to targets in the nucleus accumbens can also be seen. Lv, Figure 6. Transport of WGA-FITC following injection into the lateral ventricle and antero- Lateral ventricle; 3v, third ventricle; cx, cortex; cc, corpus callosum; Str, striatum. Scale grade transport following injection of WGA-FITC into the ventrolateral region of the VTA. a–f, bars: a–f, 100 ␮m; g, 250 ␮m; h, 100 ␮m; i, 200 ␮m; k–n,50␮m. Lennington et al. • VTA Innervates the SVZ J. Neurosci., September 14, 2011 • 31(37):13078–13087 • 13085

rons contribute to the regulation of the SVZ. It is possible that this contribution had been overlooked previously due to the use of chemical lesion models that were thought to selectively ablate SNpc A9 neurons. In particular, MPTP, which is con- verted to MPPϩ by MAO and is then preferentially transported via heightened expression of DAT into dopamine neurons (C. Y. Chung et al., 2005; Di Salvio et al., 2010), was considered to be specific for A9 SNpc neurons. In this study, we docu- ment that MPTP also depletes neurons in the ventrolateral region of the VTA. Fol- lowing exposure to MPTP, the reduction in SVZ proliferation found in ak mice was similar to the reduction found in C57BL/6 control mice. Initially, this result was sur- prising as the ak mouse is largely deficient in SNpc A9 neurons and A9 dopamine neurons were thought to be responsible for modulating SVZ proliferation (Ho¨glinger et al., 2004). Indeed, the effects of MPTP might have been expected to be of little consequence in the ak mouse due to reduced DAT expression in the re- maining midbrain cells (Hwang et al., 2009). Instead, we found that a population of dopaminergic neurons associated with the VTA is also vulnerable to MPTP. Our finding that the SNpc-deficient ak mouse is susceptible to further midbrain Figure 7. MPTP exposure depletes midbrain dopamine neurons in the ventrolateral region of the VTA and decreases SVZ dopaminergic neuron loss by MPTP em- proliferation.a–d,FollowingMPTPadministration,eightinjectionsofBrdUat6hintervalsweregiventolabeldividingcells.After phasizes that midbrain dopamine neuron 3 d, proliferation in the SVZ was significantly reduced in both control and ak mice, compared to saline-injected mice. e, f, i,In ϩ populations cannot be categorized into control mice following exposure to MPTP, the number of TH neurons in the SNpc and the VTA, including neurons in the MPTP-vulnerable and MPTP-resistant ϭ ϫ Ϫ6 ϭ ϫ Ϫ3 ϭ ventrolateral region of the VTA, were reduced (SNpc p 3.86 10 , VTA p 1.73 10 , ventrolateral VTA p 0.0134). groups based on A9 and A10 midbrain g, h, j,Inak mice, neurons in the ventrolateral region of the VTA were also reduced following MPTP injection, compared to identity alone. saline-injected controls (p ϭ 0.0462). lv, Lateral ventricle. Scale bars: a–d, 200 ␮m; e–h, 250 ␮m. In addition to the targeting of neurons in the ventrolateral region of the VTA, it is tory bulb, it is likely that dopaminergic regulation of the SVZ important to note there are other factors that may contribute to influences olfactory-based behavioral functions that fluctuate grad- the declines in SVZ proliferation observed following MPTP ex- ually over time. For example, seasonally based changes or maternal posure. These include the reported direct effects of MPTP on behavior may act to prime the olfactory system to alter long- proliferating SVZ cells due to the toxicity of MPTP and/or sec- term responsiveness to particular olfactory stimuli (Shepherd, ondary effects due to acute cell loss in regions adjacent to the SVZ 2004; Atanasova et al., 2008). (He et al., 2008; Shibui et al., 2009). While a direct effect of MPTP Retrograde tracing followed by biomarker immunoreactivity on SVZ cells has been proposed, to date neither DAT nor mono- revealed that it is actually a mixed population of midbrain neu- amine transporters have been found to regulate MPTP-induced rons that innervates the SVZ, including cells expressing GIRK2 and neuroblast apoptosis, and therefore the pathway through which Calbindin. While expression of GIRK2 and Calbindin is thought to MPTP may exert its direct effect on SVZ cells is currently un- be indicative of A9 and A10 neurons, respectively, both proteins are known (He et al., 2008; Shibui et al., 2009). In addition, there is expressed at varying levels in both neuron populations (i.e., higher reported recovery of neuroblast numbers within 24 h of MPTP concentrations of GIRK2/lower concentrations of Calbindin in treatment, making this effect very transitory and the mechanism SNpc neurons and vice versa for VTA dopaminergic neurons) (C. Y. of recovery is unclear (He et al., 2008; Shibui et al., 2009). The Chung et al., 2005). While VTA neurons contribute to the innerva- data we collected was 3 d after MPTP treatment, after the tran- tion of the SVZ in ak mice, some SNpc dopaminergic neurons may sitory period of proposed neuroblast loss through a putative also contribute to the SVZ in C57BL/6 mice. Future designation of direct MPTP mechanism. This suggests that the effects we midbrain dopaminergic neurons will need to be based on combina- observed were not due to direct MPTP toxicity. In addition, torial studies that include retrograde tracking, as well as detection of our analysis revealed significant declines in dorsal but not multiple discriminating biomarkers that take into account the com- ventral SVZ proliferating cells following MPTP treatment. plexity of this region. 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