Effects of severe dopamine depletion on dopamine neuronal impulse flow and on tyrosine hydroxylase regulation

Brain ResearchBulleth~, Vol. 6, pp. 131-134.Printed in the U.S.A.

Effects of Severe D0pamine Depletion on Dopamine Neuronal Impulse Flow and on Tyrosine Hydroxylase Regulation D. C. G E R M A N , 1 B. A. M c M I L L E N , M. K. S A N G H E R A , S. I. S A F F E R A N D P. A. S H O R E

D e p a r t m e n t s o f Physiology, Psychiatry, Pharmacology and Medical C o m p u t e r Sciences University o f Texas Health Science Center, Dallas, TX 75235 R e c e i v e d 12 S e p t e m b e r 1980 GERMAN, D. C., B. A. McMILLEN, M. K. SANGHERA, S. I. SAFFER AND P. A. SHORE. Effects of severe dopamine depletion on dopamine neuronal impulseflow and on tyrosine hydroa3,1ase regulation. BRAIN RES. BULL. 6(2) 131-134, 1981.--Reserpine depletes dopamine (DA) levels and increases tyrosine hydroxylase (TH) activity in the rat corpus striatum. TH is activated not only by enhancement of DA neuronal impulse flow, but also by cessation of impulse flow. To assist in the understanding of the relative contribution of impulse flow to the regulation of TH activity in the DA depleted neuron, we examined the consequences of severe DA depletion on substantia nigra DA neuronal impulse flow and on in vivo TH activity in the rat corpus striatum. One day after reserpine or 30 min after the reversible reserpine-like compound, Ro4-1284, striatal DA levels were severely depleted and in vivo TH activity was enhanced about three-fold. DA depletion was found to significantly increase DA neuronal impulse flow. Although the DA neuron is firing faster than normal in the DA depleted rat, because there is no DA being released it is still not clear whether the elevation in TH activity is due to the enhancement of impulse flow or to the lack of DA at presynaptic receptor sites, or both. y-Butyrolactone (GBL), causes a cessation of DA neuronal impulse flow and activates TH by a presynaptic autoreceptor mechanism. GBL inhibited by over 50 percent the elevation in TH activity produced by severe DA depletion. This finding suggests that the enhanced TH activation after DA depletion is in large part due to increased DA impulse flow. Furthermore, the TH activity seen with GBL in DA depleted rats was significantly less than that seen after GBL administration in normal rats. This finding is consistent with the hypothesis that the DA storage granule also plays a role in TH regulation. Dopamine

Tyrosine hydroxylase

Electrophysiology

EITHER enhancement or cessation of dopamine (DA) neuronal impulse flow causes an increase in striatal tyrosine hydroxylase (TH) activity. Different mechanisms, however, are thought to produce these effects [10]. Enhancement of impulse flow, produced either by neuroleptics, which reflexly increase DA neuronal impulse flow [2,4] or nigrostriatal pathway electrical stimulation, increases TH activity and alters the kinetics of the enzyme [12, 17, 18]. This increase in TH activity is thought to be due, in part, to the intraneuronai removal of end-product (i.e., DA) inhibition of TH. Likewise, cessation of DA neuronal impulse flow, produced either by nigrostriatal pathway axotomy or by use of the anesthetic, gamma-butyrolactone (GBL), also enhances TH activity [8, 15, 16, 18]. This increase in TH activity is thought to result from the lack of DA release which normally inhibits a presynaptic receptor responsible for the regulation of TH activity [6,16]. Thus both impulse flow-related end product inhibition and presynaptic receptor mechanisms can regulate TH activation in the DA neuron.

y-Butyrolactone

Reserpine

TH activity is also markedly increased shortly after rats are treated with reserpine, at a time when striatal DA levels have reached their nadir [3]. The present study sought to determine the relative contribution of DA neuronal impulse flow and/or presynaptic mechanisms to the enhancement of TH activity produced by severe DA depletion. METHOD Female Sprague-Dawley rats, 200-250 g (Holtzman), maintained on a 12-hr light/dark cycle were used in all experiments. DA depletion was accomplished by administration of reserpine (Ciba-Geigy), 2.5 mg/kg SC, and by the tetrabenazine derivative Ro4-1284 (Hoffman-LaRoche), 2.0 mg/kg IP. Other drugs used were: y-butyrolactone (GBL, Sigma Chemical Co.), 3-hydroxybenzylhydrazine dihydrochloride (NSD-1015, Regis Chemical Co.), haloperidol (McNeil Laboratories), and apomorphine (Merck). Rats were killed by chloroform asphyxiation, the brains

~Send reprint requests to: Dr. Dwight C. German, Department of Physiology, University of Texas Health Science Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235.

C o p y r i g h t © 1981 A N K H O I n t e r n a t i o n a l Inc.--0361-9230/81/020131-04500.90/0

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GERMAN E T A L .

-were rapidly removed and chilled in ice cold saline, and the corpora striata were dissected free and frozen over dry ice for later assay on the same day. Striata from one rat were used for estimation of DA by the fluorometric method [9]. Striata from two rats were pooled for assay of L-dihydroxyphenylalanine (L-DOPA) by a modification of the fluorometric method [5]. The L-DOPA assay substituted batch alumina extraction of L-DOPA for alumina columns [7]. The method involved adsorbing L-DOPA from the 0.4 M HCI04 supernatant onto alumina, elution with HCI04, followed by a second alumina adsorbtion step. The 0.2 N HCi eluate containing L-DOPA was used for the fluorophor development [5]. Recoveries were d.etermined by adding 1.0 p.g of L-DOPA in 0.1 ml to one half of a homogenate of brain tissue from rats not treated with NSD-1015 while the second half of the homogenate was used as a recovery blank. At the end of the extraction procedure a second aliquot of the 0.2 N HCI eluate was taken from the blank sample and L-DOPA standard was added as an unextracted standard. Recoveries averaged 93.5 -+ 11.5% (mean - SD). Tyrosine hydroxylase activity in vivo was measured by the 30 min accumulation of striatal L-DOPA after administration of the decarboxylase blocker, NSD-1015 (100 mg/kg IP). The accumulation of L-DOPA was linear over a 45 min period. For the electrophysiological experiments the rats were anesthetized with chloral hydrate (400 mg/kg IP) and the jugular vein was catheterized. The animal was placed in a stereotaxic apparatus and wound margins and pressure points were infiltrated with a long lasting local anesthetic (mepivacaine HCI, 2%). A glass micropipette filled with 2 M NaCI and fast green dye or a glass coated tungsten microelectrode (2-3 Mfl impedance) was hydraulically lowered into the area of the substantia nigra zona compacta (between planes A1610 and A2180/z in the Krnig and Klippel atlas) to record extracellular action potentials. Electrode potentials were passed through a high impedance amplifier (Grass P51 I, 0.3-3.0 kHz bandpass) and monitored on an oscilloscope and audio amplifier. Each action potential activated a Schmitt trigger, the output of which was counted, displayed and stored in histogram from ( M I N C l l ; bin width 10 sec). In 24 rats baseline firing rates were recorded for at least five minutes before ceils were tested for their response to the DA depictor Ro4-1284. DA cell recordings were also made in 4 rats treated with reserpine, one to two days previously (at which time TH activity is still enhanced). The recording site was marked by the iontophoretic ejection of fast green dye from the micropipette (15 rain, - 1 9 /~A), or by making a microlesion with the tungsten electrode (15 sec, + 19/zA). The animal was then deeply anesthetized and perfused transcardially with saline followed by neutral buffered Formalin. The brain was sectioned on a freezing microtome at 50/zm. The tissue was stained with cresyl violet and the recording sites were histologically verified. RESULTS Rat striatal DA concentrations were 15.1 _+ 0.31 /zg/g ( - SEM) in normal rats. Eighteen hours after reserpine (2.5 mg/kg SC) the concentration was 1.11 - 0.11 p.g/g; while 30 min after Ro4-1284 (1.0 mg/kg IP), the concentration was 0.56 _ 0.3/zg/g and DA was undetectable by 60 min. While both reserpine and Ro4-1284 treated rats were markedly sedated, the rats given Ro4-1284 showed a much greater degree of catalepsy as measured by a previously described method

[13].

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FIG. 2. Effects of Ro4-1284 or reserpine on GBL-induced activation of tyrosine hydroxylase. Ro4-1284 (2.0 mg/kg SC) or reserpine (2.5 mg/kg SC) was injected 30 min or 18 hr, respectively, before 750 mg/kg IP GBL. NSD-1015 (100 mg/kg IP) was injected 15 rain after GBL and L-DOPA was allowed to accumulate for 30 min before killing the animals. Significant differences between GBL treated rats and rats receiving saline or the DA depletors were determined by Student's t-test (**p<0.001, two-tailed). Numbers within the bars represent the number of animals in each group.

Severe DA depletion by Ro4-1284 increased DA impulse flow. Baseline firing of DA neurons is normally 2.96 _+ 1.34 Hz (mean --+ SD, N = 15). Thirty minutes or more after Ro41284 (1 mg/kg IV) DA impulse flow increased to 4.92 --- 1.8 Hz (N=20, p<0.01 Student's t-test) (Fig. 1). In the reserpine treated animals the mean DA cell firing rate was 3.91 +- 1.56 Hz (N=6, p>0.5). As is typical of DA neurons, these cells had their firing rates decreased by apomorphine and this effect was reversed by haloperidol. To be sure that GBL blocks DA impulse flow after Ro4-1284, four cells were studied (one cell per rat) and all four ceased firing 5-7 min after GBL (750 mg/kg IP). To determine whether the release

D O P A M I N E D E P L E T I O N AND TH ACTIVITY

133

of newly synthesized DA was contributing to the effects of Ro4-1284 on DA impulse flow, four cells were studied (one cell per rat) 30 min after Ro4-1284 and another 30 min after a-methyltyrosine (t~MT; 50 mg/kg IV). The elevation in impulse flow produced by Ro4-1284 was not further altered after aMT. Figure 2 shows the effects of DA depletion on the GBLinduced activation of striatal tyrosine hydroxylase. Three points are of interest here. First, both Ro4-1284 and reserpine produce about a three-fold increase in the 30 min L-DOPA accumulation, identical to that produced by GBL. Second, when G B L was combined with Ro4-1284 or reserpine, significantly less accumulation of L-DOPA was observed compared with Ro4-1284 or reserpine alone. Finally, DA depletion significantly reduce the L-DOPA accumulation produced by GBL. The amount of L-DOPA accumulation after DA depletion plus G B L was still greater than that in rats treated with NSD-1015 alone (p<0.01, Dunnett's t-test).

DISCUSSION

Striatal TH activity is markedly enhanced under conditions of severe DA depletion. Similar increases in striatal TH activity have been reported for reserpine in other laboratories [3]. During this period of elevated TH activity and lowered DA levels the animals were behaviorally cataleptic. The present study shows that when the DA neuron is depleted of its transmitter, impulse flow is enhanced. Ro41284 at 30-60 rain appeared to be a more effective DA depletor than was reserpine at 18 hours, as evidenced both by assay of striatal DA concentration and by catalepsy. Furthermore, Ro4-1284 increased DA impulse flow more than did reserpine. Since the effects of Ro4-1284 on DA neuronal firing rate could be more readily ascertained due to its rapid action, and since each cell served as its own control, Ro4-1284 more clearly illustrates the effects of severe DA depletion on DA neuronal impulse flow. As is typical of DA cell recordings [1, 2, 4] the action potentials were of long duration, the cells exhibited a slow and sometimes bursting firing pattern, the firing rates were decreased by the DA agonist, apomorphine, and the recording sites were histologically localized within the substantia nigra zona compacta area. DA neuronal impulse flow is known to be regulated by a negative feedback mechanism [1]. For example, systemic administration of stimulants (i.e., DA releasers) decrease DA impulse flow, whereas neuroleptics (i.e., DA receptor blockers) increase DA impulse flow. In the present experiment, because there is no DA to be released when the cell fires, the increase in impulse flow is consistent with a negative feedback mechanism regulating impulse flow. Since DA synthesis is markedly enhanced in the DA depleted striatum, it is possible that newly synthesized DA was being released with the enhanced impulse flow. If newly synthesized DA was being released in the Ro4-1284 treated rats, then a-MT should have further elevated the impulse flow. It seems un-

likely, however, that newly synthesized DA was being released since a-MT did not further elevate the enhancement in impulse flow already induced by Ro4-1284. Furthermore, 30-120 min after Ro4-1284 the rats were behaviorally cataleptic, suggesting that little or no DA was in the synaptic cleft. When there is no DA in the synaptic cleft, the autoreceptor which controls synthesis becomes activated and TH ~ictivity is enhanced. This would explain, at least in part, why G B L (which inhibits DA impulse flow) and severe DA depletion (as with reserpine or Ro4-1284) both markedly enhance TH activity. If the enhanced TH activity after severe DA depletion were due solely to the lack of DA at presynaptic autoreceptor sites, then the addition of GBL should not affect this enhancement. However, GBL significantly decreased the elevation in TH caused by severe DA depletion. This result suggests that GBL, by blocking DA neuronal impulse flow, eliminated the impulse flow component of the enhanced TH activity normally seen in the DA depleted rat. Thus the enhancement of TH activity in the DA depleted rat is in large part due to an enhancement of DA neuronal impulse flow. It is also of interest that G B L enhances TH activity less in the DA depleted animal than in the normal one despite the fact that G B L ' s ability to inhibit DA neuronal impulse flow occurs in both the DA depleted (see above) and in the normal animal [14]. The finding that G B L enhances TH activity less in DA depleted rats is probably due to these animals having non-functional DA storage granules due to reserpine or Ro4-1284. It has been shown that three days after reserpine, basal striatal TH activity is normal, yet the ability of GBL to enhance DA synthesis and DA levels is greatly reduced [7]. These responses recover slowly over a two week period and parallel the recovery rate of striatal DA levels (i.e., return to a full complement of functional DA storage sites). This suggests that the enhanced synthesis and accumulation of DA in striatal nerve terminals, normally induced by GBL, depends upon intact DA granular storage sites. These findings are interpreted as evidence for the DA storage granule modulating TH activity by controlling the amount of DA available for attachment to and inhibition of the TH enzyme. Thus, the fewer functional storage granules available, the less TH activation can occur. It is interesting to note that Pickel et al. [11] observed that the TH enzyme is localized ultrastructuraily in close association with DA storage granules• Thus, a mosaic of influences, including alterations of impulse flow, enzyme kinetic changes, end-produce inhibition, presynaptic autoreceptors, and DA storage function act in the regulation of'EH activity. ACKNOWLEDGEMENTS The technical assistance of Ms. Karen Johnson and Ms. Margaret Wintersole, and the preparation of this manuscript by Ms. Laura Boynton are gratefully appreciated. Thanks also go to Dr. J. D. Miller for manuscript editing. This research was supported by USPHS Grants MH-05831 and MH-33513.

REFERENCES !. Aghajanian, G. K. Feedback regulation of central monoaminergic neurons: evidence from single cell recording studies. Essays #1 Neurochemistry and Neuropharmacology. New York: John Wiley, 1977, pp. 1-32.

2. Bunney, B. S., J. R. Waiters, R. H. Roth and G. K. Aghajanian. Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity. J. Pharmac. exp. Ther. 185: 560-571, 1973.

134 3. Carlsson, A. and M. Lindqvist. Effect of reserpine on monoamine synthesis and on apparent dopaminergic behavior sensitivity in rat brain. Neuropharmacology and Behavior. New York: Plenum Press, 1978, pp. 8%102. 4. German, D. C., H. Harden, M. K. Sanghera, D. Mann, R. S. • Kiser, H. H. Miller and P. A. Shore. Dopaminergic neuronal responses to a non-amphetamine CNS stimulant. J. Neural Trans. 44: 39--49, 1979. 5. Kehr, W., A. Carlsson and M. Lindqvist. A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain. Naunyn-Schmiedeberg's Arch. Pharmac. 274: 273-280, 1972. 6. Kehr, W., A. Carlsson and M. Lindqvist. Catecholamine synthesis in rat brain after axotomy: interaction between apomorphine and haloperidol. Naunyn-Schmiedeberg's Arch. Pharmac. 297:111-117, 1977. 7. McMillen, B. A. and P. A. Shore. Role of dopamine storage function in the control of rat striatal tyrosine hydroxylase activity. Naunyn-Schmiedeberg's Arch. Pharmac. 313: 39--44, 1980. 8. Morgenroth, V. H., III, J. R. Waiters and R. H. Roth. Dopaminergic neurons-alteration in the kinetic properties of tyrosine hydroxylase after cessation of impulse flow. Biochem. Pharmac. 25: 655-661, 1976. 9. Neff, N. H. and E. Costa. The influence of monoamine oxidase inhibition on catecholamine synthesis. Life Sci. 5: 951-959, 1966. 10. Nowycky, M. C. and R. H. Roth. Dopaminergic neurons: role of presynaptic receptors in the regulation of transmitter biosynthesis. Prog. Neuropsychopharmac. 2: 13%158, 1978. 1 I. Pickel, V. M., T. H. Joh and D. J. Reis. Regional and ultrastructural localization of tyrosine hydroxylase by immunocytochemistry in dopaminergic neurons of the mesolimbic and nigroneostriatal system. Nonstriatal Doparnine Neurons. New York: Raven Press, 1977, pp. 321-328.

GERMAN ET AL. 12. Roth, R. H., V. H. Morgenroth III and L. C. Murrin. Effects of antipsychotic drugs and impulse flow on the kinetics of striatal tyrosine hydroxylase. Antipsychotic Drugs, Pharmacodynatnics and Pharmacokinetics. Wenner-Gren Center International Symposium Series. New York: Pergamon Press, Inc., 1975, 25: pp. 133-145. 13. Shore, P. A. On the role of storage granules in the functional utilization of newly synthesized dopamine.J. Neural. Trans. 39: 131-138, 1976. 14. Waiters, J. R., G. K. Aghajanian and R. H. Roth. Dopaminergic neurons: inhibition of firing by y-hydroxybutyrate. Proc. Fifth Cong Pharmac., 1972, p. 246. 15. Waiters, J. R., R. H. Roth and G. K. Aghajanian. Dopaminergic neurons: similar biochemical and histochemical effects of 3,-hydroxyburtyrate and acute lesions of the nigroneostriatal pathway. J. Pharmac. exp. Ther. 186: 630-639, 1973. 16. Waiters, J. R. and R. H. Roth. Dopaminergic neurons--alterations in the sensitivity of tyrosine hydroxylase to inhibition by endogenous dopamine after cessation of impulse flow. Biochem. Pharmac. 25: 64%654, 1976. 17. Zivkovic, B., A. Guidotti and E. Costa. Effects of neuroleptics on striatal tyrosine hydroxylase: changes in affinity for the pteridine cofactor. Molec. Pharmac. 10: 727-735, 1974. 18. Zivkovic, B., A. Guidotti and E. Costa. The regulation of striatal tyrosine hydroxylase: effects of gamma hydroxybutric acid and haloperidol. Naunyn-Schmiedeberg's Arch. Pharmac. 291: 193-200, 1975.