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Modulation of Quantal Dopamine Release by Psychostimulants
I98
Emmanuel N. Pothos and David Sulzer
References 1. Krueger, B. K. (1990). Kinetics and block of doparnine uptake in synaptosomes from rat caudate nucleus. 1. Neurochem. 55, 260-267. 2. Sonders, M. S., Zhu, S-J., Zahniser, N. R., Kavanaugh, M. P., and Amara, S. G. (1997). Multiple ionic conductances of the human doparnine transporter: The actions of doparnine and psychostimulants. 1. Neurosci. 17, 960-974. 3. Cass, W. A., and Gerhardt, G. A. (1994).Direct evidence that D2 dopamine receptors can modulate doparnine uptake. Neurosci. Lett. 176, 259-263. 4. Cass, W. A., Zahniser, N. R., Flach, K. A., and Gerhardt, G. A. (1993). Clearance of exogenous dopamine in rat dorsal striatum and nucleus accumbens: Role of metabolism and effects of locally applied uptake inhibitors. 1. Neurochem. 61, 2269-2278 5. Kelly, E., Jenner, P., and Marsden, C. D. (1985). Evidence that [3H]dopamineis taken up and released from nondoparninergic nerve terminals in the rat substantia nigra in vitro. J . Neurochem. 45, 137-144.
Emmanuel N. Pathos* and David Sulzer'f "Departments of Neurology and Psychiatry Columbia University New York, New York 10032
t
Department of Neuroscience New York State Psychiatric Institute New York, New York 10032
Modulation of Quanta1 Dopamine Release by Psychostimulants The effects of the psychostimulants cocaine and d-amphetamine (AMPH) resemble the effects of the dopamine (DA)precursor L-dihydroxyphenylalanine (L-dopa)in that they elevate extracellular DA. However, we find that while Ldopa increases the quantal size of DA release, psychostimulants reduce quantal size. Therefore, L-dopa potentiates stimulation-dependent release, whereas psychostimulants decrease stimulation-dependent release while simultaneously elevating stimulation-independent background levels via uptake blockade or promotion of reverse transport.
1. Electrochemical Methods Because postsynaptic recordings are unsuitable for observing quantal release of slow-acting neurotransmitters like DA, we used amperometry (1)to Aduancer in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589198 $25.00
Quantal DA Release by Psychostimulants
I99
directly monitor vesicle release from PC12 cells (2) and midbrain DA neurons. A +700-mV potential was applied to a 5-pm carbon-fiber electrode placed on PC12 cell bodies or on DA neuron axonal varicosities (identified by immunostaining for the vesicular monoamine transporter; VMAT2). Following stimulation of exocytosis by secretagogues or current injection, spikes of faradic current resulting from DA oxidation estimate the number of molecules released (quantal size); quantal sizes in this report are based on an assumption of 2 e- donated/molecule.
II. Variation of Quantal Size in Midbrain DA Neurons In recordings from sister cultures of rat ventral tegmental area neurons, stimulation by 80 mM KCI and 10 n M a-latrotoxin produced apparent unitary events that lasted 100-200 ps (duration at half width = 81 ? 4 ps) and represented 1800 ? 100 molecules (mean f SEM; n = 56 events; Fig. 1A). Some of these cultures were fixed and processed for tyrosine hydroxylase (TH) immunochemistry: Each process from which events were recorded was DAergic (12 of 12 neurons), although only 40% of the total neurons in these cultures are DAergic. Following exposure to 20 p M L-dopa for 30 min, the quantal size increased to 5000 C 700 molecules (n = 35 events; Fig. 1B). Because T H is the ratelimiting step in DA synthesis, this indicates that vesicles can be loaded by increasing cytosolic DA levels and may provide a functional basis for the control of TH activity by transcript regulation and phosphorylation. In the larger events, distinctions in shape such as the expression of a “foot” preceding the full spike, could be discerned (see lowest trace in Fig. 1B).
111. Variations in Quantal Size in PC I 2 Cells To observe the effects of psychostimulants on quantal release in a tractable preparation where tens to hundreds of release events can be recorded from individual cells, we used the PC12 line. Average quantal sizes, presumably representing dense core vesicles, range from 100 to 800 K molecules, depending on culture conditions. As observed in the midbrain DA neurons, L-DOPA increased quantal size, apparently by elevating cytosolic substrate ( 3 ) .Moreover, we found that lowered cytosolic DA reduced quantal size because the D2 agonist quinpirole at an exposure that inhibited TH activity by 50% also decreased quantal size by approximately 50% (E. N. Pothos, V. Davila, and D. Sulzer, in preparation). We previously used the PC12 cell line to help elucidate the mechanism of action of AMPH. Analogous to findings in isolated chromaffin granules, AMPH (10 p M for 10 min) decreased quantal size by 50%. Because weak bases that are not transporter substrates also redistribute catecholamines from isolated granules and induce DA release in cultures, this appears to follow collapse of the vesicle electrochemical gradient that provides energy for transmitter
200
A
Emmanuel N. Pothos and David Sulzer
Control
___JL___
C
D x
1.w
0.90
; 0.80 0.70
c p)
2m
0.60 0.50
0.40
2 0.30
5
0
0.20 0.10
FIGURE I Examples of mechanisms by which the vesicle free energy gradient alters quantal size. ( A ) Representative evoked amperometric spikes observed at varicosities of ventral tegmental area DA axons in monolayer culture. ( B ) Following L-dopa, which elevates cyrosolic DA levels and increases the driving potential for vesicle uptake, quantal events are larger, and variations in shape are observed that may reflect the expression of the fusion pore. ( C )Psychostimulant drugs reduce quantal size. The upper trace shows amperometric spikes following stimulation of a control PC12 cell with 80 mM of KCI. The lower traces show spikes following incubation with 54 pM of cocaine for 30 min or 10 pM of amfonelic acid for 30 min, respectively. ( D )Plots of the cumulative frequency of the quantal sizes indicate that following cocaine or amfonelic acid exposure, quantal size is decreased (p < .02, KS-Z = 1.56 for cocaine; p < .01, KS-Z = 1.84 for amfonelic acid; Kolmogorov-Smirnov two-sample test). The y axis expresses the cubed root of the quantal size, generally a normal distribution, as expected from the normal distribution of vesicle volumes (3).
accumulation. The resulting redistribution of DA to the cytosol may then initiate reverse transport across the DA transporter (DAT) (4). To examine the effects of psychostimulants that are DAT blockers, we exposed PC12 cultures to 10 pM cocaine for 30 min, which reduced the average quantal size to 50% of control levels (n = 9 cells, 109 events for cocaine; 16 cells, 393 events for controls) or to 54 p M cocaine, which reduced quantal size to 27% of controls (n = 12 cells, 100 events; traces C and D of Fig. 1). Potential mechanisms that may explain reduced quantal size following cocaine are: (1)a quinpirole-like activation of autoreceptors and resulting TH
Quantal DA Release by Psychostimulants
20 I
inhibition, (2) an AMPH-like collapse of the vesicle pH gradient by a weak base action, and ( 3 ) DAT blockade. We have ruled out autoreceptor activation as an explanation because the D2 antagonist sulpiride, used at exposures that block the effects of quinpirole, does not inhibit quantal size reduction by cocaine. We cannot rule out the possibility that collapse of the vesicle pH gradient contributes because a relatively small collapse of acidic gradients can be observed in microscopic observations using weak base vital dyes. However, we found that the DAT blocker amfonelic acid (10 p M for 30 min), which does not collapse p H gradients, reduced average quantal size to 39% of control levels (n = 14 cells, 661 events). This similar effect by a DAT blocker that is not a weak base suggests that the decreased quantal size by DAT blockade is due to a reduced cytosolic gradient. In addition to manipulations of the vesicular free energy gradient, we have identified other mechanisms that can alter the quantal size of DA release. Quantal size can be regulated by expression of the VMAT (B. C. Sun, Y. Liu, R. Edwards, and D. Sulzer, in preparation), indicating that vesicular transport can be a rate-limiting step in the accumulation of transmitter. Moreover, in PC12 cells, release evoked by a-latrotoxin rather than elevated K+ or nicotine results in a different mean quantal size, suggesting that the total vesicle contents are not generally released during exocytosis and that expression of the fusion pore can also be rate-limiting for DA flux from dense core granules (E. N. Pothos and D. Sulzer, in preparation). In summary, psychostimulants may reduce the quantal size of DA release by altering the vesicular free energy used to provide for the accumulation of neurotransmitter. This is accomplished by altering the cytosolic-intravesicular concentration or the vesicle electrochemical gradient. While reduced quantal size does not underlie the abuse potential of drugs that elevate DA overflow, inhibition of local stimulation-dependent release may disturb associated behaviors, such as working memory or classical conditioning. Indeed, while both Ldopa and psychostimulants elevate overall extracellular levels, the results indicate that the “signal to background” of local stimulation-dependent DA input is far higher for L-dopa.
Acknowledgments E. N. Pothos is an Aaron Diamond Foundation Fellow and recipient of a 1995 NARSAD Young Investigator Award. This research is supported in part by grants from the Aaron Diamond Foundation, NARSAD, the Parkinson’s Disease Foundation and NIDA.
References 1. Leszczyszyn, D. J., Jankowski, J. A., Viveros, 0. H., Diliberto, E. J. Jr., Near, J. A., and Wightman, R. M. (1991). Secretion of catecholamines from individual adrenal medullary chromaffin cells. /. Neurochem. 56, 1855-1863.
202
Beth J. Hoffman et a/.
Chen, T. K., Luo, G., and Ewing, A. G . (1994). Arnperometric monitoring of stimulated catecholamine release from rat pheochromocytoma (PC12) cells at the zeptomole level. Anal. Chem. 66, 3031-3035. Finnegan, J. M., Pihel, K., Cahill, P. S., Haung, L., Zerby, S. E., Ewing, A. G., Kennedy, R. T., and Wightman, R. M. (1996). Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic beta cells. 1.Neurosci. 66, 19141923. Pothos, E., Desmond, M., and Sulzer, D. (1996). L-3,4-Dihydroxyphenylalanineincreases the quantal size of exocytic dopamine release in vitro. J . Neurochem. 66, 629-636. Sulzer, D., Chen, T. K., Lau, Y. Y., Kristensen, H., Rayport, S., and Ewing, A. (1995). Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. 1.Neurosci. 15, 4102-4108.
Beth J. Hoffman,* Mikl6s Palkovits,*t Karel Paiak,$§ Stefan R. Hamson,* and Eva Mezey?: *Laboratory of Cell Biology NIMH, NIH Bethesda, Maryland 20892 tLaboratory of Neuromorphology Semelweis University Medical School Budapest 1094, Hungary *Clinical Neurosciences Branch NINDS, NIH Bethesda, Maryland 20892 §Department of Medicine Washington Hospital Center Washington, D.C. 200 I0
Regulation of Dopamine Transporter mRNA Levels in the Central Nervous System Dysfunction of dopamine (DA) neural systems are thought to underlie neuropsychiatric disorders and psychostimulant abuse. Abnormalities of nigrostriatal dopaminergic neurons cause motor impairment associated with Parkinson’s disease. Dysfunction of mesolimbic and mesocortical DA neurons have been implicated in schizophrenia and in drug addiction. The diencephalic DA neurons have been associated with adaptive, homeostatic responses to a variety of stressful stimuli through neuroendocrine regulation. Advances in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589198$25.00
Emmanuel N. Pothos and David Sulzer
References 1. Krueger, B. K. (1990). Kinetics and block of doparnine uptake in synaptosomes from rat caudate nucleus. 1. Neurochem. 55, 260-267. 2. Sonders, M. S., Zhu, S-J., Zahniser, N. R., Kavanaugh, M. P., and Amara, S. G. (1997). Multiple ionic conductances of the human doparnine transporter: The actions of doparnine and psychostimulants. 1. Neurosci. 17, 960-974. 3. Cass, W. A., and Gerhardt, G. A. (1994).Direct evidence that D2 dopamine receptors can modulate doparnine uptake. Neurosci. Lett. 176, 259-263. 4. Cass, W. A., Zahniser, N. R., Flach, K. A., and Gerhardt, G. A. (1993). Clearance of exogenous dopamine in rat dorsal striatum and nucleus accumbens: Role of metabolism and effects of locally applied uptake inhibitors. 1. Neurochem. 61, 2269-2278 5. Kelly, E., Jenner, P., and Marsden, C. D. (1985). Evidence that [3H]dopamineis taken up and released from nondoparninergic nerve terminals in the rat substantia nigra in vitro. J . Neurochem. 45, 137-144.
Emmanuel N. Pathos* and David Sulzer'f "Departments of Neurology and Psychiatry Columbia University New York, New York 10032
t
Department of Neuroscience New York State Psychiatric Institute New York, New York 10032
Modulation of Quanta1 Dopamine Release by Psychostimulants The effects of the psychostimulants cocaine and d-amphetamine (AMPH) resemble the effects of the dopamine (DA)precursor L-dihydroxyphenylalanine (L-dopa)in that they elevate extracellular DA. However, we find that while Ldopa increases the quantal size of DA release, psychostimulants reduce quantal size. Therefore, L-dopa potentiates stimulation-dependent release, whereas psychostimulants decrease stimulation-dependent release while simultaneously elevating stimulation-independent background levels via uptake blockade or promotion of reverse transport.
1. Electrochemical Methods Because postsynaptic recordings are unsuitable for observing quantal release of slow-acting neurotransmitters like DA, we used amperometry (1)to Aduancer in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589198 $25.00
Quantal DA Release by Psychostimulants
I99
directly monitor vesicle release from PC12 cells (2) and midbrain DA neurons. A +700-mV potential was applied to a 5-pm carbon-fiber electrode placed on PC12 cell bodies or on DA neuron axonal varicosities (identified by immunostaining for the vesicular monoamine transporter; VMAT2). Following stimulation of exocytosis by secretagogues or current injection, spikes of faradic current resulting from DA oxidation estimate the number of molecules released (quantal size); quantal sizes in this report are based on an assumption of 2 e- donated/molecule.
II. Variation of Quantal Size in Midbrain DA Neurons In recordings from sister cultures of rat ventral tegmental area neurons, stimulation by 80 mM KCI and 10 n M a-latrotoxin produced apparent unitary events that lasted 100-200 ps (duration at half width = 81 ? 4 ps) and represented 1800 ? 100 molecules (mean f SEM; n = 56 events; Fig. 1A). Some of these cultures were fixed and processed for tyrosine hydroxylase (TH) immunochemistry: Each process from which events were recorded was DAergic (12 of 12 neurons), although only 40% of the total neurons in these cultures are DAergic. Following exposure to 20 p M L-dopa for 30 min, the quantal size increased to 5000 C 700 molecules (n = 35 events; Fig. 1B). Because T H is the ratelimiting step in DA synthesis, this indicates that vesicles can be loaded by increasing cytosolic DA levels and may provide a functional basis for the control of TH activity by transcript regulation and phosphorylation. In the larger events, distinctions in shape such as the expression of a “foot” preceding the full spike, could be discerned (see lowest trace in Fig. 1B).
111. Variations in Quantal Size in PC I 2 Cells To observe the effects of psychostimulants on quantal release in a tractable preparation where tens to hundreds of release events can be recorded from individual cells, we used the PC12 line. Average quantal sizes, presumably representing dense core vesicles, range from 100 to 800 K molecules, depending on culture conditions. As observed in the midbrain DA neurons, L-DOPA increased quantal size, apparently by elevating cytosolic substrate ( 3 ) .Moreover, we found that lowered cytosolic DA reduced quantal size because the D2 agonist quinpirole at an exposure that inhibited TH activity by 50% also decreased quantal size by approximately 50% (E. N. Pothos, V. Davila, and D. Sulzer, in preparation). We previously used the PC12 cell line to help elucidate the mechanism of action of AMPH. Analogous to findings in isolated chromaffin granules, AMPH (10 p M for 10 min) decreased quantal size by 50%. Because weak bases that are not transporter substrates also redistribute catecholamines from isolated granules and induce DA release in cultures, this appears to follow collapse of the vesicle electrochemical gradient that provides energy for transmitter
200
A
Emmanuel N. Pothos and David Sulzer
Control
___JL___
C
D x
1.w
0.90
; 0.80 0.70
c p)
2m
0.60 0.50
0.40
2 0.30
5
0
0.20 0.10
FIGURE I Examples of mechanisms by which the vesicle free energy gradient alters quantal size. ( A ) Representative evoked amperometric spikes observed at varicosities of ventral tegmental area DA axons in monolayer culture. ( B ) Following L-dopa, which elevates cyrosolic DA levels and increases the driving potential for vesicle uptake, quantal events are larger, and variations in shape are observed that may reflect the expression of the fusion pore. ( C )Psychostimulant drugs reduce quantal size. The upper trace shows amperometric spikes following stimulation of a control PC12 cell with 80 mM of KCI. The lower traces show spikes following incubation with 54 pM of cocaine for 30 min or 10 pM of amfonelic acid for 30 min, respectively. ( D )Plots of the cumulative frequency of the quantal sizes indicate that following cocaine or amfonelic acid exposure, quantal size is decreased (p < .02, KS-Z = 1.56 for cocaine; p < .01, KS-Z = 1.84 for amfonelic acid; Kolmogorov-Smirnov two-sample test). The y axis expresses the cubed root of the quantal size, generally a normal distribution, as expected from the normal distribution of vesicle volumes (3).
accumulation. The resulting redistribution of DA to the cytosol may then initiate reverse transport across the DA transporter (DAT) (4). To examine the effects of psychostimulants that are DAT blockers, we exposed PC12 cultures to 10 pM cocaine for 30 min, which reduced the average quantal size to 50% of control levels (n = 9 cells, 109 events for cocaine; 16 cells, 393 events for controls) or to 54 p M cocaine, which reduced quantal size to 27% of controls (n = 12 cells, 100 events; traces C and D of Fig. 1). Potential mechanisms that may explain reduced quantal size following cocaine are: (1)a quinpirole-like activation of autoreceptors and resulting TH
Quantal DA Release by Psychostimulants
20 I
inhibition, (2) an AMPH-like collapse of the vesicle pH gradient by a weak base action, and ( 3 ) DAT blockade. We have ruled out autoreceptor activation as an explanation because the D2 antagonist sulpiride, used at exposures that block the effects of quinpirole, does not inhibit quantal size reduction by cocaine. We cannot rule out the possibility that collapse of the vesicle pH gradient contributes because a relatively small collapse of acidic gradients can be observed in microscopic observations using weak base vital dyes. However, we found that the DAT blocker amfonelic acid (10 p M for 30 min), which does not collapse p H gradients, reduced average quantal size to 39% of control levels (n = 14 cells, 661 events). This similar effect by a DAT blocker that is not a weak base suggests that the decreased quantal size by DAT blockade is due to a reduced cytosolic gradient. In addition to manipulations of the vesicular free energy gradient, we have identified other mechanisms that can alter the quantal size of DA release. Quantal size can be regulated by expression of the VMAT (B. C. Sun, Y. Liu, R. Edwards, and D. Sulzer, in preparation), indicating that vesicular transport can be a rate-limiting step in the accumulation of transmitter. Moreover, in PC12 cells, release evoked by a-latrotoxin rather than elevated K+ or nicotine results in a different mean quantal size, suggesting that the total vesicle contents are not generally released during exocytosis and that expression of the fusion pore can also be rate-limiting for DA flux from dense core granules (E. N. Pothos and D. Sulzer, in preparation). In summary, psychostimulants may reduce the quantal size of DA release by altering the vesicular free energy used to provide for the accumulation of neurotransmitter. This is accomplished by altering the cytosolic-intravesicular concentration or the vesicle electrochemical gradient. While reduced quantal size does not underlie the abuse potential of drugs that elevate DA overflow, inhibition of local stimulation-dependent release may disturb associated behaviors, such as working memory or classical conditioning. Indeed, while both Ldopa and psychostimulants elevate overall extracellular levels, the results indicate that the “signal to background” of local stimulation-dependent DA input is far higher for L-dopa.
Acknowledgments E. N. Pothos is an Aaron Diamond Foundation Fellow and recipient of a 1995 NARSAD Young Investigator Award. This research is supported in part by grants from the Aaron Diamond Foundation, NARSAD, the Parkinson’s Disease Foundation and NIDA.
References 1. Leszczyszyn, D. J., Jankowski, J. A., Viveros, 0. H., Diliberto, E. J. Jr., Near, J. A., and Wightman, R. M. (1991). Secretion of catecholamines from individual adrenal medullary chromaffin cells. /. Neurochem. 56, 1855-1863.
202
Beth J. Hoffman et a/.
Chen, T. K., Luo, G., and Ewing, A. G . (1994). Arnperometric monitoring of stimulated catecholamine release from rat pheochromocytoma (PC12) cells at the zeptomole level. Anal. Chem. 66, 3031-3035. Finnegan, J. M., Pihel, K., Cahill, P. S., Haung, L., Zerby, S. E., Ewing, A. G., Kennedy, R. T., and Wightman, R. M. (1996). Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic beta cells. 1.Neurosci. 66, 19141923. Pothos, E., Desmond, M., and Sulzer, D. (1996). L-3,4-Dihydroxyphenylalanineincreases the quantal size of exocytic dopamine release in vitro. J . Neurochem. 66, 629-636. Sulzer, D., Chen, T. K., Lau, Y. Y., Kristensen, H., Rayport, S., and Ewing, A. (1995). Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. 1.Neurosci. 15, 4102-4108.
Beth J. Hoffman,* Mikl6s Palkovits,*t Karel Paiak,$§ Stefan R. Hamson,* and Eva Mezey?: *Laboratory of Cell Biology NIMH, NIH Bethesda, Maryland 20892 tLaboratory of Neuromorphology Semelweis University Medical School Budapest 1094, Hungary *Clinical Neurosciences Branch NINDS, NIH Bethesda, Maryland 20892 §Department of Medicine Washington Hospital Center Washington, D.C. 200 I0
Regulation of Dopamine Transporter mRNA Levels in the Central Nervous System Dysfunction of dopamine (DA) neural systems are thought to underlie neuropsychiatric disorders and psychostimulant abuse. Abnormalities of nigrostriatal dopaminergic neurons cause motor impairment associated with Parkinson’s disease. Dysfunction of mesolimbic and mesocortical DA neurons have been implicated in schizophrenia and in drug addiction. The diencephalic DA neurons have been associated with adaptive, homeostatic responses to a variety of stressful stimuli through neuroendocrine regulation. Advances in Pharmacology, Volume 42 Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved. 1054-3589198$25.00