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Alterations in Nigrostriatal Dopaminergic Function within BDNF Mutant Mice
Experimental Neurology 160, 500–507 (1999) Article ID exnr.1999.7225, available online at http://www.idealibrary.com on
Alterations in Nigrostriatal Dopaminergic Function within BDNF Mutant Mice Dean E. Dluzen, Gina M. Story, Kui Xu, Jan Kucera,* and Jon M. Walro Department of Anatomy, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, P.O. Box 95, Roostown, Ohio 44272-0095; and *Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, 02118 Received May 19, 1999; accepted August 23, 1999
INTRODUCTION The influence of brain-derived neurotrophic factor (BDNF) upon the nigrostriatal dopaminergic system was evaluated in weanling and adult mice carrying a targeted inactivated BDNF gene. Regional specificity of this BDNF mutation was assessed by assaying catecholamine concentrations within the corpus striatum, hypothalamus, and olfactory bulbs. In weanling mice dopamine, but not norepinephrine, concentrations within the corpus striatum of homozygous mutant (2/2) mice were significantly reduced with levels being 54% that of the wild-type controls (1/1) and 49% that of the heterozygous mutant (1/2) mice. While no differences were obtained among the three genotypes for hypothalamic dopamine, norepinephrine concentrations of 2/2 mice were significantly lower, being 62% of 1/1 mice and 49% of 1/2 mice. The dopamine concentrations of 2/2 mice within the olfactory bulb were significantly reduced (69%) compared to the 1/2, but not 1/1 mice. Olfactory bulb norepinephrine concentrations showed a statistically significant difference among each of the three conditions with minimal levels in 2/2 mice (62% of 1/1 and 45% of 1/2). In the adults, catecholamine concentrations were measured only in 1/1 and 1/2 mice since 2/2 mice do not typically survive past 21 days. Dopamine, but not norepinephrine, concentrations within the corpus striatum were significantly increased (116%) in 1/2 compared to 1/1 mice. No other statistically significant differences were obtained in catecholamine concentrations within the hypothalamus or olfactory bulb in these adult mice. These results show that homozygous BDNF mutations produce severe depletions within the nigrostriatal dopaminergic system and substantial reductions of norepinephrine within the hypothalamus and olfactory bulb. Interestingly, maximal catecholamine concentrations for all areas sampled at both ages were observed in the 1/2 mice. These latter findings may indicate some subtle changes in catecholamine functions resulting from a heterozygous BDNF mutation. r 1999 Academic Press Key Words: nigrostriatal; norepinephrine; dopamine; brain-derived neurotrophic factor; corpus striatum; hypothalamus; olfactory bulb.
0014-4886/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
Brain-derived neurotrophic factor (BDNF) affects a number of functions involving the nigrostriatal dopaminergic (NSDA) system. For example, BDNF increases DA within culture medium, most likely through its capacity to increase tyrosine hydroxylase activity (20–22, 35, 38, 40). These effects appear to result from a direct and selective action upon dopaminergic neurons (3, 4, 21, 22). Evidence from cell culture experiments has also revealed that BDNF increases both DA release (6) and DA uptake (4, 6, 22, 25) in a dose-dependent manner. In addition to these findings using cell cultures, it has been shown that the substantia nigra expresses BDNF (1) and BDNF mRNA has been reported within the mouse (19) and rat (34) corpus striatum (CS), although not all investigators have been able to detect BDNF mRNA within the rat CS (18, 30). In spite of the absence of consistently detectable BDNF mRNA within the rat CS, BDNF levels have been measured reliably at this site (12,24), which may be the result of BDNF transport to the CS from other brain structures (2, 9). A particularly intriguing and important role for BDNF in the NSDA system is that of neuroprotectant. Results from a number of reports have shown that BDNF can function as a NSDA neuroprotectant as indicated when tested with three putative, but mechanistically different DA neuronal neurotoxins, 6-hydroxydopamine, 1-methyl-4-phenylpyridinium, and the 6-hydroxylated derivative of LDOPA -TOPA (3, 21, 25, 28, 37, 39). Such effects may have significant implications in the therapeutic use of BDNF for the treatment of Parkinson’s Disease (26). The BDNF ‘‘knock-out’’ mouse has provided an additional model in which to study the functions of this neurotrophin. BDNF null mutant (2/2) mice lack detectable levels of BDNF mRNA, are reduced in size, show severely impaired motor behavior/coordination, and typically do not survive beyond 21 days of age (10, 15). Interestingly, no discernable loss of cells or generalized changes in brain cytoarchitecture (10, 27) and both substantia nigra and CS tyrosine hydroxylase were
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reported as normal in these 2/2 mice (15, 23). Heterozygous BDNF mutants (1/2) had BDNF mRNA levels that were approximately one-half that of the wild type (1/1) and these mice exhibited no obvious changes in behavior (15). Such ‘‘gene-dosage’’ responses have been reported for other parameters of BDNF actions, including effects upon vestibular and nodose/petrosal neurons (5, 15). The apparent absence of effects upon the NSDA system in these 2/2 mice is interesting in light of the plethora of effects observed when cultured DA neurons are exposed to BDNF. A fundamental determination that has yet to be performed involves measurements of DA concentrations within the CS of these 2/2 mice. Such an approach will provide a more direct and definitive evaluation of NSDA function in mice lacking the BDNF gene. Accordingly, in Experiment 1 CS catecholamine (DA and norepinephrine-NE) concentrations were measured in 1/1, 1/2, and 2/2 weanling mice. The weanling age group was used since this represents the maximal expected life span for 2/2 mice. To assess the specificity for the absence of BDNF effects upon the NSDA system, two other brain areas that have been shown to contain detectable levels of BDNF—hypothalamus and olfactory bulb (12, 24)— were also included for assessment of catecholamine concentrations. Finally, to evaluate potential differences which may be present between adult 1/1 and 1/2 animals, in Experiment 2 these determinations were performed in 60-day-old mice. MATERIALS AND METHODS
Animals. Mice carrying a targeted inactivation of the BDNF gene (27) were housed in plastic cages. These animals were fed and watered ad libitum and maintained on a 12:12 h light:dark cycle. Heterozygous mutants (1/2) were mated to produce 1/1, 1/2, and 2/2 offspring. Mice were genotyped by PCR and 2% agarose gel of electrophoresis of tail DNA. Primers for genotyping BDNF mice were BD1 atg aaaa gaa gta aac gtc cac; BD2-cca gca gaa aga gta gag gag; and PGK1 ggg aac ttc act agg gg used in a 1:1:1 ratio. The mutant fragment (200–300 bp) is slightly larger than the wild-type fragment and can be used to genotype mice as 1/1, 1/2, and 2/2. Homozygous mutant (2/2) mice live approximately 21–24 days and show a number of neural deficits, although 1/2 mice appear indistinguishable from 1/1 mice throughout development. Mice were euthanized by decapitation at 18–21 (Experiment 1) or 57–60 (Experiment 2) days of age and selected brain regions prepared for catecholamine analysis. The Animal Care and Use Committee at NEOUCOM, in accordance with NIH guidelines for the Care and Use of
Laboratory Animals, approved all experimental procedures. Tissue preparation. At euthanasia, the corpus striatum (CS), hypothalamus, and olfactory bulbs were removed from each animal and prepared for assay of catecholamine concentrations. To dissect the CS, the brain was bisected and the ventricles pried open revealing the CS. The CS was removed from within the perimeter of the corpus callosum of both hemispheres. For dissection of the hypothalamus, two lateral cuts were made in the hypothalamic sulci, an anterior cut at the border of the optic chiasm and a posterior cut at the level of the mammillary bodies to enable removal of the hypothalamic block. For the olfactory bulb, the olfactory tracts were severed and the olfactory bulb pried off the cribriform plate. The tissues from each of these three areas were weighed and placed in vials containing 500 µl cold (4°C) 0.1 N HClO4. All samples were sonicated, centrifuged, and an aliquot was removed from the supernatant for assay of dopamine (DA) and norepinephrine (NE). Catecholamine concentrations were then expressed as picograms of DA or NE per milligram of tissue weight. Catecholamine assay. Measurements of DA and NE were performed using HPLC-EC (ESA Inc.). A 100 3 4.6-mm 5-µm C-18 reverse phase column (Biophase ODS, BAS Inc.) with an isogradient mobile phase consisting of 50 mM sodium acetate, 27.4 mM citric acid, 10 mM sodium hydroxide, 0.1 mM sodium octyl sulfate, 0.1 mM EDTA, and 7% methanol in filtered deionized water was used with this system. The final pH of 4.5 was obtained with the addition of sodium hydroxide and the mobile phase was filtered (0.45 µm, Milipore Filter) prior to use. The DA and NE standards were diluted in 0.1 N HClO4 and doses of 12.5, 25, 50, 100, 200, and 400 pg/20 µl were used to construct a standard curve. Tissue supernatant samples in 20 µl were injected into the HPLC-EC. The sensitivity of this assay was ,12.5 pg/20 µl as defined by a response reliably detectable above baseline noise. Analysis. Concentrations of DA and NE within each of the three central nervous system sites sampled were subjected to a one-way ANOVA (1/1 vs 1/2 vs 2/2) for Experiment 1. Pairwise post-hoc comparisons were performed using the Scheffe test. For Experiment 2, an unpaired t test was used to compare catecholamine concentrations between 1/1 and 1/2 mice at each site sampled. A P , 0.05 was required for results to be considered statistically significant. RESULTS
Experiment 1 Analysis of the body weights from the three groups of weanling mice revealed an overall statistically signifi-
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cant difference (F2,37 5 122.4, P , 0.001). Post-hoc comparisons of these data substantiate the impaired nature of the 2/2 mice since their body weights (Mean 6 SEM: 4.3 1 0.4 g, n 5 11) were significantly lower (P , 0.001) and approximately 40% that of the 1/1 (10.4 6 0.3 g, n 5 19) and 1/2 (9.6 6 0.2 g, n 5 10) mice. No statistically significant differences in body weights were obtained between 1/1 and 1/2 mice. The catecholamine concentrations for the CS are presented in Figs. 1A (DA) and 1B (NE). An overall statistically significant difference was obtained for CS DA concentrations (F2,37 5 18.6, P , 0.001). Post-hoc comparisons indicated that DA concentrations of the 2/2 animals were significantly lower than that of the 1/1 (P , 0.001) and the 1/2 (P , 0.001) animals. No statistically significant differences between the latter two conditions were obtained. No overall statistically significant differences in CS NE were obtained among the three groups. Hypothalamic DA concentrations are presented in Fig. 2A and analysis of these levels reveals an absence of an overall statistically significant difference. By contrast, hypothalamic NE concentrations (Fig. 2B) showed a statistically significant difference (F2,37 5 12.89, P , 0.001). Post-hoc comparisons revealed that hypothalamic NE concentrations in the 2/2 animals were significantly lower than the 1/1 (P , 0.005) and 1/2 (P , 0.001) mice. No statistically
significant differences were obtained between the 1/1 and 1/2 animals. Both olfactory bulb DA (Fig. 3A; F2,37 5 3.34, P , 0.05) and NE (Fig. 3B; F2,37 5 17.48, P , 0.001) concentrations showed an overall statistically significant difference. Post-hoc analysis of olfactory bulb DA concentrations indicated no statistically significant pair-wise differences among the 1/1, 1/2, and 2/2 mice. Posthoc analysis of olfactory bulb NE concentrations revealed that each of the three groups differed among each other: 1/1 vs 1/2 (P , 0.0003), 1/1 vs 2/2 (P , 0.019) and 1/2 vs 2/2 (P , 0.001). Experiment 2 Analysis of body weights between 1/1 and 2/2 adult mice revealed a statistically significant difference (t 5 3.64, P , 0.01). The body weights of the 1/1 mice (22.2 6 0.8 g, n 5 15) were lower than that of the 1/2 mice (27.8 6 1.5 g, n 5 9). These differences are likely due to the relatively greater number of females represented within the 1/1 group (12/15) compared with the 1/2 group (4/9). The catecholamine concentrations for the CS of adult mice are presented in Figs. 4A (DA) and 4B (NE). DA concentrations of the 1/2 mice were significantly greater (t 5 2.15, P , 0.05) than that of the 1/1 mice. No statistically significant differences in CS NE concen-
FIG. 1. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the corpus striatum of weanling controls (1/1), heterozygous (1/2), and homozygous (2/2) BDNF mutant mice. Numbers within the histograms represent the numbers of animals assayed within each genotype. Within the histograms of each panel, superscript letters are used to indicate conditions that differ significantly. Histograms with identical letters fail to differ, while those with different lettered superscripts indicate groups that differ significantly. NS (not significant) indicates an absence of statistically significant differences.
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FIG. 2. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the hypothalamus of weanling controls (1/1), heterozygous (1/2), and homozygous (2/2) BDNF mutant mice as described in the legend of Fig. 1.
trations were obtained between 1/1 and 1/2 mice. A summary of the remaining catecholamine concentrations from the 1/1 and 1/2 adult mice of Experiment 2 are presented in Table 1. No statistically significant differences between 1/1 and 1/2 adult mice were obtained for either DA or NE within the hypothalamus or olfactory bulb.
DISCUSSION
The present results indicate a number of interesting changes in central nervous system catecholamine concentrations, which occur in homozygous BDNF mutant mice. Of the areas sampled, the greatest deficits in catecholamine levels were observed for DA concentra-
FIG. 3. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the olfactory bulb of weanling controls (1/1), heterozygous (1/2), and homozygous (2/2) BDNF mutant mice as described in the legend of Fig. 1.
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FIG. 4. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the corpus striatum of adult (57–60 days) controls (1/1) and heterozygous (1/2) BDNF mutant mice as described in the legend of Fig. 1.
tions within the CS, with levels in this group being 54% of 1/1 wild-type controls in weanling mice. In addition, only CS DA concentrations of adults showed statistically significant differences with levels of 1/2 being greater than 1/1 mice. Although there was a specificity in CS catecholamine responsiveness, since no changes were obtained for CS NE levels in either weanling or adult mice, changes in catecholamine concentrations within weanling 2/2 mice were clearly not limited to the NSDA system. Notably, NE concentrations within both the hypothalamus (62% of 1/1 mice) and olfactory bulb (62% of 1/1 TABLE 1 Hypothalamic and Olfactory Bulb Dopamine and Norepinephrine Concentrations from Adult 1/1 and 1/2 BDNF Mice (mean 6 SEM, pg/mg) Hypothalamus Dopamine
Norepinephrine
1/1
1/2
503 6 27
535 6 24
1/1
1/2
2340 6 59
2410 6 101
Olfactory bulb Dopamine
Norepinephrine
1/1
1/2
1/1
1/2
162 6 6
183 6 14
256 6 16
271 6 24
Note. No statistically significant differences were obtained between 1/1 and 1/2 mice for any comparisons; however, catecholamine concentrations of 1/2 mice were consistently higher than that of 1/1 littermates.
mice) were significantly decreased in the 2/2 compared with their wild-type controls. Our data provide no indication of any classical ‘‘gene dosage’’ effects (1/1 . 1/2 . 2/2) with regard to catecholamine concentrations. However, for both catecholamines in all areas and ages sampled maximal concentrations were observed in the 1/2 animals, which may represent a subtle aspect of ‘‘gene dosage’’ resulting from the heterozygous mutation as described below. The severe deficits in CS DA concentrations coincide well with the findings from cell culture studies, which indicate that a variety of dopaminergic functions including increases in DA levels (20, 22, 35), tyrosine hydroxylase (21, 38, 40), DA release (6), and DA uptake (4, 6, 22, 25) all result from BDNF treatment. Moreover, the fact that both BDNF mRNA (19) and concentrations (12, 24) were evident in the CS further support a consequential role for this neurotrophin within the NSDA system. It is more difficult to reconcile the profound reductions in CS DA concentrations of the present report with other data from 2/2 mice, indicating no apparent change in tyrosine hydroxylase immunocytochemistry (15, 23). It should be noted that immunocytochemistry typically indicates either presence or absence of this enzyme and therefore may not be sufficiently sensitive to identify changes in amount. Moreover, while tyrosine hydroxylase can, and in many cases does, provide an index of CS DA levels, these two parameters are not perfectly correlated due to the intervening steps between tyrosine hydroxylase and DA production. Our present data would indicate that the final product of the NSDA system—DA, is significantly reduced in 2/2 mice, but this result appears not necessarily attributable to activity of tyrosine hydroxylase. There are a number of
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functions within DA neurons which could affect DA concentrations in the absence of an effect upon tyrosine hydroxylase. Since DA uptake has been shown to be increased in cell cultures treated with BDNF (4, 6, 22, 25), the potential for diminished uptake activity in 2/2 mice could have the effect of decreasing DA concentrations. In addition, a change in the sequestering of DA within vesicles of DA neurons will also affect DA concentrations as unprotected DA can be more readily metabolized. It has been demonstrated that BDNF protects DA neurons from 1-methyl-4-phenylpyridinium by increasing the sequestration of 1-methyl-4phenylpyridinium into vesicles (3). If BDNF possess the property of increasing vesicle storage, the absence of this factor in 2/2 mice could reduce vesicle storage thereby leaving more DA available for metabolism and attenuating DA concentrations. While the focus of the present report was upon the NSDA system, it is clear that weanling 2/2 mice show a relatively wide range of catecholamine deficiencies. For example, the NE levels in both the hypothalamus and olfactory bulb are significantly decreased in 2/2 compared with 1/1 mice. There are data that show that BDNF can differentially affect neuronal systems within the hypothalamus (29) and relatively high levels of BDNF, second only to the hippocampus, were observed within the hypothalamus (24). Our data would suggest that at least one potential action of this hypothalamic BDNF may be to modulate NE functioning at this site. Similarly, the effects of BDNF on the olfactory bulb NE system are also quite substantial. Like that observed for DA neurons, BDNF also acts as a survival factor for NE neurons of the locus coeruleus (18). Since the source of the olfactory bulb NE system is the locus coeruleus (31, 36), the findings that BDNF acts upon this system may serve as the basis for the present results upon the olfactory bulb NE concentrations. Although levels of CS DA were higher in 1/2 versus 1/1 weanling mice, these differences failed to achieve a statistically significant difference. These differences were accentuated in the adult with levels in 1/2 mice now being significantly greater than that of the 1/1 mice. This effect was quite specific as CS NE concentrations in these adults were not different and neither the hypothalamus or olfactory bulb showed any differences in DA or NE concentrations. The augmented DA concentrations in these 1/2 mice may be indicative of a subtle, but significant perturbation of the NSDA system. There is evidence that DA can produce neuronal damage, as shown both in culture (32) and following direct intrastriatal infusions (17). Moreover, the catabolic products of this amine can be toxic to DA neurons (7, 8, 16) and it has been suggested that such mechanisms may contribute to the DA neuronal degeneration observed in Parkinson’s disease (33). We have previously speculated that an enhanced level of basal NSDA
activity may be detrimental to NSDA functioning (14). Over the lifespan of the animal these effects can produce cumulative changes resulting in a severely impaired NSDA system. In this regard, the 1/2 BDNF mouse may represent a particularly effective model in which to study degenerative changes in the NSDA system. A final issue of the present report involves the results of the 1/2 mice. An initial review of their data would seem to indicate that these 1/2 animals failed to show a classic ‘‘gene dosage’’ effect, consisting of intermediate levels to that of the 1/1 and 2/2 mice, as has been reported in other paradigms (5, 11). Upon closer inspection of our data it is clear that we are obtaining a ‘‘gene dosage’’ effect, albeit not in the classical sense or direction of 1/1 . 1/2 . 2/2. This phenomenon of 1/2 . 1/1 was obtained for both NE and DA in all areas and ages sampled but may best be illustrated through the olfactory bulb NE data in the weanling and CS DA of the adult. NE levels at this site showed statistically significant differences among each of the three genotypes with the ordering being 1/2 . 1/1 . 2/2. The significantly increased olfactory bulb NE of the 1/2 mice may represent an altered function indicative of a deficit in this neurotransmitter system. These increased olfactory bulb NE concentrations which were obtained in the 1/2 mice are similar to that observed in aged rats. It was suggested that such augmented concentrations characterize an age related deficit, possibly involving an impaired capacity for release (13). In an analogous manner, the significantly increased olfactory bulb NE levels of the 1/2 mice may also involve a ‘‘gene dosage’’-dependent impaired release function resulting from this heterozygous mutation. Similarly, the developmental changes which occur in CS DA concentrations of 1/2 mice, with levels being higher in weanlings and significantly greater in the adults, may reflect a gene-dosage effect capable of producing degenerative changes within the NSDA system. ACKNOWLEDGMENT We thank Linda I. Anderson for her expert technical contributions to this project. This work was in part supported by OBR Research Challenge Grants from NEOUCOM to D.E.D. and J.M.W.
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Alterations in Nigrostriatal Dopaminergic Function within BDNF Mutant Mice Dean E. Dluzen, Gina M. Story, Kui Xu, Jan Kucera,* and Jon M. Walro Department of Anatomy, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, P.O. Box 95, Roostown, Ohio 44272-0095; and *Department of Neurology, Boston University School of Medicine, Boston, Massachusetts, 02118 Received May 19, 1999; accepted August 23, 1999
INTRODUCTION The influence of brain-derived neurotrophic factor (BDNF) upon the nigrostriatal dopaminergic system was evaluated in weanling and adult mice carrying a targeted inactivated BDNF gene. Regional specificity of this BDNF mutation was assessed by assaying catecholamine concentrations within the corpus striatum, hypothalamus, and olfactory bulbs. In weanling mice dopamine, but not norepinephrine, concentrations within the corpus striatum of homozygous mutant (2/2) mice were significantly reduced with levels being 54% that of the wild-type controls (1/1) and 49% that of the heterozygous mutant (1/2) mice. While no differences were obtained among the three genotypes for hypothalamic dopamine, norepinephrine concentrations of 2/2 mice were significantly lower, being 62% of 1/1 mice and 49% of 1/2 mice. The dopamine concentrations of 2/2 mice within the olfactory bulb were significantly reduced (69%) compared to the 1/2, but not 1/1 mice. Olfactory bulb norepinephrine concentrations showed a statistically significant difference among each of the three conditions with minimal levels in 2/2 mice (62% of 1/1 and 45% of 1/2). In the adults, catecholamine concentrations were measured only in 1/1 and 1/2 mice since 2/2 mice do not typically survive past 21 days. Dopamine, but not norepinephrine, concentrations within the corpus striatum were significantly increased (116%) in 1/2 compared to 1/1 mice. No other statistically significant differences were obtained in catecholamine concentrations within the hypothalamus or olfactory bulb in these adult mice. These results show that homozygous BDNF mutations produce severe depletions within the nigrostriatal dopaminergic system and substantial reductions of norepinephrine within the hypothalamus and olfactory bulb. Interestingly, maximal catecholamine concentrations for all areas sampled at both ages were observed in the 1/2 mice. These latter findings may indicate some subtle changes in catecholamine functions resulting from a heterozygous BDNF mutation. r 1999 Academic Press Key Words: nigrostriatal; norepinephrine; dopamine; brain-derived neurotrophic factor; corpus striatum; hypothalamus; olfactory bulb.
0014-4886/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.
Brain-derived neurotrophic factor (BDNF) affects a number of functions involving the nigrostriatal dopaminergic (NSDA) system. For example, BDNF increases DA within culture medium, most likely through its capacity to increase tyrosine hydroxylase activity (20–22, 35, 38, 40). These effects appear to result from a direct and selective action upon dopaminergic neurons (3, 4, 21, 22). Evidence from cell culture experiments has also revealed that BDNF increases both DA release (6) and DA uptake (4, 6, 22, 25) in a dose-dependent manner. In addition to these findings using cell cultures, it has been shown that the substantia nigra expresses BDNF (1) and BDNF mRNA has been reported within the mouse (19) and rat (34) corpus striatum (CS), although not all investigators have been able to detect BDNF mRNA within the rat CS (18, 30). In spite of the absence of consistently detectable BDNF mRNA within the rat CS, BDNF levels have been measured reliably at this site (12,24), which may be the result of BDNF transport to the CS from other brain structures (2, 9). A particularly intriguing and important role for BDNF in the NSDA system is that of neuroprotectant. Results from a number of reports have shown that BDNF can function as a NSDA neuroprotectant as indicated when tested with three putative, but mechanistically different DA neuronal neurotoxins, 6-hydroxydopamine, 1-methyl-4-phenylpyridinium, and the 6-hydroxylated derivative of LDOPA -TOPA (3, 21, 25, 28, 37, 39). Such effects may have significant implications in the therapeutic use of BDNF for the treatment of Parkinson’s Disease (26). The BDNF ‘‘knock-out’’ mouse has provided an additional model in which to study the functions of this neurotrophin. BDNF null mutant (2/2) mice lack detectable levels of BDNF mRNA, are reduced in size, show severely impaired motor behavior/coordination, and typically do not survive beyond 21 days of age (10, 15). Interestingly, no discernable loss of cells or generalized changes in brain cytoarchitecture (10, 27) and both substantia nigra and CS tyrosine hydroxylase were
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reported as normal in these 2/2 mice (15, 23). Heterozygous BDNF mutants (1/2) had BDNF mRNA levels that were approximately one-half that of the wild type (1/1) and these mice exhibited no obvious changes in behavior (15). Such ‘‘gene-dosage’’ responses have been reported for other parameters of BDNF actions, including effects upon vestibular and nodose/petrosal neurons (5, 15). The apparent absence of effects upon the NSDA system in these 2/2 mice is interesting in light of the plethora of effects observed when cultured DA neurons are exposed to BDNF. A fundamental determination that has yet to be performed involves measurements of DA concentrations within the CS of these 2/2 mice. Such an approach will provide a more direct and definitive evaluation of NSDA function in mice lacking the BDNF gene. Accordingly, in Experiment 1 CS catecholamine (DA and norepinephrine-NE) concentrations were measured in 1/1, 1/2, and 2/2 weanling mice. The weanling age group was used since this represents the maximal expected life span for 2/2 mice. To assess the specificity for the absence of BDNF effects upon the NSDA system, two other brain areas that have been shown to contain detectable levels of BDNF—hypothalamus and olfactory bulb (12, 24)— were also included for assessment of catecholamine concentrations. Finally, to evaluate potential differences which may be present between adult 1/1 and 1/2 animals, in Experiment 2 these determinations were performed in 60-day-old mice. MATERIALS AND METHODS
Animals. Mice carrying a targeted inactivation of the BDNF gene (27) were housed in plastic cages. These animals were fed and watered ad libitum and maintained on a 12:12 h light:dark cycle. Heterozygous mutants (1/2) were mated to produce 1/1, 1/2, and 2/2 offspring. Mice were genotyped by PCR and 2% agarose gel of electrophoresis of tail DNA. Primers for genotyping BDNF mice were BD1 atg aaaa gaa gta aac gtc cac; BD2-cca gca gaa aga gta gag gag; and PGK1 ggg aac ttc act agg gg used in a 1:1:1 ratio. The mutant fragment (200–300 bp) is slightly larger than the wild-type fragment and can be used to genotype mice as 1/1, 1/2, and 2/2. Homozygous mutant (2/2) mice live approximately 21–24 days and show a number of neural deficits, although 1/2 mice appear indistinguishable from 1/1 mice throughout development. Mice were euthanized by decapitation at 18–21 (Experiment 1) or 57–60 (Experiment 2) days of age and selected brain regions prepared for catecholamine analysis. The Animal Care and Use Committee at NEOUCOM, in accordance with NIH guidelines for the Care and Use of
Laboratory Animals, approved all experimental procedures. Tissue preparation. At euthanasia, the corpus striatum (CS), hypothalamus, and olfactory bulbs were removed from each animal and prepared for assay of catecholamine concentrations. To dissect the CS, the brain was bisected and the ventricles pried open revealing the CS. The CS was removed from within the perimeter of the corpus callosum of both hemispheres. For dissection of the hypothalamus, two lateral cuts were made in the hypothalamic sulci, an anterior cut at the border of the optic chiasm and a posterior cut at the level of the mammillary bodies to enable removal of the hypothalamic block. For the olfactory bulb, the olfactory tracts were severed and the olfactory bulb pried off the cribriform plate. The tissues from each of these three areas were weighed and placed in vials containing 500 µl cold (4°C) 0.1 N HClO4. All samples were sonicated, centrifuged, and an aliquot was removed from the supernatant for assay of dopamine (DA) and norepinephrine (NE). Catecholamine concentrations were then expressed as picograms of DA or NE per milligram of tissue weight. Catecholamine assay. Measurements of DA and NE were performed using HPLC-EC (ESA Inc.). A 100 3 4.6-mm 5-µm C-18 reverse phase column (Biophase ODS, BAS Inc.) with an isogradient mobile phase consisting of 50 mM sodium acetate, 27.4 mM citric acid, 10 mM sodium hydroxide, 0.1 mM sodium octyl sulfate, 0.1 mM EDTA, and 7% methanol in filtered deionized water was used with this system. The final pH of 4.5 was obtained with the addition of sodium hydroxide and the mobile phase was filtered (0.45 µm, Milipore Filter) prior to use. The DA and NE standards were diluted in 0.1 N HClO4 and doses of 12.5, 25, 50, 100, 200, and 400 pg/20 µl were used to construct a standard curve. Tissue supernatant samples in 20 µl were injected into the HPLC-EC. The sensitivity of this assay was ,12.5 pg/20 µl as defined by a response reliably detectable above baseline noise. Analysis. Concentrations of DA and NE within each of the three central nervous system sites sampled were subjected to a one-way ANOVA (1/1 vs 1/2 vs 2/2) for Experiment 1. Pairwise post-hoc comparisons were performed using the Scheffe test. For Experiment 2, an unpaired t test was used to compare catecholamine concentrations between 1/1 and 1/2 mice at each site sampled. A P , 0.05 was required for results to be considered statistically significant. RESULTS
Experiment 1 Analysis of the body weights from the three groups of weanling mice revealed an overall statistically signifi-
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cant difference (F2,37 5 122.4, P , 0.001). Post-hoc comparisons of these data substantiate the impaired nature of the 2/2 mice since their body weights (Mean 6 SEM: 4.3 1 0.4 g, n 5 11) were significantly lower (P , 0.001) and approximately 40% that of the 1/1 (10.4 6 0.3 g, n 5 19) and 1/2 (9.6 6 0.2 g, n 5 10) mice. No statistically significant differences in body weights were obtained between 1/1 and 1/2 mice. The catecholamine concentrations for the CS are presented in Figs. 1A (DA) and 1B (NE). An overall statistically significant difference was obtained for CS DA concentrations (F2,37 5 18.6, P , 0.001). Post-hoc comparisons indicated that DA concentrations of the 2/2 animals were significantly lower than that of the 1/1 (P , 0.001) and the 1/2 (P , 0.001) animals. No statistically significant differences between the latter two conditions were obtained. No overall statistically significant differences in CS NE were obtained among the three groups. Hypothalamic DA concentrations are presented in Fig. 2A and analysis of these levels reveals an absence of an overall statistically significant difference. By contrast, hypothalamic NE concentrations (Fig. 2B) showed a statistically significant difference (F2,37 5 12.89, P , 0.001). Post-hoc comparisons revealed that hypothalamic NE concentrations in the 2/2 animals were significantly lower than the 1/1 (P , 0.005) and 1/2 (P , 0.001) mice. No statistically
significant differences were obtained between the 1/1 and 1/2 animals. Both olfactory bulb DA (Fig. 3A; F2,37 5 3.34, P , 0.05) and NE (Fig. 3B; F2,37 5 17.48, P , 0.001) concentrations showed an overall statistically significant difference. Post-hoc analysis of olfactory bulb DA concentrations indicated no statistically significant pair-wise differences among the 1/1, 1/2, and 2/2 mice. Posthoc analysis of olfactory bulb NE concentrations revealed that each of the three groups differed among each other: 1/1 vs 1/2 (P , 0.0003), 1/1 vs 2/2 (P , 0.019) and 1/2 vs 2/2 (P , 0.001). Experiment 2 Analysis of body weights between 1/1 and 2/2 adult mice revealed a statistically significant difference (t 5 3.64, P , 0.01). The body weights of the 1/1 mice (22.2 6 0.8 g, n 5 15) were lower than that of the 1/2 mice (27.8 6 1.5 g, n 5 9). These differences are likely due to the relatively greater number of females represented within the 1/1 group (12/15) compared with the 1/2 group (4/9). The catecholamine concentrations for the CS of adult mice are presented in Figs. 4A (DA) and 4B (NE). DA concentrations of the 1/2 mice were significantly greater (t 5 2.15, P , 0.05) than that of the 1/1 mice. No statistically significant differences in CS NE concen-
FIG. 1. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the corpus striatum of weanling controls (1/1), heterozygous (1/2), and homozygous (2/2) BDNF mutant mice. Numbers within the histograms represent the numbers of animals assayed within each genotype. Within the histograms of each panel, superscript letters are used to indicate conditions that differ significantly. Histograms with identical letters fail to differ, while those with different lettered superscripts indicate groups that differ significantly. NS (not significant) indicates an absence of statistically significant differences.
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FIG. 2. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the hypothalamus of weanling controls (1/1), heterozygous (1/2), and homozygous (2/2) BDNF mutant mice as described in the legend of Fig. 1.
trations were obtained between 1/1 and 1/2 mice. A summary of the remaining catecholamine concentrations from the 1/1 and 1/2 adult mice of Experiment 2 are presented in Table 1. No statistically significant differences between 1/1 and 1/2 adult mice were obtained for either DA or NE within the hypothalamus or olfactory bulb.
DISCUSSION
The present results indicate a number of interesting changes in central nervous system catecholamine concentrations, which occur in homozygous BDNF mutant mice. Of the areas sampled, the greatest deficits in catecholamine levels were observed for DA concentra-
FIG. 3. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the olfactory bulb of weanling controls (1/1), heterozygous (1/2), and homozygous (2/2) BDNF mutant mice as described in the legend of Fig. 1.
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FIG. 4. Mean 6 SEM dopamine (A) and norepinephrine (B) concentrations (pg/mg) from the corpus striatum of adult (57–60 days) controls (1/1) and heterozygous (1/2) BDNF mutant mice as described in the legend of Fig. 1.
tions within the CS, with levels in this group being 54% of 1/1 wild-type controls in weanling mice. In addition, only CS DA concentrations of adults showed statistically significant differences with levels of 1/2 being greater than 1/1 mice. Although there was a specificity in CS catecholamine responsiveness, since no changes were obtained for CS NE levels in either weanling or adult mice, changes in catecholamine concentrations within weanling 2/2 mice were clearly not limited to the NSDA system. Notably, NE concentrations within both the hypothalamus (62% of 1/1 mice) and olfactory bulb (62% of 1/1 TABLE 1 Hypothalamic and Olfactory Bulb Dopamine and Norepinephrine Concentrations from Adult 1/1 and 1/2 BDNF Mice (mean 6 SEM, pg/mg) Hypothalamus Dopamine
Norepinephrine
1/1
1/2
503 6 27
535 6 24
1/1
1/2
2340 6 59
2410 6 101
Olfactory bulb Dopamine
Norepinephrine
1/1
1/2
1/1
1/2
162 6 6
183 6 14
256 6 16
271 6 24
Note. No statistically significant differences were obtained between 1/1 and 1/2 mice for any comparisons; however, catecholamine concentrations of 1/2 mice were consistently higher than that of 1/1 littermates.
mice) were significantly decreased in the 2/2 compared with their wild-type controls. Our data provide no indication of any classical ‘‘gene dosage’’ effects (1/1 . 1/2 . 2/2) with regard to catecholamine concentrations. However, for both catecholamines in all areas and ages sampled maximal concentrations were observed in the 1/2 animals, which may represent a subtle aspect of ‘‘gene dosage’’ resulting from the heterozygous mutation as described below. The severe deficits in CS DA concentrations coincide well with the findings from cell culture studies, which indicate that a variety of dopaminergic functions including increases in DA levels (20, 22, 35), tyrosine hydroxylase (21, 38, 40), DA release (6), and DA uptake (4, 6, 22, 25) all result from BDNF treatment. Moreover, the fact that both BDNF mRNA (19) and concentrations (12, 24) were evident in the CS further support a consequential role for this neurotrophin within the NSDA system. It is more difficult to reconcile the profound reductions in CS DA concentrations of the present report with other data from 2/2 mice, indicating no apparent change in tyrosine hydroxylase immunocytochemistry (15, 23). It should be noted that immunocytochemistry typically indicates either presence or absence of this enzyme and therefore may not be sufficiently sensitive to identify changes in amount. Moreover, while tyrosine hydroxylase can, and in many cases does, provide an index of CS DA levels, these two parameters are not perfectly correlated due to the intervening steps between tyrosine hydroxylase and DA production. Our present data would indicate that the final product of the NSDA system—DA, is significantly reduced in 2/2 mice, but this result appears not necessarily attributable to activity of tyrosine hydroxylase. There are a number of
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functions within DA neurons which could affect DA concentrations in the absence of an effect upon tyrosine hydroxylase. Since DA uptake has been shown to be increased in cell cultures treated with BDNF (4, 6, 22, 25), the potential for diminished uptake activity in 2/2 mice could have the effect of decreasing DA concentrations. In addition, a change in the sequestering of DA within vesicles of DA neurons will also affect DA concentrations as unprotected DA can be more readily metabolized. It has been demonstrated that BDNF protects DA neurons from 1-methyl-4-phenylpyridinium by increasing the sequestration of 1-methyl-4phenylpyridinium into vesicles (3). If BDNF possess the property of increasing vesicle storage, the absence of this factor in 2/2 mice could reduce vesicle storage thereby leaving more DA available for metabolism and attenuating DA concentrations. While the focus of the present report was upon the NSDA system, it is clear that weanling 2/2 mice show a relatively wide range of catecholamine deficiencies. For example, the NE levels in both the hypothalamus and olfactory bulb are significantly decreased in 2/2 compared with 1/1 mice. There are data that show that BDNF can differentially affect neuronal systems within the hypothalamus (29) and relatively high levels of BDNF, second only to the hippocampus, were observed within the hypothalamus (24). Our data would suggest that at least one potential action of this hypothalamic BDNF may be to modulate NE functioning at this site. Similarly, the effects of BDNF on the olfactory bulb NE system are also quite substantial. Like that observed for DA neurons, BDNF also acts as a survival factor for NE neurons of the locus coeruleus (18). Since the source of the olfactory bulb NE system is the locus coeruleus (31, 36), the findings that BDNF acts upon this system may serve as the basis for the present results upon the olfactory bulb NE concentrations. Although levels of CS DA were higher in 1/2 versus 1/1 weanling mice, these differences failed to achieve a statistically significant difference. These differences were accentuated in the adult with levels in 1/2 mice now being significantly greater than that of the 1/1 mice. This effect was quite specific as CS NE concentrations in these adults were not different and neither the hypothalamus or olfactory bulb showed any differences in DA or NE concentrations. The augmented DA concentrations in these 1/2 mice may be indicative of a subtle, but significant perturbation of the NSDA system. There is evidence that DA can produce neuronal damage, as shown both in culture (32) and following direct intrastriatal infusions (17). Moreover, the catabolic products of this amine can be toxic to DA neurons (7, 8, 16) and it has been suggested that such mechanisms may contribute to the DA neuronal degeneration observed in Parkinson’s disease (33). We have previously speculated that an enhanced level of basal NSDA
activity may be detrimental to NSDA functioning (14). Over the lifespan of the animal these effects can produce cumulative changes resulting in a severely impaired NSDA system. In this regard, the 1/2 BDNF mouse may represent a particularly effective model in which to study degenerative changes in the NSDA system. A final issue of the present report involves the results of the 1/2 mice. An initial review of their data would seem to indicate that these 1/2 animals failed to show a classic ‘‘gene dosage’’ effect, consisting of intermediate levels to that of the 1/1 and 2/2 mice, as has been reported in other paradigms (5, 11). Upon closer inspection of our data it is clear that we are obtaining a ‘‘gene dosage’’ effect, albeit not in the classical sense or direction of 1/1 . 1/2 . 2/2. This phenomenon of 1/2 . 1/1 was obtained for both NE and DA in all areas and ages sampled but may best be illustrated through the olfactory bulb NE data in the weanling and CS DA of the adult. NE levels at this site showed statistically significant differences among each of the three genotypes with the ordering being 1/2 . 1/1 . 2/2. The significantly increased olfactory bulb NE of the 1/2 mice may represent an altered function indicative of a deficit in this neurotransmitter system. These increased olfactory bulb NE concentrations which were obtained in the 1/2 mice are similar to that observed in aged rats. It was suggested that such augmented concentrations characterize an age related deficit, possibly involving an impaired capacity for release (13). In an analogous manner, the significantly increased olfactory bulb NE levels of the 1/2 mice may also involve a ‘‘gene dosage’’-dependent impaired release function resulting from this heterozygous mutation. Similarly, the developmental changes which occur in CS DA concentrations of 1/2 mice, with levels being higher in weanlings and significantly greater in the adults, may reflect a gene-dosage effect capable of producing degenerative changes within the NSDA system. ACKNOWLEDGMENT We thank Linda I. Anderson for her expert technical contributions to this project. This work was in part supported by OBR Research Challenge Grants from NEOUCOM to D.E.D. and J.M.W.
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