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Inactivation of the Dopamine Transporter Reveals Essential Roles of Dopamine in the Control of Locomotion, Psychostimulant Response, and Pituitary Function
Inactivation of DA Transporter
I79
2. Wall, S. C., Gu, H. H., and Rudnick, G. (1995). Biogenic amine flux mediated by cloned transporters stabiy expressed in cultured cell lines: Amphetamine specificity for inhibition and efflux. Mol. Pharmacol. 47, 544-550. 3. Gu, H. H., Ahn, J., Caplan, M. J., Blakely, R. D., Levey, A. I., and Ruduick, G. (1996). Cell-specific sorting of biogenic amine transporters expressed in epithelial cells. I. Biol. Chem. 271, 18100-18106. 4. Dotti, C. G., and Simons, K. (1990).Polarized sorting of viral glycoproteins to axons and dendrites of hippocampal neurons in culture. Cell 62, 63-72. 5 . Giros,B., Wang, Y., Suter, S., Mcleskey, S., Pitl, C., Caron, M. { 1994).Delineation of discrete domains for substrate, cocaine, and tricyclic antidepressant interactions using chimeric dopamine-norepinephrine transporters. J . Biol. Chem. 269, 15985-15988.
Fabio Furnagall&*Sara Jones,* Roger BOSS^,* Mohamed Jaber," Bruno Giros,* Cristina Missale,t R. Mark Wightman,$ and Marc G. Caron* "Howard Hughes Medical Institute Laboratories Departments of Cell Biology and Medicine Duke University Medical Center Durham, N o r t h Carolina 277 I0 tCurriculum in Neurobiology and Department of Chemistry University of North Carolina Chapel Hill, N o r t h Carolina 275 14
$Division of Pharmacology Department of Biomedical Sciences and Biotechnology University of Brescia Brescia, Italy
Inactivation of the Dopamine Transporter Reveals Essential Roles of Dopamine in the Control of Locomotion, Psychostimulant Response, and Pituitary Function Dopamine (DA) is an important modulator of many physiological functions. In the central nervous system (CNS), DA is involved in the control of movement, cognition, and affect, as well as neuroendocrine secretion. In the Advances in Pharmacolugy. Vulume 42 Copyright 0 1998 by Academic Press. All right, of reproduction 1054-3589198 $25.00
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any form reserved.
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periphery, DA regulates pituitary and parathyroid hormone synthesis and secretion, in addition to modulating certain retinal, cardiovascular, and kidney functions. DA is also thought to influence gastrointestinal physiology (1, 2). In the CNS, the DA transporter (DAT) presumably plays an important role in terminating dopaminergic transmission by uptaking DA from extracellular spaces back into presynaptic terminals. The role of the DAT in the CNS has been largely inferred from the clinical and psychosocial effects of drugs such as antidepressants and psychostimulants. These drugs interfere with the function of DAT. However, the involvement of DAT in the peripheral as well as the hypothalamic-neuroendocrine actions of DA has remained more enigmatic. DAT belongs to the large family of Na+- and C1--dependent transporters containing 12 putative transmembrane domains. Other members of this family include transporters for norepinephrine, serotonin, gamma aminobutyric acid, glycine, creatine, and betaine. In an attempt to develop an animal model in which the role of the DAT in the control of dopaminergic neurotransmission and in other physiological functions of DA could be directly examined, we have inactivated the mouse DAT gene by homologous recombination (3). Homozygous DAT knockout (-/-) animals are viable. However, adult (-/--) DAT animals show a pronounced weight deficit (50-70% of normal animals). (-/-) DAT mothers have a markedly reduced ability to nurse. The most obvious behavioral characteristic of these mice is a five- sixfold increase in spontaneous locomotor activity when tested in an open field. The level of activity of the (-/-) DAT animals is nearly as high as that achieved by treatment of wild-type animals with psychostimulants. Indeed, treatments of ( -/- ) DAT mice with high doses of cocaine or amphetamine yielded no further overall increase in locomotor activity, indicating that the locomotor effects of these drugs is mediated by interactions with DAT. These findings suggest that, in the absence of DA uptake, ( - I - ) DAT animals are hyperdopaminergic. In an attempt to examine whether this hyperdopaminergic behavioral phenotype was reflected at the biochemical level, several parameters were examined. Messenger RNAs for genes that are normally under dopaminergic control, such as preproenkephalin and dynorphin, showed variations indicative of increased dopaminergic tone. Similarly mRNAs for pre- and postsynaptic D2 dopamine receptors, as well as striatal D1 receptors, were markedly (>50%) diminished. As assessed by autoradiography, D1 and D2 receptor numbers in the striatal and ventral midbrain areas were also decreased by more than 50%, presumably reflecting downregulation of these receptors in response to the elevated dopaminergic tone of these animals. Even more interestingly, at the neurochemical level, these animals displayed properties reminiscent of what might be expected of parkinsonian animals. When striatal levels of DA were measured by high-performance liquid chromatography, they were found to be 5-10% of those of normal animals. Although at the gross morphological level the dopaminergic cell bodies of the substantia nigra-ventral tegmental area and the terminal fields of the striatum appeared essentially normal, in situ hybridization and Western blots for tyrosine hydroxylase (TH)in these areas revealed important adaptive changes. A marked decrease in the levels of TH protein was observed in these regions with no significant
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181
changes in TH mRNA, suggesting that posttranslational control mechanisms might be important in regulating TH activity in dopaminergic neurons (MJ, SJ, RB, BG, and MGC, in preparation). Thus, in the absence of a functional DAT, the (-/-) DAT mice have undergone major adaptive changes in their biochemical and neurochemical mediators of dopaminergic transmission in an attempt to dampen the presumed increased DA signal. This situation is schematized in Figure 1. However, none of these changes appear sufficient to effectively quench the increased signal, because the ( -/-) DAT animals still display spontaneous hyperlocomotion. This paradox may be explained when one considers that, as measured by fast cyclic voltammetry, DA persists at the synapse of (-/-.) DAT animals about 100-300 times longer than in normal animals (SJ, R. G, MJ, BG, RMW, and MGC, in preparation). These findings suggest that the DAT plays a most crucial role in maintaining neuronal dopaminergic tone. Additional striking features of the DAT knockout mice include their weight deficit and lack of appropriate lactation of nursing mothers. These properties suggest that a hypothalamo-pituitary dysfunction might exist in these mice. Although low amounts of DAT mRNA are present in the cell bodies of the tuberoinfundibular system (arcuate nucleus and zona incerta), the functional role of DAT in this area of the brain has remained obscure (4).DA released from hypothalamic nuclei is known to modulate synthesis and secretion of prolactin from anterior pituitary lactotrophs. In rodents, hypothalamic DA also
FIGURE I Adaptive changes in biochemical and neurochemical mediators of DA transmission in DAT knockout mice. Rectangles represent pre- and postsynaptic receptors; triangles depict stored and released DA; circles represent vesicles within presynaptic terminals; tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, is abbreviated TH, and the difference in size of the symbols reflects different amounts of TH. In the normal mice, after stimulation, DA is released from presynaptic terminals. Once in the synaptic cleft, DA interacts with preand postsynaptic receptors to elicit its effects and is then rapidly carried back into nerve terminals by the DAT, which regulates synaptic levels of DA. In the DAT knockout mice, due to the absence of transporter, DA is not cleared away and remains in the synapse 300 times longer than normal. Despite lower amounts of released DA ( 3 ) , absence of DAT leads to increased functional DA, which presumably will occupy pre- and postsynaptic receptors, resulting in downregulation of receptor numbers, a decrease in DA content, and ultimately a decrease in TH. Modified from Science 271, 909, 1996.
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modulates the secretion of a-melanocyte-stimulating hormone (a-MSH) from the intermediate lobe. These effects of DA are mediated via D2 DA receptors on anterior pituitary lactotrophs and a-MSH-secreting cells of the intermediate lobe (5). Interestingly, (-/-) DAT mice show a marked hypopituitarism, with glands of knockout animals being about half the size of those of normal animals. (-/-) DAT mice show a marked decrease in the size of both the anterior and intermediate lobes of the pituitary, whereas the posterior lobe, which is known not to be under dopaminergic control, is unaffected. Autoradiographic studies reveal that the density of the D2 DA receptors is reduced by more than 7 5 % , in both anterior and intermediate lobes. Using radioimmunoassays, the pituitary content of both prolactin and, unexpectedly, growth hormone was decreased by 85 and 70%, respectively in (-/-) DAT animals compared with wild-type littermates. The extent of the changes in the content of prolactin and growth hormone may explain both the growth and nursing deficits of these animals. Taken together, these findings provide direct evidence for a role of DAT in the tuberoinfundibular DA system. Moreover, these results establish the importance of DA and its hypothalamic re-uptake mechanism in the function of the pituitary gland. Further elucidation of the mechanisms underlying this pituitary phenotype should reveal whether DA also plays a role in pituitary development.
References 1. Giros, B., and Caron, M. G. (1993). Molecular characterization of the dopamine transporter. Trends Pharmacol. Sci. 14, 43-49. 2. Lackovic, Z., and Relja, M. (1983).Evidence for a widely distributed peripheral dopaminergic system. Fed. Proc. 42, 3000-3004. 3. Giros, B., Jaber, M., Jones, S. R., Wightman, R. M., Caron, M. G. (1996).Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606-612. 4. Meister, B., Elde, R. (1993). Dopamine transporter mRNA in neurons of the rat hypothalamus. Neuroendocrinology 58, 388-395. 5 . Gingrich, J. A,, Caron, M. G. (1993).Recent advances in the molecular biology of dopamine receptors. Annu. Rev. Neurosci. 16, 299-321.
I79
2. Wall, S. C., Gu, H. H., and Rudnick, G. (1995). Biogenic amine flux mediated by cloned transporters stabiy expressed in cultured cell lines: Amphetamine specificity for inhibition and efflux. Mol. Pharmacol. 47, 544-550. 3. Gu, H. H., Ahn, J., Caplan, M. J., Blakely, R. D., Levey, A. I., and Ruduick, G. (1996). Cell-specific sorting of biogenic amine transporters expressed in epithelial cells. I. Biol. Chem. 271, 18100-18106. 4. Dotti, C. G., and Simons, K. (1990).Polarized sorting of viral glycoproteins to axons and dendrites of hippocampal neurons in culture. Cell 62, 63-72. 5 . Giros,B., Wang, Y., Suter, S., Mcleskey, S., Pitl, C., Caron, M. { 1994).Delineation of discrete domains for substrate, cocaine, and tricyclic antidepressant interactions using chimeric dopamine-norepinephrine transporters. J . Biol. Chem. 269, 15985-15988.
Fabio Furnagall&*Sara Jones,* Roger BOSS^,* Mohamed Jaber," Bruno Giros,* Cristina Missale,t R. Mark Wightman,$ and Marc G. Caron* "Howard Hughes Medical Institute Laboratories Departments of Cell Biology and Medicine Duke University Medical Center Durham, N o r t h Carolina 277 I0 tCurriculum in Neurobiology and Department of Chemistry University of North Carolina Chapel Hill, N o r t h Carolina 275 14
$Division of Pharmacology Department of Biomedical Sciences and Biotechnology University of Brescia Brescia, Italy
Inactivation of the Dopamine Transporter Reveals Essential Roles of Dopamine in the Control of Locomotion, Psychostimulant Response, and Pituitary Function Dopamine (DA) is an important modulator of many physiological functions. In the central nervous system (CNS), DA is involved in the control of movement, cognition, and affect, as well as neuroendocrine secretion. In the Advances in Pharmacolugy. Vulume 42 Copyright 0 1998 by Academic Press. All right, of reproduction 1054-3589198 $25.00
111
any form reserved.
I80
Fabio Fumagalli et a/.
periphery, DA regulates pituitary and parathyroid hormone synthesis and secretion, in addition to modulating certain retinal, cardiovascular, and kidney functions. DA is also thought to influence gastrointestinal physiology (1, 2). In the CNS, the DA transporter (DAT) presumably plays an important role in terminating dopaminergic transmission by uptaking DA from extracellular spaces back into presynaptic terminals. The role of the DAT in the CNS has been largely inferred from the clinical and psychosocial effects of drugs such as antidepressants and psychostimulants. These drugs interfere with the function of DAT. However, the involvement of DAT in the peripheral as well as the hypothalamic-neuroendocrine actions of DA has remained more enigmatic. DAT belongs to the large family of Na+- and C1--dependent transporters containing 12 putative transmembrane domains. Other members of this family include transporters for norepinephrine, serotonin, gamma aminobutyric acid, glycine, creatine, and betaine. In an attempt to develop an animal model in which the role of the DAT in the control of dopaminergic neurotransmission and in other physiological functions of DA could be directly examined, we have inactivated the mouse DAT gene by homologous recombination (3). Homozygous DAT knockout (-/-) animals are viable. However, adult (-/--) DAT animals show a pronounced weight deficit (50-70% of normal animals). (-/-) DAT mothers have a markedly reduced ability to nurse. The most obvious behavioral characteristic of these mice is a five- sixfold increase in spontaneous locomotor activity when tested in an open field. The level of activity of the (-/-) DAT animals is nearly as high as that achieved by treatment of wild-type animals with psychostimulants. Indeed, treatments of ( -/- ) DAT mice with high doses of cocaine or amphetamine yielded no further overall increase in locomotor activity, indicating that the locomotor effects of these drugs is mediated by interactions with DAT. These findings suggest that, in the absence of DA uptake, ( - I - ) DAT animals are hyperdopaminergic. In an attempt to examine whether this hyperdopaminergic behavioral phenotype was reflected at the biochemical level, several parameters were examined. Messenger RNAs for genes that are normally under dopaminergic control, such as preproenkephalin and dynorphin, showed variations indicative of increased dopaminergic tone. Similarly mRNAs for pre- and postsynaptic D2 dopamine receptors, as well as striatal D1 receptors, were markedly (>50%) diminished. As assessed by autoradiography, D1 and D2 receptor numbers in the striatal and ventral midbrain areas were also decreased by more than 50%, presumably reflecting downregulation of these receptors in response to the elevated dopaminergic tone of these animals. Even more interestingly, at the neurochemical level, these animals displayed properties reminiscent of what might be expected of parkinsonian animals. When striatal levels of DA were measured by high-performance liquid chromatography, they were found to be 5-10% of those of normal animals. Although at the gross morphological level the dopaminergic cell bodies of the substantia nigra-ventral tegmental area and the terminal fields of the striatum appeared essentially normal, in situ hybridization and Western blots for tyrosine hydroxylase (TH)in these areas revealed important adaptive changes. A marked decrease in the levels of TH protein was observed in these regions with no significant
Inactivation of DA Transporter
181
changes in TH mRNA, suggesting that posttranslational control mechanisms might be important in regulating TH activity in dopaminergic neurons (MJ, SJ, RB, BG, and MGC, in preparation). Thus, in the absence of a functional DAT, the (-/-) DAT mice have undergone major adaptive changes in their biochemical and neurochemical mediators of dopaminergic transmission in an attempt to dampen the presumed increased DA signal. This situation is schematized in Figure 1. However, none of these changes appear sufficient to effectively quench the increased signal, because the ( -/-) DAT animals still display spontaneous hyperlocomotion. This paradox may be explained when one considers that, as measured by fast cyclic voltammetry, DA persists at the synapse of (-/-.) DAT animals about 100-300 times longer than in normal animals (SJ, R. G, MJ, BG, RMW, and MGC, in preparation). These findings suggest that the DAT plays a most crucial role in maintaining neuronal dopaminergic tone. Additional striking features of the DAT knockout mice include their weight deficit and lack of appropriate lactation of nursing mothers. These properties suggest that a hypothalamo-pituitary dysfunction might exist in these mice. Although low amounts of DAT mRNA are present in the cell bodies of the tuberoinfundibular system (arcuate nucleus and zona incerta), the functional role of DAT in this area of the brain has remained obscure (4).DA released from hypothalamic nuclei is known to modulate synthesis and secretion of prolactin from anterior pituitary lactotrophs. In rodents, hypothalamic DA also
FIGURE I Adaptive changes in biochemical and neurochemical mediators of DA transmission in DAT knockout mice. Rectangles represent pre- and postsynaptic receptors; triangles depict stored and released DA; circles represent vesicles within presynaptic terminals; tyrosine hydroxylase, the rate-limiting enzyme of DA synthesis, is abbreviated TH, and the difference in size of the symbols reflects different amounts of TH. In the normal mice, after stimulation, DA is released from presynaptic terminals. Once in the synaptic cleft, DA interacts with preand postsynaptic receptors to elicit its effects and is then rapidly carried back into nerve terminals by the DAT, which regulates synaptic levels of DA. In the DAT knockout mice, due to the absence of transporter, DA is not cleared away and remains in the synapse 300 times longer than normal. Despite lower amounts of released DA ( 3 ) , absence of DAT leads to increased functional DA, which presumably will occupy pre- and postsynaptic receptors, resulting in downregulation of receptor numbers, a decrease in DA content, and ultimately a decrease in TH. Modified from Science 271, 909, 1996.
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Fabio Fumagalli et a/.
modulates the secretion of a-melanocyte-stimulating hormone (a-MSH) from the intermediate lobe. These effects of DA are mediated via D2 DA receptors on anterior pituitary lactotrophs and a-MSH-secreting cells of the intermediate lobe (5). Interestingly, (-/-) DAT mice show a marked hypopituitarism, with glands of knockout animals being about half the size of those of normal animals. (-/-) DAT mice show a marked decrease in the size of both the anterior and intermediate lobes of the pituitary, whereas the posterior lobe, which is known not to be under dopaminergic control, is unaffected. Autoradiographic studies reveal that the density of the D2 DA receptors is reduced by more than 7 5 % , in both anterior and intermediate lobes. Using radioimmunoassays, the pituitary content of both prolactin and, unexpectedly, growth hormone was decreased by 85 and 70%, respectively in (-/-) DAT animals compared with wild-type littermates. The extent of the changes in the content of prolactin and growth hormone may explain both the growth and nursing deficits of these animals. Taken together, these findings provide direct evidence for a role of DAT in the tuberoinfundibular DA system. Moreover, these results establish the importance of DA and its hypothalamic re-uptake mechanism in the function of the pituitary gland. Further elucidation of the mechanisms underlying this pituitary phenotype should reveal whether DA also plays a role in pituitary development.
References 1. Giros, B., and Caron, M. G. (1993). Molecular characterization of the dopamine transporter. Trends Pharmacol. Sci. 14, 43-49. 2. Lackovic, Z., and Relja, M. (1983).Evidence for a widely distributed peripheral dopaminergic system. Fed. Proc. 42, 3000-3004. 3. Giros, B., Jaber, M., Jones, S. R., Wightman, R. M., Caron, M. G. (1996).Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606-612. 4. Meister, B., Elde, R. (1993). Dopamine transporter mRNA in neurons of the rat hypothalamus. Neuroendocrinology 58, 388-395. 5 . Gingrich, J. A,, Caron, M. G. (1993).Recent advances in the molecular biology of dopamine receptors. Annu. Rev. Neurosci. 16, 299-321.