At low doses, harmaline increases forelimb tremor in the rat

Neuroscience Letters 241 (1998) 41–44

At low doses, harmaline increases forelimb tremor in the rat John A. Stanford a, Stephen C. Fowler a , b ,* a

Department of Human Development, 4011 Dole Center, University of Kansas, Lawrence, KS 66045, USA b Department of Pharmacology and Toxicology, The Schiefelbusch Institute for Life Span Studies, University of Kansas, Lawrence, KS 66045, USA

Received 29 September 1997; received in revised form 10 December 1997; accepted 10 December 1997

Abstract A behavioral preparation especially sensitive to low-dose drug effects on fine motor behavior in rats was used to assess the tremorogenic effects of harmaline, an indole alkaloid and b-carboline derivative. Rats that were trained to press downward on a force transducer for water reinforcement were initially administered harmaline (0.5 and 1.0 mg/kg) in an acute dosing regime. Immediately following the day of initial acute exposure to 1 mg/kg, 3 consecutive days at this dose ensued, providing for a 4-day, repeated-dosing analysis. Harmaline did not significantly suppress task engagement during either acute or repeated dosing. Acute administration of harmaline dose-dependently increased power in the high-frequency (10–25 Hz) band of the power spectrum (tremor) without affecting overall forelimb force output. Upon continued administration, tremor remained significantly elevated above vehicle values. Harmaline also slowed the rats’ licking frequency, an effect that did not diminish with repeated dosing. Harmaline increased the durations of individual responses during acute dosing and continued to exert this effect with repeated dosing. The effects reported in the present study may represent low-dose harmaline-induced alterations in the olivocerebellar system.  1998 Elsevier Science Ireland Ltd.

Keywords: Harmaline; b-Carbolines; Indole alkaloids; Tremor; Rats; Operant; Olivo-cerebellar

There has been increasing interest in the anti-addictive properties of the beta carboline indole alkaloids, including harmaline and ibogaine ([7]; U.S. Patents 4, 499, 096, Feb. 12, 1985; 4, 587, 243, May 6, 1986; 5, 591, 738, Jan. 7, 1997). Potential side-effects of these agents, including tremor, remain an important issue. The propensity for harmaline to produce visually observable tremor with a frequency between 8 to 14 Hz in a variety of mammalian species is well known [8,9,13,17,19]. Harmaline is believed to produce tremor by increasing the rhythmic activity of inferior olivary cells which, via the climbing fiber pathway, induce rhythmic activation of cerebellar Purkinje cells, ultimately co-activating alpha- and gamma- motor neurons [3,10]. Upon repeated administration, the tremorogenic effect of harmaline has been shown to exhibit rapid pharmacological tolerance, with the tolerance generally attributed to persistent alterations in olivo-cerebellar function [16,17]. * Corresponding author. Tel.: +1 785 8640715; fax: +1 785 8645202; e-mail: [email protected]

Although harmaline-induced tremor has been studied extensively, there is scant information on the drug’s lowdose effects on operant (purposeful, skilled) behavior, with previous studies producing conflicting results [1,18,26]. Furthermore, in these operant behavior studies, there was no mention of tremor produced by the drug, probably because the doses used in operant studies were low compared to the doses used in experiments observing gross tremor (see below for reported dose ranges). In the present study, we used a sensitive technique for measuring forelimb tremor within an operant behavioral paradigm that has been shown to reveal low-dose tremor effects in the rat [6,22]. Twenty male Sprague–Dawley rats (average body weight 455 g) were trained with water reinforcement to press and hold a flat horizontal disk that was attached to a force-sensing transducer. The operandum and reinforcement dipper were arranged so that the rat extended a single forelimb to exert pressure on the operandum and was able at the same time to drink water from a solenoid-operated dipper presented at about floor level (see [6] for a diagram). Computer

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00974- 9

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J.A. Stanford, S.C. Fowler / Neuroscience Letters 241 (1998) 41–44

software recorded at 100 samples/s the force output of the transducer/operandum and required an initial response force of 20 g or greater for dipper presentation. The dipper remained within reach of the rat’s tongue as long as the force on the operandum was kept above 6.7 g. When force fell below 6.7 g the dipper dropped back into the water reservoir. Apparatus and procedural details have been published [5,6]. After training and the attainment of stable task performance, Harmaline HCl (Sigma, St. Louis, MO, USA, USA) was administered intraperitoneally 30 min pre-session in saline volumes of 1 ml/kg. The dosing regimen consisted of an acute dosing phase (vehicle, 0.5 mg/kg, 1.0 mg/kg) followed by repeated dosing with the higher dose (1.0 mg/ kg) as follows: day 1, vehicle; day 2, 0.5 mg/kg; day 3, no injection; day 4, vehicle; day 5, 1.0 mg/kg; day 6, 1.0 mg/ kg; day 7, 1.0 mg/kg; day 8, 1.0 mg/kg. Experimental sessions for each rat lasted approximately 8 min and were analyzed by computer programs which provided five measures of behavior. The first measure was one of overall task engagement: time on task (TOT; i.e. the time a rat spent applying at least 1 g of force to the transducer during the session). Each rat’s raw data was parsed into individual waveforms (identified by computer as response bouts consisting of at least 4.36 s of 20 g or greater force output). The mean duration of each bout greater than 4.36 s was calculated and served as a dependent variable. Individual waveforms were ensemble averaged in the time domain for the first 4.36 s of the event; from these averaged waveforms we computed the maximum or ‘peak’ force attained by the rat during the first second, as well as the mean or ‘hold’ force sustained during the remaining 3.36 s of the response. The 3.36 s hold portion of each individual force-time waveform was then subjected to a prime factor fast Fourier transformation (Alligator Technologies, Fountain Valley, CA, USA) to yield separate power spectra for each event. The spectra were then ensemble averaged in the frequency domain for each subject. The averaged power spectra for each rat provided the basis for the tremor measure which was defined as the power integrated across the 10–25 Hz frequency band. Tremor was defined in terms of the 10–25 Hz frequency band because previous work [22] indicated that frequencies below 10 Hz in this task contain

power attributable to the rat’s licking behavior as it consumes water from the dipper. For the acute comparisons, dependent variables were analyzed using repeated measures one-way analyses of variance (ANOVAs) [25] comparing vehicle, 0.5 mg/kg, and the first 1.0 mg/kg day, with vehicle data averaged across the 2 vehicle days serving as the vehicle value for each dependent variable. To test for possible tolerance effects, dependent variables were also analyzed for the repeated dosing phase using repeated measures one-way ANOVAs, with the 4 days of exposure to harmaline 1.0 mg/kg being compared. As shown in Table 1, neither acute nor repeated harmaline exposure significantly affected TOT, which shows that the doses used were too low to affect motivation to engage in the task. Harmaline did produce a dose-dependent rhythm (LICK in Table 1) slowing, an effect that did not diminish with repeated exposure to the higher dose. Integrated power in the 10–25 Hz frequency band (tremor) was dose-dependently increased by harmaline. This increase in tremor, which with repeated dosing remained significantly higher than vehicle values, appeared to be limited to a narrow frequency band between 8 and 14 Hz (see Fig. 1). Inspection of the power spectra for each of the 20 rats after the first dose of 1.0 mg/kg of harmaline revealed that 19 rats exhibited a slowing of the lick frequency (peak in the 5–6 Hz region of the spectrum) and 19 rats showed an increase in power in the 10–12 Hz region as depicted in Fig. 1. Harmaline affected neither of the forelimb force measures (peak force, hold force) but did affect individual response durations dose-dependently, as these were significantly lengthened. Like the other affected variables, the durationlengthening effect did not diminish with repeated dosing (see Table 1). Our finding that harmaline had negligible effects on task engagement at the doses administered are in general agreement with earlier studies examining the effect of low doses of harmaline on schedule controlled operant behavior ([1,18], see also [26] for an exception). Although [26] suggests that harmaline’s lack of task decrement (as reported by [18]) resulted from a time interval between harmaline injection and behavioral testing that was too long (60 min versus the 40 min interval in [26]), results reported by [1], in which

Table 1 Group means and standard errors of the mean for the indicated dependent variables as a function of harmaline dose Dose (mg/kg)

TOT (s)

Vehicle 0.5 1.0 (Day 1.0 (Day 1.0 (Day 1.0 (Day

337 276 326 287 306 345

1) 2) 3) 4)

± + ± ± ± ±

9 30 13 24 19 8

LICK (Hz) 6.54 5.87 5.50 5.54 5.57 5.62

± ± ± ± ± ±

0.15 0.1** 0.1** 0.1 0.07 0.13

DUR (s) 6.34 6.99 7.77 7.87 7.65 7.60

± ± ± ± ± ±

0.24 0.28** 0.25** 0.29 0.25 0.28

HIGH 4.49 4.96 5.01 4.97 4.86 4.90

± ± ± ± ± ±

0.21 0.21* 0.25* 0.21 0.22 0.21

TOT, time-on-task; LICK, dominant or lick frequency; DUR, response durations; HIGH, power in the high-frequency band (tremor) expressed in arbitrary log10 units. Acute ANOVA comparisons (vehicle versus 0.5 and 1.0 mg/kg): *P , 0.01; **P , 0.001.

J.A. Stanford, S.C. Fowler / Neuroscience Letters 241 (1998) 41–44

Fig. 1. Averaged power spectra from all twenty rats illustrating the tremorogenic effects of 0.5 and 1.0 mg/kg harmaline. Note also the dose related, leftward shift in the peaks around 6 Hz. This shift represents the slowing effect of harmaline on the lick frequency in this task.

harmaline produced no behavioral suppression of shock avoidance (at doses comparable to those in [26]) during the 30 min following injection, suggest otherwise. Caution should be exercised, however, when comparing the results of studies in which different contingencies and schedules of reinforcement are utilized to assess behavioral suppressing properties of drugs [20]. Given the substantial literature regarding harmalineinduced tremor, our finding of increased tremor was somewhat expected, although in the present study, harmaline produced tremor at doses that ranged from 1 to 11% of those reported previously (generally ranging from 9 to 50 mg/kg in the rat; [4,7,16,17,21]). In addition, the tremor produced by harmaline appeared qualitatively different from the tremor produced by physostigmine using these measures [22]. The relatively narrow-band increase in power produced by harmaline in this task corresponds with the drug’s putative mechanism of action: an amplification of natural rhythmic activity governed by the olivo-cerebellar system. That the increase in tremor in the present study remained significantly above vehicle values may be attributed to either: (1) our cessation of testing before tolerance could occur (although 4 days is within the reported time frame of tolerance onset, with tolerance developing sooner for lower doses; [17]), or (2) our doses being too low to produce the reported persisting effects of repeated harmaline treatment on the olivo-cerebellar system [17]. It is possible that harmaline’s effects on lick frequency and on response hold duration may be related to the role of the inferior olive as the principal generator of rhythmic activity in skeletal muscles [13,14,24], and of the olivocerebellar system (particularly the climbing fibers) as a modulator of muscle movement onset [12]. Regarding the frequency effect (slowing), unpublished observations from

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our laboratory demonstrated that the dominant frequency measure is a reflection of the rat’s lick rhythm being mechanically transmitted through the forelimb musculature. Since rats’ licking is highly rhythmic, with this rhythmicity governed partially by the inferior olive [24], and since it has been shown that microinjecting harmaline into the inferior olive slows the oscillatory frequency of the neurons there [23], our finding of a dose-dependent slowing of frequency is likely to be associated with harmaline’s actions at the inferior olive. The dose-dependent lengthening of response durations may be related to the role of the olivo-cerebellar system in the phasic activation of opposing muscles during a motor act. It has been suggested that the olivo-cerebellar system, by way of governing the ‘switching’ between activation of motoneurons and input from mossy fibers, is instrumental in ballistic motor control [13]. Although the present task may not be considered particularly ballistic, harmaline may disrupt the olivo-cerebellar system’s contribution to the cessation of the forelimb hold response (the observed duration-lengthening effect) as part of the system’s wider role in motor sequence timing [11,15]. The findings reported here demonstrate subtle behavioral manifestations of harmaline’s low-dose effects on the olivocerebellar system, effects which may accompany the use of such drugs as anti-addictive agents. It has been suggested that disruption of central pacemakers, including the olivocerebellar system, may result in not only tremor, but also in other disorders affecting learned motor sequences and postural control [2]. An additional implication of the results is that these methods may prove valuable in assessing the sideeffects of beta carboline indole alkaloids (e.g. ibogaine) hypothesized to have potential in treating human drug addiction. This work was supported by NIMH Grant MH43429. [1] Bocknik, S.E., Hingtgen, J.N., Hughes, F.W. and Forney, R.B., Harmaline effects on tetrabenazine depression of avoidance responding in rats, Life Sci., 7 (1968) 1189–1201. [2] DeLong, M.R., Possible involvement of central pacemakers in clinical disorders of movement, Fed. Proc., 37 (1978) 2171– 2175. [3] de Montigny, C. and Lamarre, Y., Rhythmic activity induced by harmaline in the olivo-cerebello-bulbar system of the cat, Brain Res., 53 (1973) 81–95. [4] Eblen, R., Loschmann, P.-A., Wullner, U., Turski, L. and Klockgether, T., Effects of 7-nitroindazole, NG-nitro-L-arginine, and D-CPPene on harmaline-induced postural tremor, Nmethyl-D-aspartate-induced seizures, and lisuride-induced rotations in rats with nigral 6-hydroxydopamine lesions, Eur. J. Pharmacol., 299 (1996) 9–16. [5] Fowler, S.C., Davison, K.H. and Stanford, J.A., Unlike haloperidol, clozapine slows and dampens rats’ forelimb force oscillation and decreased force output in a press-while-licking behavioral task, Psychopharmacology, 116 (1994) 19–25. [6] Fowler, S.C., Liao, R.M. and Skjoldager, P.D., A new rodent model for neuroleptic-induced pseudoparkinsonism: low doses of haloperidol increase forelimb tremor in the rat, Behav. Neurosci., 104 (1990) 449–456.

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