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Neurotrophic factors BDNF and GDNF protect embryonic chick spinal cord motoneurons from ethanol neurotoxicity in vivo
Developmental Brain Research 112 Ž1999. 99–106
Research report
Neurotrophic factors BDNF and GDNF protect embryonic chick spinal cord motoneurons from ethanol neurotoxicity in vivo a,b,c,d
Douglas M. Bradley
, Francesca D. Beaman d , D. Blaine Moore Marieta Barrow Heaton a,b,c,d,)
a,b,c,d
, Kara Kidd
a,b,c
,
a
d
UniÕersity of Florida Brain Institute, GainesÕille, FL 32610-0244, USA b Department of Neuroscience, GainesÕille, FL 32610-0244, USA c Center for Alcohol Research, GainesÕille, FL 32610-0244, USA UniÕersity of Florida College of Medicine, GainesÕille, FL 32610-0244, USA Accepted 20 October 1998
Abstract Maternal consumption of ethanol is widely recognized as a leading cause of mental and physical deficits. Many populations of the central nervous system are affected by the teratogenic effects of ethanol. Neurotrophic factors ŽNTFs. have been shown to protect against ethanol neurotoxicity in culture, although there have been no demonstrations of such protection in vivo, in specific neuronal populations. Previous studies have demonstrated that ethanol is toxic to developing chick embryo motoneurons when administered from embryonic day 10 ŽE10. to E15. NTFs such as brain-derived neurotrophic factor ŽBDNF. and glial cell line-derived neurotrophic factor ŽGDNF. have been shown to support developing spinal cord motoneurons, and when exogenously applied, decrease naturally occurring cell death, and protect against axotomy. The concurrent delivery of BDNF or GDNF with ethanol to the embryonic chick from E10 to E15 was designed to examine the capacity of these NTFs to provide in vivo neuroprotection for this ethanol-sensitive motoneuron population. Analysis of motoneuron numbers indicated that both BDNF and GDNF provided protection to developing spinal cord motoneurons from ethanol toxicity, restoring motoneuron numbers to control levels. This study represents the first demonstration of in vivo neuroprotection from ethanol toxicity with respect to specific neuronal populations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: BDNF; GDNF; Fetal alcohol syndrome; Neuroprotection; Ethanol
1. Introduction Over the last 25 years, considerable evidence has been generated regarding the deleterious effects ethanol exerts in the developing nervous system. Fetal alcohol syndrome ŽFAS. is diagnosed in 1–2 out of every 1000 live births in the United States and is characterized by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ w1,24,61x. Neuronal populations that are known to be affected by developmental ethanol exposure include the cerebellum w12,68x, the septohippocampal system w2x, cerebral cortex w42x, the substantia nigra w59x, chief sensory trigeminal nucleus w43x, red nucleus w72x, inferior olivary nucleus w49x, striatum w20x and )
Corresponding author. University of Florida College of Medicine, Department of Neuroscience, P.O. Box 100244, Health Science Center, Gainesville, FL 32610-0244, USA. Fax: q1-352-392-8347; E-mail: [email protected]
motoneurons of the spinal cord w4,17x. The present study focuses on the motor system of the developing chick embryo. Spinal cord motoneurons have been shown to be susceptible to the toxic effects of ethanol both in culture w13,17x and in vivo w4,17x. Ethanol reduces motoneuron number when administered to chick embryos from E4 to E11 Žduring the period of natural cell death; w17x or from E10 to E15 Žafter the cell death period. w4x. Developing motoneurons have access to and are responsive to a number of neurotrophic factors ŽNTFs., some of which have been hypothesized to play a role in their normal ontogeny. Two of the NTFs which these neurons normally encounter are brain-derived neurotrophic factor ŽBDNF. and glial-derived neurotrophic factor ŽGDNF.. BDNF is a member of the neurotrophin family of NTFs which includes nerve growth factor ŽNGF., neurotrophin-3, and neurotrophin-4r5. BDNF binds with high affinity to tropomyosin receptor kinase B ŽtrkB. w28x, and has been shown to be produced in developing skeletal muscle, the
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 5 5 - 2
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D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
spinal cord motoneuron target tissue w15,21x. GDNF is a distant and atypical member of the transforming growth factor beta ŽTGF-b . superfamily w35x. Recent studies suggest that GDNF and its receptors, GDNF-Ra and c-ret, form a complex that allows c-ret to transduce the signals from GDNF w23,56,63x. During embryogenesis of the rat, GDNF mRNA is expressed in developing skeletal muscle beginning at E15 w50,64,70x. Populations that are responsive to GDNF express c-ret, including spinal cord motoneurons w53,65x. Studies of gene-deleted Ž‘knockout’. mice have added to the understanding of the relative importance and developmental role of various NTFs and their receptors. In the BDNF knockout, there is no loss of spinal cord motoneurons w25x, although the trkB gene deletion does produce a reduction in motoneuron number w29x. Conversely, GDNF-deficient animals exhibit a significant loss of spinal cord motoneurons Žy22% w46x; y31% w57x., while similar reductions were not reported in c-ret knockouts w37x, although it should be noted that with the c-ret mutants, specific motoneuron counts were not made. These results suggest that the receptors that are responsible for BDNF signal transduction are important for proper motoneuron development, while the GDNF ligand is similarly important. The observation that trkB but not the BDNF knockout results in motoneuron loss suggests that there may be some redundancy in the NTFs that can activate trkB, as well as redundancy in NTFs which can support the development of this population. The reduction in motoneuron number in the GDNF loss-of-function mutation implies an important role for this NTF in normal motoneuron development. The explanation for the apparent stability of motoneuron number in the c-ret knockout, if it should be confirmed by cell counts, is not clear, but again may be indicative of redundancy of neurotrophic support, or GDNF signaling via alternative pathways. Perhaps consistent with this possibility is the observation that GDNF reduces motoneuron normally occurring cell death when administered in the chick embryo from E5 to E10, despite the fact that c-ret expression is quite weak in this population during this period w47,51x. Neuroprotection by NTFs has been investigated extensively in recent years, using a variety of neurotrophic substances, including BDNF and GDNF. In the developing nervous system, both BDNF and GDNF have been shown to be potent in reducing spinal cord motoneuron naturally occurring cell death ŽNOCD. w51,52x, and in protecting against motoneuron degeneration following axotomy w22,51,58,71x. Neurotrophic factors have also been demonstrated to afford specific protection against ethanol cytotoxicity in a number of tissue culture studies. NGF, for example, protects cultured dorsal root ganglion neurons, septal neurons, and cerebellar granule cells from ethanol toxicity w18,19,36x. Basic fibroblast growth factor ŽbFGF. also provides neuroprotection to cultured septal, hippocampal, and cerebellar granule neurons w19,36x. In addition,
BDNF mitigates the neurotoxicity of ethanol and ethanol combined with hypoxic conditions in cultured hippocampal cells w44x and GDNF protects Purkinje cells in organotypic explants from ethanol-induced cell death w40x. In vivo neuroprotection from ethanol toxicity within specific neuronal populations, however, has not been demonstrated. The objective of the present experiment was to extend the in vitro studies of NTF neuroprotection against ethanol cytotoxicity, and to determine whether exogenous NTFs could provide similar protection in vivo. For this study, the embryonic chick motoneuron system at E10–E15 was chosen, since, as noted above, developing motoneurons during this period have been shown to be both vulnerable to ethanol, and responsive to several NTFs during adverse conditions. Since both BDNF and GDNF have been demonstrated to provide potent support for developing motoneurons, and to be normally expressed within their environment, the concurrent delivery of each of these NTFs with ethanol was designed to determine their capacity to provide neuroprotection for this ethanol-sensitive population. Analysis of motoneuron number indicated that both BDNF and GDNF protected against ethanol toxicity in this developing motoneuron population. 2. Materials and methods 2.1. Subjects White leghorn chick eggs were obtained from the University of Florida Poultry Science Department. Eggs were placed in a Marsh incubator and maintained at 378C and 70% relative humidity until E4. At that time, the eggs were moved to a forced draft turning incubator, maintained at the same conditions indicated above, and divided into groups. Six experimental groups were used in this study: Ethanol, Saline, BDNF, BDNFq Ethanol, GDNF, and GDNFq Ethanol. Embryos received daily injections of ethanol, saline, an NTF, or a combination of ethanol and an NTF from E10 to E15. At E16, embryos were removed from the eggs, sacrificed by decapitation, and the lumbar section of the spinal cord removed and prepared for histology. 2.2. Injections Ethanol and saline injections were administered daily from E10 through E15. These dates were chosen to replicate a previous study in which ethanol was shown to reduce motoneuron number in the lumbar spinal cord w4x, and because NOCD, which occurs in the chick spinal cord from E5 through E9 w55x, is completed at this time. Since NOCD is completed when injections begin, any change in motoneuron number observed is attributable to treatment per se and not an interaction of treatment with NOCD. Ethanol embryos received 150 ml of 30% wrv ethanol Ž45 mg ethanol per day., dissolved in a 0.9% wrv nonpyro-
D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
genic saline vehicle, through a pinhole in the shell into the airspace. We have determined that this concentration of ethanol produces blood ethanol concentrations that peak between 250 and 300 mgrdl w4x. Saline embryos received 150 ml of the 0.9% wrv nonpyrogenic saline vehicle. NTF injection wGDNF ŽAmgen. or BDNF ŽRegeneron.x, which also occurred from E10 through E15, involved creating a pinhole directly over the embryo in addition to the pinhole created in the airspace. The airspace was then allowed to shift to a position superior to the embryo and 50 ml of 0.2 mgrml NTF was injected into that space above the embryo directly onto the vascularized chorioallantoic membrane Ž10 mg BDNF or GDNF per day.. These levels of BDNF and GDNF administration were previously shown to partially rescue motoneurons from NOCD without being toxic to the embryo w51,52x. BDNFq Ethanol and GDNFq Ethanol embryos were given ethanol and NTF injections from E10 to E15 as described above. Ethanol injections preceded NTF injections and the embryos were allowed to sit in an incubator for 1 h between injections. This delay in injection time was necessary to ensure that the ethanol was absorbed through the inner shell membrane within the airspace before the eggs were turned on their side for NTF administration. Also, the two injections administered in this study represent a significant volume Ž200 ml. for the embryonic system to incorporate on a daily basis. The delay between injections, therefore, allowed absorption of the volume of ethanol before the NTF solution was presented to the embryo. Pinholes created by the injection process were sealed with paraffin immediately following injection to prevent evaporation andror leakage of the solutions. The eggs were then returned to the turning incubator. 2.3. Dissections and histological procedures Embryos of all experimental groups were sacrificed by decapitation on E16 and the lumbar section of the spinal cord removed. The vertebrae of the spinal cords were cut along the dorsal surface to expose the nervous tissue and allow the fixative to adequately penetrate the tissue. Following dissection, the E16 spinal cords were placed in Bouin’s Fixative for 14 to 21 days to allow the vertebrae to decalcify w33x. The tissue was then embedded in paraffin, cut into 12 mm coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. 2.4. Motoneuron size and spinal cord length Motoneuron size and spinal cord length were measured to determine whether ethanol alters any general characteristics of the motoneuron system. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostral–caudal position of the region of each embryonic spinal cord with an eyepiece micrometer. The section exactly 2.4 mm following the beginning of the
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lumbar spinal cord was sampled. Three embryos from each experimental condition were analyzed for a total of 30 cells per condition. Spinal cord length was determined by counting the number of sections present in each embryo following determination of the boundaries of the lumbar spinal cord by the anatomical methods described below and multiplying this number by the section thickness Ž12 mm.. 2.5. Motoneuron counts Motoneuron counts were completed following methods described previously w16,17x. Briefly, a uniform area encompassed by six DRG was noted in each embryo. This procedure ensured that a similar area was counted in each subject. Starting from the most rostral section included in the six DRG region, motoneurons in the lateral motor column of one side of every tenth section were marked onto paper using a camera lucida, with a magnification of 400 = . The counting procedure employed was the method pioneered by Hamburger w16x, which has been utilized in most subsequent studies in which spinal cord motoneuron enumeration has been undertaken. For this procedure, neurons were counted only if they exhibited the following morphological features: a large soma, a clear nucleus bounded by an intact nuclear membrane, and a distinct nucleolus. This neuronal counting procedure has been empirically demonstrated to be as accurate and reliable as those using correction factors or stereology with this spinal cord motoneuron population, as well as with other neuronal populations w10,34x. Laterality was maintained throughout each individual embryo, but chosen at random before beginning the counting process. Previous studies have shown that there is no difference between the number of motoneurons contained in the right and left sides of the spinal cord w55x. All counts were done by DMB and FDB. Each embryo was coded so that the experimenter had no knowledge of its experimental treatment until the study was completed. The number of embryos utilized for each condition was as follows: Saline, N s 11; Ethanol, N s 7; BDNF, N s 5; BDNFq Ethanol, N s 6; GDNF, N s 7; GDNFq Ethanol, N s 5. 2.6. Statistical analyses The analysis of variance ŽANOVA. was performed using the Stat View Program on a Macintosh computer. When applicable, individual differences between groups were tested using Fisher’s Protected Least Significant Difference ŽPLSD. post-hoc analyses.
3. Results Neither BDNF nor GDNF administration appeared to be deleterious. Embryonic survival was 82% in the BDNF
D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
102 Table 1 Motoneuron size and spinal cord length Group
Cell size Žmm.
Spinal cord length Žmm.
Saline Ethanol GDNF GDNFqEthanol BDNF BDNFqEthanol
18.6"0.525 18.9"0.508 18.7"0.514 18.4"0.502 18.6"0.433 18.6"0.438
6020"69.0 5966"137.9 5820"154.2 5856"148.9 5904"88.2 5900"153.1
All values are means"S.E.M. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostral–caudal position of the region of three embryonic spinal cords with an eyepiece micrometer. Therefore, N s 30 for each group. Spinal cord length was computed by determining the number of sections present in a given spinal cord and then multiplying by section thickness Ž12 mm.. For spinal cord length, Saline N s12, Ethanol N s 7, GDNF N s6, GDNFqEthanol N s 5, BDNF N s 5, and BDNFqEthanol N s6.
group and 65% in the BDNFq Ethanol group. The GDNF group survived at a rate of 93% and the GDNFq Ethanol group survived at a rate of 69%. These rates compare favorably with control survival rates, with 90% of the Saline group and 60% of the Ethanol group surviving. 3.1. Motoneuron size and spinal cord length As noted above, motoneuron size and spinal cord length analyses were performed to determine whether treatments utilized in these studies altered the gross morphology of the embryonic spinal cord. The ANOVA indicated that
embryos from these experimental groups exhibited no differences in motoneuron size due to treatment Ž F s 0.13, df s 209, P ) 0.9.. That is, the motoneuron size of ethanol-treated animals was unchanged from that of neurotrophin-treated or control animals. The ANOVA also revealed that overall lumbar spinal cord length was unaltered by treatment Ž F s 0.44, df s 40, P ) 0.8.. There were no significant differences in the length of the spinal cord region counted among the experimental groups. Table 1 displays the data from this portion of the study. Taken together, these results suggest that the treatments administered in this study did not adversely affect the basic anatomy of the spinal cord. 3.2. Motoneuron number The ANOVA for motoneuron number indicated a significant treatment effect Ž F s 3.79, df s 40, P s 0.0075.. Post hoc testing showed that while there was a significant reduction in motoneurons in the Ethanol-treated embryos compared to their Saline controls Ž P s 0.0052., co-treatment with either BDNF or GDNF eliminated this ethanolinduced motoneuron loss. Thus, neither the BDNF nor the GDNF motoneuron numbers differed from their ethanoltreated counterparts, and the NTF q Ethanol groups also did not differ from the Saline controls. Both GDNF and GDNFq Ethanol differed significantly from the Ethanolalone group Ž P s 0.0002 and 0.0104, respectively., and while the BDNFq Ethanol group did not differ from
Fig. 1. Chick embryos were injected in ovo with Saline, Ethanol in saline vehicle, BDNF, BDNFq Ethanol Žconcurrently., GDNF, or GDNFq Ethanol Žconcurrently., daily from E10 to E15. Counts of lumbosacral spinal cord motoneurons were subsequently made. Both BDNF and GDNF administrated with ethanol abolished ethanol-induced motoneuron loss Žas seen in the Ethanol group. and restored motoneuron counts to control Žsaline or neurotrophic factor-wNTFx alone. levels. Motoneuron counts are presented as meansq S.E.M. Data represent actual counts obtained from the lumbosacral spinal cord and are not population estimates. Ža. Significantly greater than the Ethanol group Ž P s 0.0052.; Žb. difference from the Ethanol group approached significance Ž P s 0.0559.; Žc. significantly greater than the Ethanol group Ž P s 0.0002.; Žd. significantly greater than the Ethanol group Ž P s 0.0104.. Neither the BDNFq Ethanol nor the GDNFq Ethanol counts differed from their respective NTF-only controls, and neither differed from the Saline controls.
D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
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Fig. 2. Photomicrographs of sections from spinal cord showing lateral motor column of E16 chick embryos which were treated from E10–E15 with ŽA. Saline, ŽB. Ethanol, ŽC. BDNFq Ethanol, or ŽD. GDNFq Ethanol. Note the reduction of motoneurons in the Ethanol embryo ŽB. compared to the other conditions. Bar represents 180 mm.
Ethanol-alone, the BDNF group compared to Ethanol approached significance Ž P s 0.0559.. The most relevant comparisons, however, between Saline vs. Ethanol, BDNF vs. BDNFq Ethanol, and GDNF vs. GDNFq Ethanol, clearly show neuroprotection in the two NTF-supplemented groups. These results are presented in Fig. 1. Fig. 2 shows photomicrographs of the lateral motor column from Saline, Ethanol, BDNFq Ethanol, and GDNFq Ethanol E16 embryos.
4. Discussion This study has demonstrated that both BDNF and GDNF can protect spinal cord motoneurons against ethanol toxicity in the intact animal. When ethanol was administered alone to E10–E15 embryonic chicks, a significant loss of motoneurons ensued. But when BDNF or GDNF were co-administered with ethanol, this cell loss was abolished, and the motoneuron counts were restored to control Ži.e., Saline and NTF-alone. levels. The GDNF effect tended to be somewhat more striking than that seen with BDNF: when GDNF was combined with ethanol, motoneuron numbers were significantly greater than those found in the Ethanol group, while the BDNFq Ethanol embryos did not differ significantly from Ethanol embryos, and the
BDNF group only approached significance in comparison to the Ethanol group. In the most salient comparisons, however, both NTFs protected against ethanol neurotoxicity: Thus, motoneuron numbers in GDNFq Ethanol embryos did not differ from those found in GDNF embryos, and, similarly, the motoneuron complement in BDNFq Ethanol embryos did not differ from that assessed in BDNF embryos. The fact that treatment with either BDNF or GDNF alone did not significantly alter motoneuron number above Saline control levels strongly suggests that the amelioration observed was attributable to an attenuation of ethanol neurotoxicity, and not to a shift in baseline motoneuron number. This point is also supported by the fact that ethanol and NTFs were administered after the normal cell death period. While ethanol administration did have an adverse effect on the motoneuron population, it did not change the overall morphology of the spinal cord, since lumbar cord length, an indicator of volume, was unchanged following ethanol treatment. Therefore, volumetric alterations did not contribute to the enhanced motoneuron survival when BDNF or GDNF accompanied ethanol administration. These observations represent the first demonstration of in vivo neurotrophic factor protection from ethanol toxicity with respect to a specific neuronal population. The findings in some respects resemble those
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D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
of Brodie and Vernadakis w5x, who found that while in ovo treatment of the chick embryo from E1–E3 with ethanol produced a decrease in choline acetyltransferase activity, co-treatment with either NGF or epidermal growth factor prevented this reduction. Both BDNF and GDNF have previously been shown to protect against a number of adverse in vivo conditions. For example, both NTFs reduce death of spinal cord motoneurons during the normal cell death period in the chick embryo w22,51x, and both protect against motoneuron death following axotomy in the rat w14,66,71x. Intrastriatal or intraventricular administration of BDNF or GDNF, respectively, attenuates motor deficits and rescues dopaminergic neurons following 6-hydroxydopamine- or MPTP-induced trauma, and thus have therapeutic implications for Parkinson’s disease w3,9,27,32,60,62x. BDNF also affords protection against neonatal hypoxic–ischemic brain injury w8x, and GDNF protects against ischemic cortical injury induced by middle cerebral artery occlusion w67x. GDNF also provides protection against kainic acid-mediated seizures w38x, and in animal models of Huntington’s Disease, using the NMDA receptor agonist quinolinate, GDNF protects a sub-population of striatal calbindin neurons from excitotoxic damage w54x. In addition, BDNF has been demonstrated to reduce the loss of basal forebrain cholinergic neurons following fimbria–fornix transection w30x. Our results of BDNF and GDNF protection against ethanol insult are also consistent with the recent observation showing that BDNF can protect cultured hippocampal neurons from ethanol as well as ethanol combined with hypoxic conditions w44x, and the GDNF rescue of cerebellar Purkinje cells in organotypic explant cultures from ethanol-induced apoptosis w40x. As noted earlier, both BDNF and GDNF are available to developing motoneurons, and motoneuron expression of both trkB and c-ret, the high affinity receptors responsible for signal transduction of BDNF and GDNF, respectively, begins during embryonic development. trkB mRNA expression is first detectable in the chick embryo at E8 and its level of expression increases throughout the embryonic period w41x. At E16, trkB mRNA is highly expressed in the lateral motor column of the chick spinal cord w41x. c-ret mRNA expression is also detectable in chick embryo spinal cord motoneurons. This expression is relatively weak from E5 to E10, but is intense between E17 and adulthood w47x. Support by these NTFs could be either direct or indirect, possibly acting via increased gliogenesis or upregulation of some other supportive molecules. A number of mechanisms and events resulting from developmental ethanol exposure have been postulated to be involved in the subsequent neurotoxicity Žsee review in Ref. w69x.. It may be significant that many of these same events have been shown to be modulated by neurotrophic factors, including BDNF and GDNF, and such modulation could be involved in amelioration of ethanol neurotoxicity such as that seen in the present study. BDNF, for example,
has been found to protect against hypoxia, ischemia w6x and hypoglycemia w6,7,48x. It can also regulate calcium homeostasis w7x, increase antioxidative enzyme levels w39x, and mitigate neuronal apoptosis w31x. Similarly, GDNF protects against ischemia w67x and excitotoxic cellular damage w54x, and can modulate apoptosis w11x and MPTP toxicity w62x. The possibility that alterations in expression of cell death effector or repressor genes Že.g., of the bcl-2 family. may contribute to ethanol-related neurotoxicity has also been proposed, and recent evidence supports this concept w45x. Certain NTFs Že.g., NGF. have been demonstrated to regulate expression of these genes w26x suggesting another possible route of NTF protection. NTFs have proven to be versatile molecules with the ability to sustain neurons in the presence of a variety of potentially lethal insults or conditions. Following the present demonstration of in vivo neuroprotection from ethanol toxicity, additional investigations should assess the protective potential of other NTFs andror combinations of NTFs, and should investigate the mechanism of this protection, with particular emphasis on the signaling pathways involved. In addition, experiments should be designed to determine whether other ethanol-sensitive populations can be similarly protected by NTFs in vivo. Such protection against ethanol-induced neuronal damage in the intact animal might eventually have therapeutic applicability to prevent or ameliorate the neurological damage seen in FAS.
Acknowledgements The authors thank Michael Paiva and Scarlet Raby for their excellent technical assistance. We also thank Amgen for their generous contribution of the GDNF and Regeneron for their generous contribution of the BDNF. This study was supported by NIAAA Grant AA09128. Douglas M. Bradley was supported by an NSF Predoctoral Fellowship and NIAAA Grant T32-AA07561, Francesca Beaman was supported by NIH Grant T35-HL07489, and D. Blaine Moore was supported by NIAAA Grants T32-AA07561 and AA05502.
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Research report
Neurotrophic factors BDNF and GDNF protect embryonic chick spinal cord motoneurons from ethanol neurotoxicity in vivo a,b,c,d
Douglas M. Bradley
, Francesca D. Beaman d , D. Blaine Moore Marieta Barrow Heaton a,b,c,d,)
a,b,c,d
, Kara Kidd
a,b,c
,
a
d
UniÕersity of Florida Brain Institute, GainesÕille, FL 32610-0244, USA b Department of Neuroscience, GainesÕille, FL 32610-0244, USA c Center for Alcohol Research, GainesÕille, FL 32610-0244, USA UniÕersity of Florida College of Medicine, GainesÕille, FL 32610-0244, USA Accepted 20 October 1998
Abstract Maternal consumption of ethanol is widely recognized as a leading cause of mental and physical deficits. Many populations of the central nervous system are affected by the teratogenic effects of ethanol. Neurotrophic factors ŽNTFs. have been shown to protect against ethanol neurotoxicity in culture, although there have been no demonstrations of such protection in vivo, in specific neuronal populations. Previous studies have demonstrated that ethanol is toxic to developing chick embryo motoneurons when administered from embryonic day 10 ŽE10. to E15. NTFs such as brain-derived neurotrophic factor ŽBDNF. and glial cell line-derived neurotrophic factor ŽGDNF. have been shown to support developing spinal cord motoneurons, and when exogenously applied, decrease naturally occurring cell death, and protect against axotomy. The concurrent delivery of BDNF or GDNF with ethanol to the embryonic chick from E10 to E15 was designed to examine the capacity of these NTFs to provide in vivo neuroprotection for this ethanol-sensitive motoneuron population. Analysis of motoneuron numbers indicated that both BDNF and GDNF provided protection to developing spinal cord motoneurons from ethanol toxicity, restoring motoneuron numbers to control levels. This study represents the first demonstration of in vivo neuroprotection from ethanol toxicity with respect to specific neuronal populations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: BDNF; GDNF; Fetal alcohol syndrome; Neuroprotection; Ethanol
1. Introduction Over the last 25 years, considerable evidence has been generated regarding the deleterious effects ethanol exerts in the developing nervous system. Fetal alcohol syndrome ŽFAS. is diagnosed in 1–2 out of every 1000 live births in the United States and is characterized by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ w1,24,61x. Neuronal populations that are known to be affected by developmental ethanol exposure include the cerebellum w12,68x, the septohippocampal system w2x, cerebral cortex w42x, the substantia nigra w59x, chief sensory trigeminal nucleus w43x, red nucleus w72x, inferior olivary nucleus w49x, striatum w20x and )
Corresponding author. University of Florida College of Medicine, Department of Neuroscience, P.O. Box 100244, Health Science Center, Gainesville, FL 32610-0244, USA. Fax: q1-352-392-8347; E-mail: [email protected]
motoneurons of the spinal cord w4,17x. The present study focuses on the motor system of the developing chick embryo. Spinal cord motoneurons have been shown to be susceptible to the toxic effects of ethanol both in culture w13,17x and in vivo w4,17x. Ethanol reduces motoneuron number when administered to chick embryos from E4 to E11 Žduring the period of natural cell death; w17x or from E10 to E15 Žafter the cell death period. w4x. Developing motoneurons have access to and are responsive to a number of neurotrophic factors ŽNTFs., some of which have been hypothesized to play a role in their normal ontogeny. Two of the NTFs which these neurons normally encounter are brain-derived neurotrophic factor ŽBDNF. and glial-derived neurotrophic factor ŽGDNF.. BDNF is a member of the neurotrophin family of NTFs which includes nerve growth factor ŽNGF., neurotrophin-3, and neurotrophin-4r5. BDNF binds with high affinity to tropomyosin receptor kinase B ŽtrkB. w28x, and has been shown to be produced in developing skeletal muscle, the
0165-3806r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 5 5 - 2
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D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
spinal cord motoneuron target tissue w15,21x. GDNF is a distant and atypical member of the transforming growth factor beta ŽTGF-b . superfamily w35x. Recent studies suggest that GDNF and its receptors, GDNF-Ra and c-ret, form a complex that allows c-ret to transduce the signals from GDNF w23,56,63x. During embryogenesis of the rat, GDNF mRNA is expressed in developing skeletal muscle beginning at E15 w50,64,70x. Populations that are responsive to GDNF express c-ret, including spinal cord motoneurons w53,65x. Studies of gene-deleted Ž‘knockout’. mice have added to the understanding of the relative importance and developmental role of various NTFs and their receptors. In the BDNF knockout, there is no loss of spinal cord motoneurons w25x, although the trkB gene deletion does produce a reduction in motoneuron number w29x. Conversely, GDNF-deficient animals exhibit a significant loss of spinal cord motoneurons Žy22% w46x; y31% w57x., while similar reductions were not reported in c-ret knockouts w37x, although it should be noted that with the c-ret mutants, specific motoneuron counts were not made. These results suggest that the receptors that are responsible for BDNF signal transduction are important for proper motoneuron development, while the GDNF ligand is similarly important. The observation that trkB but not the BDNF knockout results in motoneuron loss suggests that there may be some redundancy in the NTFs that can activate trkB, as well as redundancy in NTFs which can support the development of this population. The reduction in motoneuron number in the GDNF loss-of-function mutation implies an important role for this NTF in normal motoneuron development. The explanation for the apparent stability of motoneuron number in the c-ret knockout, if it should be confirmed by cell counts, is not clear, but again may be indicative of redundancy of neurotrophic support, or GDNF signaling via alternative pathways. Perhaps consistent with this possibility is the observation that GDNF reduces motoneuron normally occurring cell death when administered in the chick embryo from E5 to E10, despite the fact that c-ret expression is quite weak in this population during this period w47,51x. Neuroprotection by NTFs has been investigated extensively in recent years, using a variety of neurotrophic substances, including BDNF and GDNF. In the developing nervous system, both BDNF and GDNF have been shown to be potent in reducing spinal cord motoneuron naturally occurring cell death ŽNOCD. w51,52x, and in protecting against motoneuron degeneration following axotomy w22,51,58,71x. Neurotrophic factors have also been demonstrated to afford specific protection against ethanol cytotoxicity in a number of tissue culture studies. NGF, for example, protects cultured dorsal root ganglion neurons, septal neurons, and cerebellar granule cells from ethanol toxicity w18,19,36x. Basic fibroblast growth factor ŽbFGF. also provides neuroprotection to cultured septal, hippocampal, and cerebellar granule neurons w19,36x. In addition,
BDNF mitigates the neurotoxicity of ethanol and ethanol combined with hypoxic conditions in cultured hippocampal cells w44x and GDNF protects Purkinje cells in organotypic explants from ethanol-induced cell death w40x. In vivo neuroprotection from ethanol toxicity within specific neuronal populations, however, has not been demonstrated. The objective of the present experiment was to extend the in vitro studies of NTF neuroprotection against ethanol cytotoxicity, and to determine whether exogenous NTFs could provide similar protection in vivo. For this study, the embryonic chick motoneuron system at E10–E15 was chosen, since, as noted above, developing motoneurons during this period have been shown to be both vulnerable to ethanol, and responsive to several NTFs during adverse conditions. Since both BDNF and GDNF have been demonstrated to provide potent support for developing motoneurons, and to be normally expressed within their environment, the concurrent delivery of each of these NTFs with ethanol was designed to determine their capacity to provide neuroprotection for this ethanol-sensitive population. Analysis of motoneuron number indicated that both BDNF and GDNF protected against ethanol toxicity in this developing motoneuron population. 2. Materials and methods 2.1. Subjects White leghorn chick eggs were obtained from the University of Florida Poultry Science Department. Eggs were placed in a Marsh incubator and maintained at 378C and 70% relative humidity until E4. At that time, the eggs were moved to a forced draft turning incubator, maintained at the same conditions indicated above, and divided into groups. Six experimental groups were used in this study: Ethanol, Saline, BDNF, BDNFq Ethanol, GDNF, and GDNFq Ethanol. Embryos received daily injections of ethanol, saline, an NTF, or a combination of ethanol and an NTF from E10 to E15. At E16, embryos were removed from the eggs, sacrificed by decapitation, and the lumbar section of the spinal cord removed and prepared for histology. 2.2. Injections Ethanol and saline injections were administered daily from E10 through E15. These dates were chosen to replicate a previous study in which ethanol was shown to reduce motoneuron number in the lumbar spinal cord w4x, and because NOCD, which occurs in the chick spinal cord from E5 through E9 w55x, is completed at this time. Since NOCD is completed when injections begin, any change in motoneuron number observed is attributable to treatment per se and not an interaction of treatment with NOCD. Ethanol embryos received 150 ml of 30% wrv ethanol Ž45 mg ethanol per day., dissolved in a 0.9% wrv nonpyro-
D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
genic saline vehicle, through a pinhole in the shell into the airspace. We have determined that this concentration of ethanol produces blood ethanol concentrations that peak between 250 and 300 mgrdl w4x. Saline embryos received 150 ml of the 0.9% wrv nonpyrogenic saline vehicle. NTF injection wGDNF ŽAmgen. or BDNF ŽRegeneron.x, which also occurred from E10 through E15, involved creating a pinhole directly over the embryo in addition to the pinhole created in the airspace. The airspace was then allowed to shift to a position superior to the embryo and 50 ml of 0.2 mgrml NTF was injected into that space above the embryo directly onto the vascularized chorioallantoic membrane Ž10 mg BDNF or GDNF per day.. These levels of BDNF and GDNF administration were previously shown to partially rescue motoneurons from NOCD without being toxic to the embryo w51,52x. BDNFq Ethanol and GDNFq Ethanol embryos were given ethanol and NTF injections from E10 to E15 as described above. Ethanol injections preceded NTF injections and the embryos were allowed to sit in an incubator for 1 h between injections. This delay in injection time was necessary to ensure that the ethanol was absorbed through the inner shell membrane within the airspace before the eggs were turned on their side for NTF administration. Also, the two injections administered in this study represent a significant volume Ž200 ml. for the embryonic system to incorporate on a daily basis. The delay between injections, therefore, allowed absorption of the volume of ethanol before the NTF solution was presented to the embryo. Pinholes created by the injection process were sealed with paraffin immediately following injection to prevent evaporation andror leakage of the solutions. The eggs were then returned to the turning incubator. 2.3. Dissections and histological procedures Embryos of all experimental groups were sacrificed by decapitation on E16 and the lumbar section of the spinal cord removed. The vertebrae of the spinal cords were cut along the dorsal surface to expose the nervous tissue and allow the fixative to adequately penetrate the tissue. Following dissection, the E16 spinal cords were placed in Bouin’s Fixative for 14 to 21 days to allow the vertebrae to decalcify w33x. The tissue was then embedded in paraffin, cut into 12 mm coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. 2.4. Motoneuron size and spinal cord length Motoneuron size and spinal cord length were measured to determine whether ethanol alters any general characteristics of the motoneuron system. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostral–caudal position of the region of each embryonic spinal cord with an eyepiece micrometer. The section exactly 2.4 mm following the beginning of the
101
lumbar spinal cord was sampled. Three embryos from each experimental condition were analyzed for a total of 30 cells per condition. Spinal cord length was determined by counting the number of sections present in each embryo following determination of the boundaries of the lumbar spinal cord by the anatomical methods described below and multiplying this number by the section thickness Ž12 mm.. 2.5. Motoneuron counts Motoneuron counts were completed following methods described previously w16,17x. Briefly, a uniform area encompassed by six DRG was noted in each embryo. This procedure ensured that a similar area was counted in each subject. Starting from the most rostral section included in the six DRG region, motoneurons in the lateral motor column of one side of every tenth section were marked onto paper using a camera lucida, with a magnification of 400 = . The counting procedure employed was the method pioneered by Hamburger w16x, which has been utilized in most subsequent studies in which spinal cord motoneuron enumeration has been undertaken. For this procedure, neurons were counted only if they exhibited the following morphological features: a large soma, a clear nucleus bounded by an intact nuclear membrane, and a distinct nucleolus. This neuronal counting procedure has been empirically demonstrated to be as accurate and reliable as those using correction factors or stereology with this spinal cord motoneuron population, as well as with other neuronal populations w10,34x. Laterality was maintained throughout each individual embryo, but chosen at random before beginning the counting process. Previous studies have shown that there is no difference between the number of motoneurons contained in the right and left sides of the spinal cord w55x. All counts were done by DMB and FDB. Each embryo was coded so that the experimenter had no knowledge of its experimental treatment until the study was completed. The number of embryos utilized for each condition was as follows: Saline, N s 11; Ethanol, N s 7; BDNF, N s 5; BDNFq Ethanol, N s 6; GDNF, N s 7; GDNFq Ethanol, N s 5. 2.6. Statistical analyses The analysis of variance ŽANOVA. was performed using the Stat View Program on a Macintosh computer. When applicable, individual differences between groups were tested using Fisher’s Protected Least Significant Difference ŽPLSD. post-hoc analyses.
3. Results Neither BDNF nor GDNF administration appeared to be deleterious. Embryonic survival was 82% in the BDNF
D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
102 Table 1 Motoneuron size and spinal cord length Group
Cell size Žmm.
Spinal cord length Žmm.
Saline Ethanol GDNF GDNFqEthanol BDNF BDNFqEthanol
18.6"0.525 18.9"0.508 18.7"0.514 18.4"0.502 18.6"0.433 18.6"0.438
6020"69.0 5966"137.9 5820"154.2 5856"148.9 5904"88.2 5900"153.1
All values are means"S.E.M. Motoneuron size was determined by measuring the diameter of 10 random cells in the same rostral–caudal position of the region of three embryonic spinal cords with an eyepiece micrometer. Therefore, N s 30 for each group. Spinal cord length was computed by determining the number of sections present in a given spinal cord and then multiplying by section thickness Ž12 mm.. For spinal cord length, Saline N s12, Ethanol N s 7, GDNF N s6, GDNFqEthanol N s 5, BDNF N s 5, and BDNFqEthanol N s6.
group and 65% in the BDNFq Ethanol group. The GDNF group survived at a rate of 93% and the GDNFq Ethanol group survived at a rate of 69%. These rates compare favorably with control survival rates, with 90% of the Saline group and 60% of the Ethanol group surviving. 3.1. Motoneuron size and spinal cord length As noted above, motoneuron size and spinal cord length analyses were performed to determine whether treatments utilized in these studies altered the gross morphology of the embryonic spinal cord. The ANOVA indicated that
embryos from these experimental groups exhibited no differences in motoneuron size due to treatment Ž F s 0.13, df s 209, P ) 0.9.. That is, the motoneuron size of ethanol-treated animals was unchanged from that of neurotrophin-treated or control animals. The ANOVA also revealed that overall lumbar spinal cord length was unaltered by treatment Ž F s 0.44, df s 40, P ) 0.8.. There were no significant differences in the length of the spinal cord region counted among the experimental groups. Table 1 displays the data from this portion of the study. Taken together, these results suggest that the treatments administered in this study did not adversely affect the basic anatomy of the spinal cord. 3.2. Motoneuron number The ANOVA for motoneuron number indicated a significant treatment effect Ž F s 3.79, df s 40, P s 0.0075.. Post hoc testing showed that while there was a significant reduction in motoneurons in the Ethanol-treated embryos compared to their Saline controls Ž P s 0.0052., co-treatment with either BDNF or GDNF eliminated this ethanolinduced motoneuron loss. Thus, neither the BDNF nor the GDNF motoneuron numbers differed from their ethanoltreated counterparts, and the NTF q Ethanol groups also did not differ from the Saline controls. Both GDNF and GDNFq Ethanol differed significantly from the Ethanolalone group Ž P s 0.0002 and 0.0104, respectively., and while the BDNFq Ethanol group did not differ from
Fig. 1. Chick embryos were injected in ovo with Saline, Ethanol in saline vehicle, BDNF, BDNFq Ethanol Žconcurrently., GDNF, or GDNFq Ethanol Žconcurrently., daily from E10 to E15. Counts of lumbosacral spinal cord motoneurons were subsequently made. Both BDNF and GDNF administrated with ethanol abolished ethanol-induced motoneuron loss Žas seen in the Ethanol group. and restored motoneuron counts to control Žsaline or neurotrophic factor-wNTFx alone. levels. Motoneuron counts are presented as meansq S.E.M. Data represent actual counts obtained from the lumbosacral spinal cord and are not population estimates. Ža. Significantly greater than the Ethanol group Ž P s 0.0052.; Žb. difference from the Ethanol group approached significance Ž P s 0.0559.; Žc. significantly greater than the Ethanol group Ž P s 0.0002.; Žd. significantly greater than the Ethanol group Ž P s 0.0104.. Neither the BDNFq Ethanol nor the GDNFq Ethanol counts differed from their respective NTF-only controls, and neither differed from the Saline controls.
D.M. Bradley et al.r DeÕelopmental Brain Research 112 (1999) 99–106
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Fig. 2. Photomicrographs of sections from spinal cord showing lateral motor column of E16 chick embryos which were treated from E10–E15 with ŽA. Saline, ŽB. Ethanol, ŽC. BDNFq Ethanol, or ŽD. GDNFq Ethanol. Note the reduction of motoneurons in the Ethanol embryo ŽB. compared to the other conditions. Bar represents 180 mm.
Ethanol-alone, the BDNF group compared to Ethanol approached significance Ž P s 0.0559.. The most relevant comparisons, however, between Saline vs. Ethanol, BDNF vs. BDNFq Ethanol, and GDNF vs. GDNFq Ethanol, clearly show neuroprotection in the two NTF-supplemented groups. These results are presented in Fig. 1. Fig. 2 shows photomicrographs of the lateral motor column from Saline, Ethanol, BDNFq Ethanol, and GDNFq Ethanol E16 embryos.
4. Discussion This study has demonstrated that both BDNF and GDNF can protect spinal cord motoneurons against ethanol toxicity in the intact animal. When ethanol was administered alone to E10–E15 embryonic chicks, a significant loss of motoneurons ensued. But when BDNF or GDNF were co-administered with ethanol, this cell loss was abolished, and the motoneuron counts were restored to control Ži.e., Saline and NTF-alone. levels. The GDNF effect tended to be somewhat more striking than that seen with BDNF: when GDNF was combined with ethanol, motoneuron numbers were significantly greater than those found in the Ethanol group, while the BDNFq Ethanol embryos did not differ significantly from Ethanol embryos, and the
BDNF group only approached significance in comparison to the Ethanol group. In the most salient comparisons, however, both NTFs protected against ethanol neurotoxicity: Thus, motoneuron numbers in GDNFq Ethanol embryos did not differ from those found in GDNF embryos, and, similarly, the motoneuron complement in BDNFq Ethanol embryos did not differ from that assessed in BDNF embryos. The fact that treatment with either BDNF or GDNF alone did not significantly alter motoneuron number above Saline control levels strongly suggests that the amelioration observed was attributable to an attenuation of ethanol neurotoxicity, and not to a shift in baseline motoneuron number. This point is also supported by the fact that ethanol and NTFs were administered after the normal cell death period. While ethanol administration did have an adverse effect on the motoneuron population, it did not change the overall morphology of the spinal cord, since lumbar cord length, an indicator of volume, was unchanged following ethanol treatment. Therefore, volumetric alterations did not contribute to the enhanced motoneuron survival when BDNF or GDNF accompanied ethanol administration. These observations represent the first demonstration of in vivo neurotrophic factor protection from ethanol toxicity with respect to a specific neuronal population. The findings in some respects resemble those
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of Brodie and Vernadakis w5x, who found that while in ovo treatment of the chick embryo from E1–E3 with ethanol produced a decrease in choline acetyltransferase activity, co-treatment with either NGF or epidermal growth factor prevented this reduction. Both BDNF and GDNF have previously been shown to protect against a number of adverse in vivo conditions. For example, both NTFs reduce death of spinal cord motoneurons during the normal cell death period in the chick embryo w22,51x, and both protect against motoneuron death following axotomy in the rat w14,66,71x. Intrastriatal or intraventricular administration of BDNF or GDNF, respectively, attenuates motor deficits and rescues dopaminergic neurons following 6-hydroxydopamine- or MPTP-induced trauma, and thus have therapeutic implications for Parkinson’s disease w3,9,27,32,60,62x. BDNF also affords protection against neonatal hypoxic–ischemic brain injury w8x, and GDNF protects against ischemic cortical injury induced by middle cerebral artery occlusion w67x. GDNF also provides protection against kainic acid-mediated seizures w38x, and in animal models of Huntington’s Disease, using the NMDA receptor agonist quinolinate, GDNF protects a sub-population of striatal calbindin neurons from excitotoxic damage w54x. In addition, BDNF has been demonstrated to reduce the loss of basal forebrain cholinergic neurons following fimbria–fornix transection w30x. Our results of BDNF and GDNF protection against ethanol insult are also consistent with the recent observation showing that BDNF can protect cultured hippocampal neurons from ethanol as well as ethanol combined with hypoxic conditions w44x, and the GDNF rescue of cerebellar Purkinje cells in organotypic explant cultures from ethanol-induced apoptosis w40x. As noted earlier, both BDNF and GDNF are available to developing motoneurons, and motoneuron expression of both trkB and c-ret, the high affinity receptors responsible for signal transduction of BDNF and GDNF, respectively, begins during embryonic development. trkB mRNA expression is first detectable in the chick embryo at E8 and its level of expression increases throughout the embryonic period w41x. At E16, trkB mRNA is highly expressed in the lateral motor column of the chick spinal cord w41x. c-ret mRNA expression is also detectable in chick embryo spinal cord motoneurons. This expression is relatively weak from E5 to E10, but is intense between E17 and adulthood w47x. Support by these NTFs could be either direct or indirect, possibly acting via increased gliogenesis or upregulation of some other supportive molecules. A number of mechanisms and events resulting from developmental ethanol exposure have been postulated to be involved in the subsequent neurotoxicity Žsee review in Ref. w69x.. It may be significant that many of these same events have been shown to be modulated by neurotrophic factors, including BDNF and GDNF, and such modulation could be involved in amelioration of ethanol neurotoxicity such as that seen in the present study. BDNF, for example,
has been found to protect against hypoxia, ischemia w6x and hypoglycemia w6,7,48x. It can also regulate calcium homeostasis w7x, increase antioxidative enzyme levels w39x, and mitigate neuronal apoptosis w31x. Similarly, GDNF protects against ischemia w67x and excitotoxic cellular damage w54x, and can modulate apoptosis w11x and MPTP toxicity w62x. The possibility that alterations in expression of cell death effector or repressor genes Že.g., of the bcl-2 family. may contribute to ethanol-related neurotoxicity has also been proposed, and recent evidence supports this concept w45x. Certain NTFs Že.g., NGF. have been demonstrated to regulate expression of these genes w26x suggesting another possible route of NTF protection. NTFs have proven to be versatile molecules with the ability to sustain neurons in the presence of a variety of potentially lethal insults or conditions. Following the present demonstration of in vivo neuroprotection from ethanol toxicity, additional investigations should assess the protective potential of other NTFs andror combinations of NTFs, and should investigate the mechanism of this protection, with particular emphasis on the signaling pathways involved. In addition, experiments should be designed to determine whether other ethanol-sensitive populations can be similarly protected by NTFs in vivo. Such protection against ethanol-induced neuronal damage in the intact animal might eventually have therapeutic applicability to prevent or ameliorate the neurological damage seen in FAS.
Acknowledgements The authors thank Michael Paiva and Scarlet Raby for their excellent technical assistance. We also thank Amgen for their generous contribution of the GDNF and Regeneron for their generous contribution of the BDNF. This study was supported by NIAAA Grant AA09128. Douglas M. Bradley was supported by an NSF Predoctoral Fellowship and NIAAA Grant T32-AA07561, Francesca Beaman was supported by NIH Grant T35-HL07489, and D. Blaine Moore was supported by NIAAA Grants T32-AA07561 and AA05502.
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