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responses by deprenyl (Selegiline): an electrophysiological and behavioral study in the rat relevant to Parkinson's disease. Ann Neurol 1998;43:613-617.

Modification of Levodopa Responses by Deprenyl (Selegiline): An klectrobhysiological and Behavioral Study in the Rat Relevant to Parkinson's Disease Nicola B. Mercuri, MD, Mariangela Scarponi, BNS, Mauro Federici, BNS, Antonello Bonci, MD, Antonio Siniscalchi, MD, and Giorgio Bernardi, MD

From using in vitro intracellular recordings from mesencephalic neurons and monoamine-depleted rats, we report that the functions of levodopa in the brain are greatly enhanced and prolonged by high doses of the monoamine oxidase (MAO) inhibitor deprenyl. Dopaminergic neurons were hyperpolarized and inhibited by levodopa application. These effects of levodopa were largely potentiated by pretreatment with nonselective doses of deprenyl. Furthermore, when locomotor aaivity induced by levodopa was examined on a rodent model of Parkinson's disease, pretreatment of the animals with nonselective doses of deprenyl caused an enhancement of the antiparkinsonian action of levodopa. The great increase in levodopa responses by deprenyl suggests a likely therapeutic use of this dopamine precursor with a higher dosage of the MA0 inhibitor, to reduce effectively the daily levodopa requirements in Parkinson's disease patients.

Mercuri NB, Scarponi M, Federici M, Bonci A, Siniscalchi A, Bernardi G. Modification of levodopa responses by deprenyl (Selegiline): an electrophysiological and behavioral study in the rat relevant to Parkinson's disease. Ann Neurol 1998;43:613-617

The therapeutic use of the irreversible monoamine oxidase B (MA0 B) inhibitor deprenyl, as adjuvant to levodopa in Parkinson's disease, is based mainly on the following rationale. The inhibition of MA0 B should lead to diminished metabolism of dopamine (DA) in the nigrostriatal system and a significant increase in the concentration of the neurotransmitrer formed from levodopa.1-3 Despite this, therapy with deprenyl in Parkinson's disease has been characterized by different clinical result^.^-^ In fact, although this drug has been shown initially to be useful in the treatment of wearing off, it does not clearly ameliorate motor fluctuations, freezing, and psychiatric side effects that often occur in complicated patients treated with levodopa for several years.627These negative findings could be explained by the fact that the selective blockade of M A 0 B by low concentrations of deprenyl' might not be a sufficient factor to enhance the effectiveness of levodopa in the brain. In accord with this, it has recently been demonstrated that a pure inhibition of MA0 B enzymes does not prolong the effects of DA on a single cell of the mammalian central nervous system.'

The objective of the present study, then, was to evaluate experimentally whether deprenyl could really enhance the antiparkinsonian action of levodopa. To do this we used either an in vitro electrophysiological neuronal model or a behavioral model of Parkinson's disease.

From the IRCCS Santa Lucia and Clinica Neurologica, University of Tor Vergata, Rome, Italy.

Address correspondence to Dr Mercuri, IRCCS Ospedale Santa Lucia, Via Ardeatina 306, 00179 Rome, Italy.

Materials and Methods Elertrophysiological Experiments The method used has been described previously." In brief, male Wistar rats (150-250 g) (Morini, Reggio Emilia, Italy) were anesthetized with halothane and killed. The brain was removed and horizontal slices (300 pm thick) were cut by a Vibratome, starting from the ventral surface of the midbrain. A single slice, containing the substantia nigra and the ventral tegmental area (VTA), was then transferred into a recording chamber and submerged completely in an artificial cerebrospinal fluid with a continuously flowing (2.5 ml/min) solution at 35°C (pH 7.4). This solution contained (mM): NaCI, 126; KCI, 2.5; MgCI,, 1.2; NaH,P04, 1.2; CaCl,, 2.4; glucose, 11; and NaHCO,, 20; and was gassed with 95% 0, and 5% CO,. The recording electrodes (Clark 1; 1.5 mm, thick wall), pulled by Narishige vertical and horizontal pullers, were filled with 2 M KC1 and had a tip re-

Received Sep 2, 1997, and in revised form Nov 27. Accepted for publication Dec 3, 1997.

Copyright 0 1998 by the American Neurological Association


sistance of 40 to 80 MO. The signals were obtained by an amplifier (Axoclamp-2A, Axon Instruments) and were displayed by a pen recorder (Gould 2400 S) and on a digital oscilloscope (Tektronix) or were saved on an IBMcomparible computer by the Axotape software (Axon Instruments) for off-line analysis. The tips of the recording electrodes were placed in the substantia nigra pars compacta and VTA by using a dissecting microscope. Drugs were made in stock solutions and bath applied at known concentrations via a three-way tap system. A complete exchange of the solution in the recording chamber occurred in about 1 minute. Levodopa was superfused on the cells by changing to a solution that contained 100 (*M agonist for 70 seconds. This was done to induce a standard hyperpolarization and firing inhibition. The changes in firing rate caused by levodopa in the dopaminergic cells were normalized as percentages of control. In other experiments, the slices were preincubated for at least 2 hours with deprenyl (300 nM to 10 (*M) to allow sufficient time for steady-state M A 0 inhibition to develop." Then the membrane responses to levodopa were evaluated.

Behavioral Experiments For the behavioral experiments, the rats were placed in the animal house (temperature, 22 ? 2°C) and maintained on a 12-hour lighddark cycle during which they were allowed free access to drinking water and food. Reserpine 12 mglkg was injected subcutaneously 14 to I 6 hours before behavioral testing. Reserpine treatment caused a profound akinetic rigid state that was stable for at least 3 days. The horizontal motor activity was measured by an automatic activity cage (Basile) provided with a time-related counter of events (n/30 min). Each animal was used only once. All the substances except reserpine were administered intraperitoneally. The rodents were treated with 25 mg carbidopa 60 minutes before levodopa to inhibit peripheral levodopa decarboxylation. Behavioral testing was performed at around 9 AM. The automatic scoring of motor activity started 1 hour before the first intraperitoneal injection (deprenyl or saline); no acclimatization period was necessary for reserpine-treated rats.I2 The rats were divided into three groups of 5 animals, as follows: Group 1 received saline 2 hours before the injection of levodopa 150 mg/kg (saline, carbidopa, levodopa 150); group 2 received deprenyl 1 mg/kg and then 2 hours later levodopa 150 mg/kg (deprenyl 1 mg/kg, carbidopa, levodopa 150); and group 3 received deprenyl 15 mg/kg and then levodopa 150 mg/kg (deprenyl 15 mg/kg, carbidopa, levodopa 150). Results were expressed as mean ? SEM values and were analyzed by Student's t rest or analysis of variance. Significance was set at p < 0.05.

Drugs The following substances were used: deprenyl, which is more selective for type B MA01"13 (Research Biochemicals), L-sulpiride (Ravizza), L-dihydroxyphenylalanine (levodopa), levodopa methyl ester and reserpine (Sigma), carbidopa (Merck Sharpe & Dohme). Reserpine was dissolved in a drop of glacial acetic acid and diluted with distilled water.

614 Annals of Neurology Vol 43 No 5 May 1998

Results Electrophysiological Experiment3 RESPONSES TO LEVODOPA. Intracehlar recordings were obtained from 10 spontaneously firing presumed dopaminergic cells of the rat mesencephalon, T h e neurons were identified by their characteristic electrophysiological properties. 10,14,15 T h e rate of firing was 1.64 ? 0.09 Hz. Bath-applied levodopa (100 pM) determined a reversible membrane hyperpolarization (- 13.2 ? 1.9 mV) and block of firing in all the tested cells" (n = 10; Fig 1).



already reported, l7 the pretreatment of the dopaminergic cells with deprenyl u p to 10 p,M did not modify the spontaneous firing activity (1.6 ? 0.1 n = 7). Although concentrations of deprenyl below 3 p M did not change the cellular responses to levodopa application, higher concentrations (3-10 pM) induced a long-term prolongation of levodopa effects that did not wash out even after 2 hours (see Fig 1). T h e amplitude of the membrane hyperpolarization caused by levodopa was also increased by deprenyl (24 2 7% of control; n = 6 with 10 pM). T h e long-lasting inhibition caused by levodopa application on the deprenyl-treated cells could be readily reversed by the D2 receptor antagonist L-sulpiride (1-3 FM) (see Fig 1).

Motor Activity To test whether the enhancement of levodopa responses caused by deprenyl o n a single cell was also evident in the whole animal, we performed behavioral observations in rats rendered parkinsonian by reserpine. INTRAPERITONEAL ADMINISTRATION OF LEVODOPA AND DEPRENYL. W e used one dose of levodopa methyl es-

ter in reserpinized rats, 150 mg/kg, and two doses of deprenyl, 1 mglkg (which is selective for M A 0 B)18 and 15 mg/kg (which also causes substantial inhibition of M A 0 A).18213T h e dose of 150 mg/kg levodopa did not cause an increase of the motor activity (group 1) (Fig 2A). T h e injection of 150 mglkg levodopa in rats pretreated with 1 mg/kg deprenyl did not change the spontaneous motor activity (group 2) (see Fig 2B). In contrast, the injection of 15 mg/kg deprenyl and 150 mg/kg levodopa attenuated the reserpine-induced locomotor depression (group 3) for at least 350 minutes (see Fig 2C).

Discussion In the present study, from either in vitro electrophysiological experiments or behavioral observations, we have shown a clear augmentation and prolongation of levodopa response caused by a high dose of deprenyl that produces inhibition of both M A 0 A and M A 0 B.

Fig 1. Potentiating effects of deprenyl on the electrophysiological responses caused by levodopa in mesencephalic dopaminergic cells. (A) The top trace shows a typical response to a short application of levodopa (100 pM). Note the reversible hyperpolarization and inhibition o f j r i n g caused by the amino acid. The bottom trace shows the typical pattern of cellular response to levodopa after the pretreatment of the neurons with deprenyl (10 pM). The membrane response to levodopa application did not recover during washout, and the cells were enabled to $re again by the supe$sion of the 0 2 receptor antagonist sulpiride. Note that the time scale was changed (see X below) to show individual action potentials. (B) The graph shows the normalized time course of j r i n g inhibition produced by 100 pM levodopa under control conditions and after the treatment with deprenyl in the dopaminergic cells. Each point is an average o f f i u r to seven observations.

This phenomenon may have important implications for parkinsonian patients, because it suggests an appropriate therapeutic regimen of levodopa and deprenyl, which should increase the effectiveness of a single dose of levodopa in the brain.

In Vitro Electrophysiological Experiments Membrane hyperpolarization and inhibition of the spontaneous firing of dopaminergic cells caused by levodopa is very likely due to an elevated extracellular

Fig 2. Potentiating effect of deprenyl on the antiparkinsonian effects of levodopa in resevpinized rats. (A) Group I . A subthreshold dose of intraperitoneal levodopa methyl ester (150 mg/kg) does not activate motor activity. (B) Group 2. Pretreatment of the animals with 1 mg/kg deprenyl does not induce motor activation after the injection of levodopa. (C) Group 3. Pretreatment of the animals with 15 mg/kg deprenyl enables levodopa to reverse the parkinsonian symptoms.

efflux of newly synthesized DA and not to a direct agonist effect of this DA In fact, we have already demonstrated that levodopa does not affect the dopaminergic neurons when synthesis of DA is reduced by the dopa decarboxylase inhibitor carbidopa. l 6 Thus, after levodopa superfusion, there is an increased concentration of DA in the ventral mesencephalic tissue, which then activates the inhibitory D2 autoreceptors located on the soma and the dendrites of the dopaminergic cells. This is also confirmed by the antagonism of levodopa effects obtained with the D2 receptor antagonist sulpiride. It is noteworthy that only high concentrations (3-10 pM) of deprenyl, which cause substantial M A 0 A and B inhibition,' prolong for a relatively sustained period the effects of levodopa on a single cell. Thus, the

Mercuri et al: Synergistic Interaction between Deprenyl and Levodopa 615

blocked degradation of DA by deprenyl would protract the permanence of DA on its receptors and, in turn, increase the amount of catecholamine available for release after levodopa loading. In accordance, it has been recently reported that a pure blockade of M A 0 B enzymes is not a sufficient factor to enhance significantly the electrophysiological responses caused by the exogenous application of DA on the dopaminergic cells.9 Furthermore, the inhibition of M A 0 B enzymes does not affect DA metabolism formed from exogenous levodopa in the striatum of hemiparkinsonian rats.21 Another piece of information obtained from the in vitro experiments is that deprenyl influences the neurons directly and not by its peripheral transformation in L-methamphetamine and amphetamine.22

Behavioral Observations It has been recognized for a long time that levodopa reverses parkinsonian symptoms in animals treated with reserpine. As observed from the electrophysiological experiments in which high concentrations of deprenyl potentiated the effects of levodopa on a single cell, this compound increased the ability of levodopa to reverse motor depression in monoamine-depleted animals. It is worth noting that only the concentrations of deprenyl that were able to block both M A 0 A and MA0 B enzymes were effective. In fact, MA0 B-selective doses of deprenyl (1 mg/kg) neither potentiated the electrophysiological nor the behavioral effects of levodopa. The behavioral enhancement of levodopa responses after nonselective deprenyl treatment can be explained by a prolonged activation of central dopaminergic receptors caused by new-formed, noncatabolized DA.23

Clinical Imp lications Although there is a general agreement that a constant and smooth stimulation of DA receptors is preferable when it is necessary to control the symptoms of Parkinson’s disease by l e ~ o d o p a , ~ the * , ~approaches ~ that have been attempted so far, to protract the therapeutic effects of levodopa in the brain, have not yet caused consistent clinical benefits.26 In addition, despite the fact that MA0 B is more abundant than M A 0 A in human brain, a pure deprenyl-induced inhibition of M A 0 B enzymes might not be sufficient to increase the sensitivity of neurons to DA formed from levodopa, because both MA0 A and MA0 B participate in the deamination of this catecholamine. 9323,27 This can, at least in part, explain the modest beneficial effects of MA0 B inhibition in Parkinson’s disease caused by the currently used selective doses of deprenyl (5-10 mg/day).26 It is noteworthy that the present results show that the central effects of levodopa are markedly enhanced by a mixed inhibition of M A 0 A and B enzymes; and

616 Annals of Neurology Vol 43 No 5 May 1998

based on electrophysiological and behavioral evidence, we suggest a combined therapeutic use of levodopa and high doses of deprenyl in Parkinson’s disease. Thus, in order to obtain a continuous activation of central dopaminergic receptors and to reduce the doses of levodopa required to control parkinsonian symptoms, an appropriate treatment with higher doses of deprenyl and levodopa might be attempted in patients. It has recently been reported that relatively high doses of deprenyl (40- 60 mg/day) have antidepressive actions and are well tolerated in the elderly.28 Notwithstanding that control of parkinsonian symptoms derived from the use of levodopa has been attributed to its decarboxylation to DA in the ~triaturn,~’ the overall physiological changes induced by this amino acid on neurons of the ventral mesencephalon might also participate in its therapeutic a ~ t i o n . ~ ’ - ~ ~ Conclusion In conclusion, considering these results, the idea of administering levodopa and nonselective concentrations of deprenyl to parkinsonian patients appears to be based on a sound pharmacological and physiological rationale.

The technical support of Giuseppe Gattoni is appreciated.

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Mercuri et al: Synergistic Interaction between Deprenyl and Levodopa