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Effects of benzodiazepines on single unit activity in the substantia nigra pars reticulata
Life Sciences, Vol. 31, pp. 1025-1035 Printed in the U.S.A.
Pergamon Press
EFFECTS OF BENZODIAZEPINES ON SINGLE UNIT ACTIVITY IN THE SUBSTANTIA NIGRA PARS RETICULATA Richard 3. Ross, M.D., Ph.D., Barbara L. Waszczak, Ph.D., Eun Kyu Lee and 3udith R. Waiters, Ph.D. Clinical Psychobiology Branch, National Institute of Mental Health and Experimental Therapeutics Branch National Institute of Neurological and Communicative Disorders and Stroke 9000 Rockville Pike, Bethesda, Maryland 20205 USA (Received in final form June 21, 1982)
Summary Intravenous administration of two benzodiazepines, flurazepam and diazepam, had an inhibitory effect on the firing rates of neurons of the substantia nigra pars reticulata, a brain region with an identified GABAergic innervation. Diazepam was more potent than flurazepam. Bicuculline and picrotoxin, two drugs which block GABAergic transmission, and caffeine and theophylline, two methylxanthines which inhibit benzodiazepine binding, all reversed the inhibition produced by diazepam. The action of theophylline was less consistent than that of caffeine. Similarly, Ro 15-17gg, an imidazodiazepine which putatively functions as a specific benzodiazepine antagonist, reversed the diazepam-induced inhibition. These findings are consistent with previous reports which suggest that the benzodiazepines may act through a GABAergic mechanism. In a separate group of experiments, caffeine or Ro 15=17gg was administered alone. While caffeine excited all reticulata cells tested, Ro 15-17gg, the more specific benzodiazepine antagonist, generally had l i t t l e excitatory effect. These results suggest: l) that cells of the substantia nigra pars reticulata may not receive a substantial, tonic inhibition mediated by an endogenous benzodiazepine-like substance; and 2) that the methylxanthines may increase reticulata cell firing, at least in part, through mechanisms unrelated to the blockade of benzodiazepine receptors. Much attention has been devoted to elucidating the mechanism of action of the benzodiazepines. Recently it has been demonstrated that the mammalian brain contains receptors which bind members of this class of drugs stereo-specifically and with high a f f i n i t y (1,2). It has been suggested that the benzodiazepine receptor forms a part of a GABA-benzodiazepine receptor-chloride ionophore complex (3) and that the benzodiazepines exert their numerous physiological and behavioral effects by f a c i l i t a t i n g GABAergic transmission in the central nervous system (4,5). This concept is supported by the observations that GABA enhances the a f f i n i t y of benzodiazepine binding (3,6) and that, in many neuronal systems, the benzodiazepines potentiate the electrophysiological effects of GABA (7,8). The SN is a brain region with significant benzodiazepine binding (9) and high concentrations of GABA (10). In immunocytochemical studies, it is seen to contain numerous terminals which stain positively for glutamic acid decarboxylase ( l l ) . Classically it has been hypothesized that the SN receives a substantial GABAergic input from the striatum and that this pathway serves as a "feedback loop" regulating 0024-3205/82/101025-11503.00/0
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nigrostriatal dopaminergic function (12,13). The inhibitory striatonigral GABAergic pathway appears to also directly influence cells of the SN pars reticulata. Consistent with this view is the observation that these cells, which are inhibited by iontophoretically applied GABA and muscimol and systemically administered GABAmimetic agents ([4), become supersensitive to these GABAergic agents following destruction of the striatonigral pathway (15). In the present study, we have investigated whether the benzodiazepines might influence the activity of these GABA-sensitive substantia nigra pars r e t i c u l a t a neurons. In addition, the ability of drugs of several different classes to act as antagonists was tested. These included Ro lS-17gg, an imidazodiazepine which putatively acts as a specific benzodiazepine antagonist (16,17), bicuculline and picrotoxin, two compounds which are known to interfere with GABAergic transmission, and caffeine and theophyiline, two methylxanthines. These latter drugs have central stimulating properties which contrast with the anxiolytic3and anticonvulsant properties of the benzodiazepines, and they competitively inhibit H-diazepam binding (lg). In addition, in considering the possibility that an endogenous benzodiazepine-like substance might mediate a tonic inhibition of neurons in the pars r e t i c u l a t a region, the effect of systemically administered Ro 15-17gg on the firing rates of r e t i c u l a t a cells was also studied. It was reasoned that any demonstrated change might provide evidence for the presence of an endogenous benzodiazepine-like compound with a role in regulating r e t i c u l a t a cell activity. The effect of i.v. caffeine was compared with that of Ro 1517gg in order to explore whether the methylxanthines might influence r e t i c u l a t a cell firing through a mechanism related to the benzodiazepine receptor. Methods Male, Sprague-Dawley rats (Taconic Farms, Germantown, NY), generally weighing 250-300 g, were anesthetized with chloral hydrate, 400 mg/kg, i.p. They were mounted in a standard stereotaxic apparatus, and a needle was inserted in a tail vein. Additional doses of chloral hydrate were injected i.v. throughout the experiment, as needed to maintain a degree of anesthesia sufficient to dampen the flexion response to hindpaw pinch. Body temperature was generally maintained at 36-3g ° C. A burr hole, approximately 3 mm in diameter, was drilled in the skull at a point 2.0 mm lateral to lambda and 3.0 mm anterior to the lambdoid suture. Microelectrodes were mounted in a hydraulic microdrive and lowered through the hole into the region of the SN. The ceils which were recorded were located within the boundaries of the stereotaxic coordinates, anterior, 1760-2580 ]Jm; lateral, 1.7-2.5 ram; and ventral - 1.5 to -2.5 ram, according to K6nig and Klippel (19). The single barrel recording microelectrodes were pulled from 2.0 mm glass capillary tubing (1.0 mm internal diameter). Under microscopic observation, the tips were broken back to a diameter of approximately I-2 lain. The micropipettes were then filled with a solution of i% Pontamine Sky Blue (GURR, High Wycombe, Bucks, UK) in 2M NaCI. Electrodes used in recording had resistances ranging from 3.g to 6.0 Mfl (measured at 135 Hz). Electrical signals were passed through a high input impedance micro-probe amplifier and led into an amplitude/time discriminator with a variable gain, variable bandwidth input amplifier. The amplified signals were displayed on an oscilloscope and also passed into a counter which registered each time a spike was discriminated. Counts were integrated over I0 sec intervals and displayed in histograms plotted by a strip chart recorder. Cells of the SN were identified as belonging to either the pars compacta (dopamine-containing neurons) or pars r e t i c u l a t a on the basis of several generally accepted criteria (1%20). In brief, extracellularly recorded neurons of the pars compacta region have longer duration action potentials and exhibit lower frequencies of firing than pars r e t i c u l a t a neurons. Neurons recorded extracellularly in the pars r e t i c u l a t a region have action potential durations of 0.5-0.7 msec (mean 0.63 + 0.02 msec) and typically exhibit frequencies of firing of 10-40 spikes/sec. The pars r e t i c u l a t a is located ventral to the pars compacta, and a r e t i c u l a t a neuron was generally not recorded unless a dopamine neuron was located more superficially in the same track or a neighboring track. A baseline period of at least 5 min of unit activity was recorded
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before the first drug injection. In some e x p e r i m e n t s , drugs were a d m i n i s t e r e d i.v. through the tail vein at 2 rain intervals, in increasing increments, such t h a t each successive dose doubled the previous cumulative dose. In other studies, single drug doses were administered, and p o t e n t i a l antagonists were injected subsequently. In all e x p e r i m e n t s , only one cell was recorded in each animal. At the conclusion of the e x p e r i m e n t , the recording site was m a r k e d by passing a 15-20 IJA anodal c u r r e n t through the recording e l e c t r o d e for I5-20 rain. Each animal was perfused with 10% buffered formalin phosphate. Brains were sectioned, mounted, and stained, and recording sites could be verified by identifying the Pontamine Sky Blue deposit. The e f f e c t of a drug on the firing r a t e of a neuron was quantified by taking the mean of the number of spikes over successive 10 sec intervals within a p a r t i c u l a r dosage period and dividing t h a t by the mean baseline r a t e , to obtain a p e r c e n t a g e change. The drugs used in these e x p e r i m e n t s were chloral hydrate, bicuculline HCI, picrotoxin, caffeine, theophylline, and Tween 80 (polyoxyethylene sorbitan mono-oleate) (all from Sigma Chemical Co., St. Louis, Mo.); muscimol (Regis Chemical Co., Morton Grove, Ill.); and d i a z e p a m , diazepam vehicle, f l u r a z e p a m dihydrochloride, and Ro 151788 (ethyl $ - f l u o r o - 5 , 6 - d i h y d r o - 5 - m e t h y l - 6 - o x o - $ H - i m i d a z o [1,5-a ] [1,4 ] benzod i a z e p i n e - 3 - c a r b o x y l a t e ) (all from Hoffmann LaRoche, Inc., Nutley, N.3.). For i.v. injections, Ro 15-1788 was suspended in an aqueous solution of Tween 80 (2 drops of Tween per 10 ml water). Where applicable, drug doses are given in t e r m s of the weight of the salt or on a molar basis. Results F l u r a z e p a m , a d m i n i s t e r e d i.v. in increasing increments to a c u m u l a t i v e dose of g.0 mg/kg, had a modest inhibitory e f f e c t on SN pars r e t i c u l a t a neurons (n:16; Figs. 1,2). There was considerable v a r i a b i l i t y in the responses of cells in the population. Sixty-nine p e r c e n t of cells were inhibited by at least 20%. No cells were inhibited by as much as 80%. A c u m u l a t i v e dose of 0.5 mg/kg did not produce a significant inhibition, while a c u m u l a t i v e dose of 8.0 mg/kg reduced firing by 39% (n=ll). Cells which were insensitive to f l u r a z e p a m could still be e f f e c t i v e l y inhibited by i.v. muscimol 3.2-6.t¢ mg/kg (n=4).
FLUR (cumulative doses to 8 m g / k g )
DIAZ (cumulative doses to 8 m g / k g )
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Figure 1 E f f e c t s of increasing Lv. doses of flurazepam (FLUR) and diazepam (DIAZ) on the firing r a t e s of s u b s t a n t i a nigra pars r e t i c u l a t a neurons. Both drugs were a d m i n i s t e r e d so t h a t each dose doubled the previously a d m i n i s t e r e d c u m u l a t i v e dose (see text). Doses of flurazepam were .125, .125, .25, .50, 1.0, 2.0 and t~.0 mg/kg; (total dose, 8.0 mg/kg). Doses of diazepam were .031, .031, .063, .125, .25, .50, 1.0, 2.0, and to.0 mg/kg; (total dose g.0 mg/kg).
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Figure 2 Cumulative log dose-response curves of the effects of increasing i.v. doses of FLUR (O) n--16) and DIAZ (O, n=17) on the f i r i n g rates of substantia nigra pars reticulata neurons. Points represent the mean firing rates in the 2 minute intervals following each dose. Vertical lines represent the standard errors of the means.
Diazepam had a more potent inhibitory effect) although there was still considerable variability in the sensitivity of individual neurons (Figs. 192). When this drug was administered i.v. in increasing increments to a cumulative dose of g.0 mg/kg, 87% of cells were inhibited by at least 20%. Twenty-seven percent of cells were inhibited by 80% or more. The average degree of inhibition following a cumulative dose of 0.5 mg/kg was 45% (n=17); following a cumulative dose of 8.0 mg/kg) cells were inhibited by an average of 53% (n=12). A f t e r a single 0.5 mg/kg dose, cells were inhibited by an average of 49% (n=18); their f i r i n g rates did not return to baseline until 30-50 minutes had elapsed (n=5). When administered alone, the diazepam vehicle produced either no change or a modest excitation (n= 12). In contrast to its ability to inhibit pars reticulata neurons, diazepam (doses up to 8.0 mg/kg) i.v.) did not consistently alter the firing of pars compacta dopamine neurons. Of the 10 cells tested) 5 cells were moderately but significantly excited, 3 cells were unaffected, and 2 cells were inhibited. Two blockers of GABAergic transmission, bicuculline and picrotoxin, two methylxanthines, caffeine and theophylline, and Ro 15-1788 could all reverse the inhibition produced by a single 0.5 mg/kg i.v. dose of diazepam (Figs. 3,4). Firing was restored to near baseline rates by bicuculline) at doses of 0.04-1.0 mg/kg, Lv. (n=5). Reversals by picrotoxin occurred at doses of 5-7 mg/kg i.v. (n=5). Caffeine)
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administered i.v. to a cumulative dose of 80 mg/kg, reversed the diazepam-induced inhibition in each of the 10 neurons studied. Theophylline, administered i.v. to a cumulative dose of 100 mg/kg, less consistently reversed the inhibition produced by diazepam (6 of 9 neurons tested). The specific benzodiazepine antagonist Ro I5-1788, administered as a single 0.53 mg/kg dose, consistently reversed the inhibitory effect of diazepam (n=$). On a tJmol/kg basis, this dose of Ro 15-178g was equivalent to the dose of diazepam previously given (Fig. 4). The Tween 80 vehicle, administered alone in control studies, failed to reverse the inhibition (n= 4).
DIAZ (1.76 ~mol/kg)
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200
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Ro 15-1788 (cumulative doses to 8 5 m g / k g )
300
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Figure t~ Up: Action of i.v. Ro 15-1788 in reversing the inhibition of a substantia nigra pars reticulata neuron produced by an equimelar i.v. dose of DIAZ (0.5 mg/kg). Down: Effect of i.v. Ro 15-1788 on the firing rate of a substantia nigra pars reticulata neuron. The drug was administered so that each dose doubled the previously administered cumulative dose. Doses were equivalent, on a molar basis, to the doses described previously for diazepam (see Fig. l).
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In a separate group of experiments, we also studied the e f f e c t s of caffeine and Ro 15-175g, administered alone, on r e t i c u l a t a cell firing. Caffeine, given to a cumulative dose of 80 mg/kg, excited all neurons tested (n=$). However, Ro 15-1755, suspended in an aqueous solution of Tween 80 and given to a cumulative dose of g.52 mg/kg (equivalent on a ]amol/kg basis to the total dose of diazepam administered above), significantly excited only 1796 of neurons studied. This drug had little e f f e c t on the remaining cells tested (total n=l$; Fig.0). When the Tween gO aqueous solution was administered alone in control studies, it was found that this vehicle had an e f f e c t comparable to that of the Ro 15-17gg - Tween 80 combination; i.e. 14% of the cells were excited, and the remainder were u n a f f e c t e d (n--7).
Discussion Our findings demonstrate that the systemic administration of either of two benzodiazepines is able to inhibit the firing of neurons of the SN pars reticulata. While flurazepam and diazepam were both effective, the latter was more potent. This difference in potency, which may reflect differences in lipid solubility and kinetics of distribution, remained when the dosages were compared on a lamol/kg basis. One possible explanation for the difference is diazepam's greater a f f i n i t y for the benzodiazepine receptor, but i t seems unlikely that the disparity between the two K. values obtained in binding studies (9 nM vs 16 nM) (7) is sufficiently great to account fo~ the inequivalent physiological effects found here. In any case, the different potencies observed in the rat parallel the relative clinical potencies of these two drugs in man (21). In contrast to its inhibition of SN pars reticulata neurons, an equivalent i.v. dose of diazepam did not consistently alter the firing rates of SN pars compacta dopamine neurons. While most dopamine cells monitored were unaffected or moderately stimulated by the drug, a result similar to that observed after i.v. doses of the GABA agonist muscimoI (1%22,23), a few do,amine neurons were inhibited by i.v. diazepam. During the 5 rain interval following the systemic administration of a single 0.5 mg/kg dose of diazepam, the average effect on reticulata cell firing was inhibition by ~9%. This is of a similar magnitude to the previously reported inhibitory effect of diazepam on neurons of the locus coeruleus recorded in the anesthetized rat (2#). In that study, no changes in blood pressure were observed after the systemic administration of doses of diazepam up to 5 mg/kg, and a hemodynamic explanation of the decrease in unit a c t i v i t y was therefore considered unlikely. In the cat, i t has been shown that a large part of the hypotensive response to an i.v. infusion of diazepam (2 mg/kg) can be attributed to the propylene glycol vehicle; a slow infusion of diazepam (0.5 mg/kg/min) produced only a small drop in blood pressure which rapidly reverted to normal (25). No effect of the diazepam vehicle, injected alone, was demonstrated in this study. It therefore seems unlikely that either a change in blood pressure or some direct effect of the vehicle can explain the inhibition of reticulata cell a c t i v i t y which was observed. It is possible that the effects of the benzodiazepines may not be mediated entirely through a GABAergic mechanism. For example9 i t has been argued that the benzodiazepines block the reuptake of adenosine, thereby potentiating the depressant action of this endogenous inhibitory substance (35). However, there is substantial evidence that the physiological effects of the benzodiazepines are mediated through a modulation of GABAergic transmission and that the respective receptor mechanisms are tightly coupled (3,%5). In considering the possibility that the benzodiazepines produced their effect on reticulata cell firing at least in part through a GABAergic mechanism, we studied the actions of both bicuculline and picrotoxin in reversing the diazepam-mediated inhibition. Both drugs, a GABA receptor antagonist and a blocker of the GABA-associated Cl-ionophore, respectively, antagonized the effect of a single 0.5 mg/kg dose of diazepam. These findings are compatible with, but not specific proof
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of, a common locus of action of the benzodiazepines and GABA on r e t i c u l a t a neurons. Also supportive of this hypothesis is the finding t h a t p r e t r e a t m e n t with a low, 50 g g / k g i.v. dose of d i a z e p a m , which only minimally a l t e r s neuronal a c t i v i t y , significantly p o t e n t i a t e s the inhibitory e f f e c t of Lv. muscimol on r e t i c u l a t a cell firing (Waszczak, unpublished observations). Our results a r e consistent with other observations concerning the e f f e c t s of the benzodiazepines in the substantia nigra. Although they did not s p e c i f i c a l l y l o c a t e their recording sites to the pars r e t i c u l a t a , Wolf e t al. also r e p o r t e d t h a t cells in the SN a r e inhibited by s y s t e m i c a l l y a d m i n i s t e r e d benzodiazepines (26). In evoked potential studies in the c a t , Haefely e t al. found t h a t a nigral response to s t r i a t a l stimulation, which was inhibited by bicuculIine, was r e s t o r e d by diazepam (5). There are numerous examples from other electrophysiological studies of this presumed i n t e r a c t i o n between the benzodiazepines and GABA (27,25,29,30,31,32,33). For example, Gallager (34) r e p o r t e d t h a t neurons of the rat dorsal raphe nucleus were inhibited by s y s t e m i c a l l y a d m i n i s t e r e d diazepam a f t e r p r e t r e a t m e n t with the GABAt r a n s a m i n a s e inhibitor AOAA; iontophoretically applied chlordiazepoxide and f l u r a z e p a m themselves had no inhibitory e f f e c t , but they did p o t e n t i a t e the inhibition produced by GABA. Geller e t al. (8) found t h a t diazepam enhanced the e l e c t r i c a l l y evoked, G A B A - m e d i a t e d inhibition of cultured tuberal hypothalamic neurons at doses which failed to e x e r t a d i r e c t depressant e f f e c t ; f u r t h e r m o r e , both bicuculline and picrotoxin antagonized the inhibition which had been produced by the benzodiazepines. Although pars r e t i c u l a t a cells were variably a f f e c t e d by the benzodiazepines, neurons which were minimally inhibited by f l u r a z e p a m were still inhibited by muscimol 3.2-6.~ mg/kg (n=4). Waszczak e t al. (14) found t h a t all r e t i c u l a t a neurons t e s t e d could be t o t a l l y inhibited by the i.v. administration of muscimol (12.8 mg/kg or lower). This implies t h a t some r e t i c u l a t a neurons a r e highly sensitive to GABA and considerably less sensitive to the benzodiazepines, and t h e r e b y raises the possibility t h a t GABA r e c e p t o r s on r e t i c u l a t a neurons may not always be linked to benzodiazepine r e c e p t o r s . This i n t e r p r e t a t i o n ~. consistent with the findings in a r e c e n t autoradiographic study in the r a t t h a t : l) H - f l u n i t r a z e p a m binding is uniformly s t i m u l a t e d by exogenous GABA, suggesting t h a t most or all benzodiazepine r e c e p t o r s a r e coupled to a form of the GABA r e c e p t o r ; and 2) not all GABA r e c e p t o r s , perhaps only a low a f f i n i t y subset, are linked to benzodiazepine sites (36). There a r e , in f a c t , known to be regional differences, within the SN in the density of benzodiazepine binding, with the pars l a t e r a I i s exhibiting a p a r t i c u l a r l y high level c o m p a r e d to the medial pars r e t i c u l a t a , where most of the cells r e c o r d e d in t h e s e studies were l o c a t e d (9). Caffeine and theophylline were able to r e v e r s e the diazepam-induced inhibition of firing. These. methylxanthlnes, which a r e m e t h y l a t e d purines, a c t as c o m p e t i t i v e inhibitors of H-diazepam binding (18). Because they have stimulating p r o p e r t i e s and, in high doses, cause convulsions (21), it has been p o s t u l a t e d t h a t they might e x e r t their pharmacologic action a t the site of the benzodiazepine r e c e p t o r , perhaps by blocking an endogenous Iigand. C o m p a t i b l e with this idea are: I) clinical observations t h a t the benzodiazepines have potent anxiolytic and anticonvulsant properties; and 2) the suggestion t h a t several purine nucleosides might function as natural Iigands for the benzodiazepine r e c e p t o r (18). Our finding t h a t two methylxanthines, c a f f e i n e and theophyUine, reversed the inhibitory e f f e c t of diazepam on r e t i c u l a t a cell firing is consistent with the hypothesis t h a t all of these drugs might a c t a t a common site. While r e l a t i v e l y high doses of c a f f e i n e (up to 80 mg/kg) were required to r e v e r s e the d i a z e p a m - i n d u c e d inhibition, it has been pointed out t h a t , in various m a m m a l i a n systems, the antagonism of benzodiazepine-induced e f f e c t s also occurs a t high doses, ranging up to 180 mg/kg, p.o. (37). Similarly, binding studies d e m o n s t r a t e a low a f f i n i t y of the methylxanthines for the benzodiazepine r e c e p t o r (18). However, while t h e methylxanthines may function as benzodiazepine r e c e p t o r blockers, these compounds a r e also known to have a wide range of e f f e c t s a t the cellular level which a r e not r e l a t e d to their ability to c o m p e t i t i v e l y inhibit benzodiazepine binding. These other actions, which include phosphodiesterase inhibition, mobilization of intraceUular calcium, and antagonism of a d e n o s i n e - m e d i a t e d neurotransmission (21), could
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conceivably have played some role in the ability of these drugs to increase the a c t i v i t y of pars reticulata neurons alter the administration of diazepam. However, since theophylline is a considerably more potent phosphodiesterase inhibitor than caffeine, its lesser potency in antagonizing the inhibitory effect of diazepam on reticulata ceils argues against a mechanism involving cyclic nucIeotide accumulation (35). Analogous reasoning suggests that the blockade of adenosine receptors is not involved (39). The relative potencies of caffeine and theophyHine in inhibiting benzodiazepine binding do, however, parallel their potencies in restoring reticulata cell firing (18). This suggests that an action of the methylxanthines at the benzodiazepine receptor might be involved in their ability to reverse the diazepam-induced inhibition of neuronal firing. If such were the case, the observation that caffeine, administered alone, excited reticulata neurons would raise the possibility that an endogenous ligand for the benzodiazepine receptor were being displaced. Our studies with Ro 15-1788, discussed below, provided additional insight into this possibility. Various approaches have been used in investigating whether there might exist a naturally occurring compound which functions as a physiological ligand for the benzodiazepine receptor. In binding studies, several putative agonists have been identified through their abilities to serve as competitive inhibitors (t0). In an electrophysiological study, the effects of several of these substances on cultured spinal neurons have been observed, and the findings have been compared with those obtained from the application of a benzodiazepine (01). In the present study, the effects of systemically administered Ro 15-1788 on reticulata cell a c t i v i t y were examined in order to gain some insight into whether these benzodiazepine-sensitive cells are tonically influenced by an endogenous benzodiazepine-like ~ompound. Ro 15-1788 is an imidazodiazepine with a potent inhibitory effect on H-diazepam binding in brain (16). It acts as a specific antagonist of many behavioral, electrophysiological, and biochemical effects of the typical benzodiazepines and by itself generally lacks the tranquilizing properties of these benzodiazepines (17). Consistent with these reports, Ro 15-178g readily reversed the inhibitory effect of diazepam on reticulata cell firing. Given alone, Ro 15-I788 generally had l i t t l e excitatory effect. While the few instances of stimulation of cell firing by Ro 15-1758 may suggest a minor role for an endogenous benzodiazepine-like inhibitor, i t seems quite likely that these cases reflected a response of cells to the aqueous Tween gO vehicle in which the Ro 15-178g was suspended. Consequently, it would appear that reticulata neurons are not subject to a significant tonic inhibitory control mediated by an endogenous ligand for the benzodiazepine receptor. These findings then also imply that the excitation produced by caffeine, the much less specific benzodiazepine antagonist, most likely reflects a mechanism of action unrelated to the blockade of benzodiazepine sites. This is consistent with other evidence suggesting that the opposite actions of the benzodiazepines and the methylxanthines do not necessarily reflect an interaction at benzodiazepine receptors (K¢2,~3). It is of interest to speculate about possible correlations between the electrophysiological effects of the benzodiazepines on the SN and certain documented behavioral effects of these drugs. Recently i t has been reported that both muscimol and gamma-vinyl GABA (an irreversible GABA-transaminase inhibitor), injected bilaterally into the SN, protected against bicuculline-induced seizures. On this basis, i t has been suggested that GABAergic systems in the SN might be involved in limiting the propagation of seizure a c t i v i t y ( ~ ) . The possibility exists that the anticonvulsant action of the benzodiazepines involves a facilitation of GABAergic transmission within the SN and a reduction in a c t i v i t y in an efferent pathway from the pars reticulata. Other workers have reported that oral diazepam significantly affects saccadic e y e movements in humans (45,~(;). Since two important efferent projection sites of the SN pars reticulata are the superior colliculus and the reticular formation, both areas presumably involved in the generation of saccadic eye movements, i t might be hypothesized that the benzodiazepines influence saccades through an action on nigral pars reticulata neurons which project to either or both of these regions.
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Acknowledgements We wish to thank William E. Scott, Ph.D. (Hoffmann-La Roche, Inc., Nutley, N.J.) tor supplying the benzodiazepines and Ro 17-1788 used. We also acknowledge Steven M. Paul, M.D. and Frederick K. Goodwin, M.D. for helpful discussions during the course of these studies.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
29. 30. 31. 32. 33. 34. 35.
R.F. SQUIRES and C. BRAESTRUP, Nature 266 732-734 (1977). H. MOHLER and T. OKADA, Science 198 849-851 (1977). 3.F. TALLMAN, 3.W. THOMAS and D.W. GALLAGER, Nature 274 383-385 (1978). E. COSTA, A. GUIDOTTI, C.C. MAO and A. SURIA, Life Sci. 17 167-186 (1975). W. HAEFELY, A. KULCSAR, H. MC)HLER, L. PIERI) P. POLC and R. SCHAFFNER, in Advances in Biochemical Psychopharmacology, Vol. 12, pp. 131151, E. COSTA and P. GREENGARD (Eds.), Raven Press, New York (1975). M. KAROBATH and G. SPERK, Proc. Natl. Acad. Sci. 76 1004-1006 (1979). 3.F. TALLMAN, S.M. PAUL, P. SKOLNICK and D.W. GALLAGER, Science 207 274-281 (1980). H.M. GELLER, B.3. HOFFER and D.A. TAYLOR, Fed. Proc. 39 3016-3023 (1980). W.S. YOUNG, III and M.3. KUHAR, J. Pharmacol. Exp. Ther. 2"-72 337-346 (1980). G.3. BALCOM, R.H. LENOX and 3.L. MEYERHOFF, 3. Neurochem. 24 609-613 (1975). E.C. RIBAK, 3.E. VAUGHN, K. SAITO, R. BARBER and E. ROBERTS, Brain Res. 116 287-298 (1976). A. CARLSSON and M. LINDQVIST, Acta Pharmacol. et Toxicol. 20 140-144 (1963). 3.S. KIM, 1.3. BAK, R. HASSLER and Y. OKADA, Exp. Brain Res. 14 95-10t~ (1971). B.L. WASZCZAK, N. ENG and 3.R. WALTERS, Brain Res. 188 185-197 (1980). B.L. WASZCZAK, C. HUME and 3.R. WALTERS, Life Sci. 28 2411-2420 (1981). W. HUNKELER, H. MC)HLER, L. PIERI, P. POLC, E.P. B"'ONETTI, R. CUMIN, R. SCHAFFNER and W. HAEFELY, Nature 290 514-516 (1981). P. POLC, 3.P. LAURENT, R. SCHERSCHLICHT and W. HAEFELY, NaunynSchmied. Arch. Pharmacol. 316 317-325 (1981). P.3. MARANGOS, S.M. PAUL, A.M. PARMA, F.K. GOODWIN, P. SYAPIN and P. SKOLN1CK, Life Sci. 24 851-858 (1979). 3.F.R. K6NIG and R-'~'A. KLIPPEL, The Rat Brain: A Stereotaxic Atlas, R.E. Krieger Publ., Huntington, N.Y. (1970). B.S. BUNNEY, 3.R. WALTERS, R.H. ROTH and G.K. AGHA3ANIAN, J. Pharmacol. Exp. Ther. 185 560-571 (1973). A.G. GILMAN, L.S. GOODMAN and A. GILMAN, The Pharmacological Basis of Therapeutics, Macmillan Publ., New York (1980). D. MacNEIL, M. GOWER and I. SAYMANSKA, Brain Res. 154 401-403 (1978). A.A. GRACE and B.S. BUNNEY, Eur. 3. Pharmacol. 59 211-218 (1979). S.3. GRANT, Y.H. HUANG and D.E. REDMOND, 3r., Life Sci. 27 2231-2236 (1980). L. SHARER and H. KUTT, Arch. Neurol. 2.~4169-175 (1971). P. WOLF and H.L. HAAS, Naunyn-Schmied. 299 211-218 (1977). W. SCHLOSSER~.Arch. Int. Pharmacodyn. 194 93-102 (1971). P. POLC, H. MOHLER and W. HAEFELY, Naunyn-Schmied. Arch. Pharmacol. 284 319-337 (1974). D.R. CURTIS, D. LODGE, G.A.R. 3OHNSTON and S.3. BRAND, Brain Res. 118 344-347 (1976). T. TSUCHIYA and H. FUKUSHIMA, Eur. 3. Pharmacol. 48 421-424 (1978). R.L. MACDONALD and 3.L.BARKER, Brain Res. 167 323-'--336 (1979). 3.N. NESTOROS and A. NISTRI, Can. 3. Physiol. Pharmacol. 57 1324-1329 (1979). M.A. SIMMONDS, Nature 284 558-560 (19S0). D.W. GALLAGER, Eur. 3. Plaarmacol. 49 133-143 (1978). 3.W. PHILLIS and P.H. WU, Le 3ournal Canadien des Sciences Neurologiques 7 247-249 (1980).
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3.R. UNNERSTALL, M.3. KUHAR, D.L. NIEHOFF and 3.M. PALACIOS, 3. Pharmacol. Exp. Ther. 21g 797-$04 {1981}. P. POLC, E.P. BONETTI, L. PIERI, R. CUMIN, R.M. ANGIOI, H. M()HLER and W.E. HAEFELY, Life Sci. 2$ 2265-2275 (19$1). R.W. BUTCHER and E.W. S--UTHERLAND, 3. Biol. Chem. 237 1244=1250 (1962). A. SATTIN and T.W. RALL, Mol. Pharmacol. 6 13-23 (1970-~-. P.3. MARANGOS, S.M. PAUL and F.K. GOOD-WIN, Life Sci. 2__551093-1102 (1979). 3.F. MacDONALD, 3.L. BARKER, S.M. PAUL, P.3. blARANGOS and P. SKOLNICK, Science 205 715-717 (1979). 3.W. DALY, R.F. BRUNS and S.H. SNYDER, Life Sci. 2g 2083-2097 (1981). S.H. SNYDER, 3.3. KATIMS, Z. ANNAU, R.F. BRUNS and 3.W. DALY, Proc. Natl. Acad. Sci. 7g 3260-3264 (1981). K. GALE, N-eurosci. Abst. 7 591 (19gl). S.3. ROTHENBERG and D. SELKOE, Psychopharmacology 7.._.4~232-236 (1981). P.R.M. BITTENCOURT, P. WADE, A.T. SMITH and A. RICHENS, Br. 3. Clin. Pharmacol. 12 523-533 (19gl).
Pergamon Press
EFFECTS OF BENZODIAZEPINES ON SINGLE UNIT ACTIVITY IN THE SUBSTANTIA NIGRA PARS RETICULATA Richard 3. Ross, M.D., Ph.D., Barbara L. Waszczak, Ph.D., Eun Kyu Lee and 3udith R. Waiters, Ph.D. Clinical Psychobiology Branch, National Institute of Mental Health and Experimental Therapeutics Branch National Institute of Neurological and Communicative Disorders and Stroke 9000 Rockville Pike, Bethesda, Maryland 20205 USA (Received in final form June 21, 1982)
Summary Intravenous administration of two benzodiazepines, flurazepam and diazepam, had an inhibitory effect on the firing rates of neurons of the substantia nigra pars reticulata, a brain region with an identified GABAergic innervation. Diazepam was more potent than flurazepam. Bicuculline and picrotoxin, two drugs which block GABAergic transmission, and caffeine and theophylline, two methylxanthines which inhibit benzodiazepine binding, all reversed the inhibition produced by diazepam. The action of theophylline was less consistent than that of caffeine. Similarly, Ro 15-17gg, an imidazodiazepine which putatively functions as a specific benzodiazepine antagonist, reversed the diazepam-induced inhibition. These findings are consistent with previous reports which suggest that the benzodiazepines may act through a GABAergic mechanism. In a separate group of experiments, caffeine or Ro 15=17gg was administered alone. While caffeine excited all reticulata cells tested, Ro 15-17gg, the more specific benzodiazepine antagonist, generally had l i t t l e excitatory effect. These results suggest: l) that cells of the substantia nigra pars reticulata may not receive a substantial, tonic inhibition mediated by an endogenous benzodiazepine-like substance; and 2) that the methylxanthines may increase reticulata cell firing, at least in part, through mechanisms unrelated to the blockade of benzodiazepine receptors. Much attention has been devoted to elucidating the mechanism of action of the benzodiazepines. Recently it has been demonstrated that the mammalian brain contains receptors which bind members of this class of drugs stereo-specifically and with high a f f i n i t y (1,2). It has been suggested that the benzodiazepine receptor forms a part of a GABA-benzodiazepine receptor-chloride ionophore complex (3) and that the benzodiazepines exert their numerous physiological and behavioral effects by f a c i l i t a t i n g GABAergic transmission in the central nervous system (4,5). This concept is supported by the observations that GABA enhances the a f f i n i t y of benzodiazepine binding (3,6) and that, in many neuronal systems, the benzodiazepines potentiate the electrophysiological effects of GABA (7,8). The SN is a brain region with significant benzodiazepine binding (9) and high concentrations of GABA (10). In immunocytochemical studies, it is seen to contain numerous terminals which stain positively for glutamic acid decarboxylase ( l l ) . Classically it has been hypothesized that the SN receives a substantial GABAergic input from the striatum and that this pathway serves as a "feedback loop" regulating 0024-3205/82/101025-11503.00/0
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nigrostriatal dopaminergic function (12,13). The inhibitory striatonigral GABAergic pathway appears to also directly influence cells of the SN pars reticulata. Consistent with this view is the observation that these cells, which are inhibited by iontophoretically applied GABA and muscimol and systemically administered GABAmimetic agents ([4), become supersensitive to these GABAergic agents following destruction of the striatonigral pathway (15). In the present study, we have investigated whether the benzodiazepines might influence the activity of these GABA-sensitive substantia nigra pars r e t i c u l a t a neurons. In addition, the ability of drugs of several different classes to act as antagonists was tested. These included Ro lS-17gg, an imidazodiazepine which putatively acts as a specific benzodiazepine antagonist (16,17), bicuculline and picrotoxin, two compounds which are known to interfere with GABAergic transmission, and caffeine and theophyiline, two methylxanthines. These latter drugs have central stimulating properties which contrast with the anxiolytic3and anticonvulsant properties of the benzodiazepines, and they competitively inhibit H-diazepam binding (lg). In addition, in considering the possibility that an endogenous benzodiazepine-like substance might mediate a tonic inhibition of neurons in the pars r e t i c u l a t a region, the effect of systemically administered Ro 15-17gg on the firing rates of r e t i c u l a t a cells was also studied. It was reasoned that any demonstrated change might provide evidence for the presence of an endogenous benzodiazepine-like compound with a role in regulating r e t i c u l a t a cell activity. The effect of i.v. caffeine was compared with that of Ro 1517gg in order to explore whether the methylxanthines might influence r e t i c u l a t a cell firing through a mechanism related to the benzodiazepine receptor. Methods Male, Sprague-Dawley rats (Taconic Farms, Germantown, NY), generally weighing 250-300 g, were anesthetized with chloral hydrate, 400 mg/kg, i.p. They were mounted in a standard stereotaxic apparatus, and a needle was inserted in a tail vein. Additional doses of chloral hydrate were injected i.v. throughout the experiment, as needed to maintain a degree of anesthesia sufficient to dampen the flexion response to hindpaw pinch. Body temperature was generally maintained at 36-3g ° C. A burr hole, approximately 3 mm in diameter, was drilled in the skull at a point 2.0 mm lateral to lambda and 3.0 mm anterior to the lambdoid suture. Microelectrodes were mounted in a hydraulic microdrive and lowered through the hole into the region of the SN. The ceils which were recorded were located within the boundaries of the stereotaxic coordinates, anterior, 1760-2580 ]Jm; lateral, 1.7-2.5 ram; and ventral - 1.5 to -2.5 ram, according to K6nig and Klippel (19). The single barrel recording microelectrodes were pulled from 2.0 mm glass capillary tubing (1.0 mm internal diameter). Under microscopic observation, the tips were broken back to a diameter of approximately I-2 lain. The micropipettes were then filled with a solution of i% Pontamine Sky Blue (GURR, High Wycombe, Bucks, UK) in 2M NaCI. Electrodes used in recording had resistances ranging from 3.g to 6.0 Mfl (measured at 135 Hz). Electrical signals were passed through a high input impedance micro-probe amplifier and led into an amplitude/time discriminator with a variable gain, variable bandwidth input amplifier. The amplified signals were displayed on an oscilloscope and also passed into a counter which registered each time a spike was discriminated. Counts were integrated over I0 sec intervals and displayed in histograms plotted by a strip chart recorder. Cells of the SN were identified as belonging to either the pars compacta (dopamine-containing neurons) or pars r e t i c u l a t a on the basis of several generally accepted criteria (1%20). In brief, extracellularly recorded neurons of the pars compacta region have longer duration action potentials and exhibit lower frequencies of firing than pars r e t i c u l a t a neurons. Neurons recorded extracellularly in the pars r e t i c u l a t a region have action potential durations of 0.5-0.7 msec (mean 0.63 + 0.02 msec) and typically exhibit frequencies of firing of 10-40 spikes/sec. The pars r e t i c u l a t a is located ventral to the pars compacta, and a r e t i c u l a t a neuron was generally not recorded unless a dopamine neuron was located more superficially in the same track or a neighboring track. A baseline period of at least 5 min of unit activity was recorded
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Benzodiazepines and SN Pars Reticulata
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before the first drug injection. In some e x p e r i m e n t s , drugs were a d m i n i s t e r e d i.v. through the tail vein at 2 rain intervals, in increasing increments, such t h a t each successive dose doubled the previous cumulative dose. In other studies, single drug doses were administered, and p o t e n t i a l antagonists were injected subsequently. In all e x p e r i m e n t s , only one cell was recorded in each animal. At the conclusion of the e x p e r i m e n t , the recording site was m a r k e d by passing a 15-20 IJA anodal c u r r e n t through the recording e l e c t r o d e for I5-20 rain. Each animal was perfused with 10% buffered formalin phosphate. Brains were sectioned, mounted, and stained, and recording sites could be verified by identifying the Pontamine Sky Blue deposit. The e f f e c t of a drug on the firing r a t e of a neuron was quantified by taking the mean of the number of spikes over successive 10 sec intervals within a p a r t i c u l a r dosage period and dividing t h a t by the mean baseline r a t e , to obtain a p e r c e n t a g e change. The drugs used in these e x p e r i m e n t s were chloral hydrate, bicuculline HCI, picrotoxin, caffeine, theophylline, and Tween 80 (polyoxyethylene sorbitan mono-oleate) (all from Sigma Chemical Co., St. Louis, Mo.); muscimol (Regis Chemical Co., Morton Grove, Ill.); and d i a z e p a m , diazepam vehicle, f l u r a z e p a m dihydrochloride, and Ro 151788 (ethyl $ - f l u o r o - 5 , 6 - d i h y d r o - 5 - m e t h y l - 6 - o x o - $ H - i m i d a z o [1,5-a ] [1,4 ] benzod i a z e p i n e - 3 - c a r b o x y l a t e ) (all from Hoffmann LaRoche, Inc., Nutley, N.3.). For i.v. injections, Ro 15-1788 was suspended in an aqueous solution of Tween 80 (2 drops of Tween per 10 ml water). Where applicable, drug doses are given in t e r m s of the weight of the salt or on a molar basis. Results F l u r a z e p a m , a d m i n i s t e r e d i.v. in increasing increments to a c u m u l a t i v e dose of g.0 mg/kg, had a modest inhibitory e f f e c t on SN pars r e t i c u l a t a neurons (n:16; Figs. 1,2). There was considerable v a r i a b i l i t y in the responses of cells in the population. Sixty-nine p e r c e n t of cells were inhibited by at least 20%. No cells were inhibited by as much as 80%. A c u m u l a t i v e dose of 0.5 mg/kg did not produce a significant inhibition, while a c u m u l a t i v e dose of 8.0 mg/kg reduced firing by 39% (n=ll). Cells which were insensitive to f l u r a z e p a m could still be e f f e c t i v e l y inhibited by i.v. muscimol 3.2-6.t¢ mg/kg (n=4).
FLUR (cumulative doses to 8 m g / k g )
DIAZ (cumulative doses to 8 m g / k g )
300-
0. I 5 MIN
Figure 1 E f f e c t s of increasing Lv. doses of flurazepam (FLUR) and diazepam (DIAZ) on the firing r a t e s of s u b s t a n t i a nigra pars r e t i c u l a t a neurons. Both drugs were a d m i n i s t e r e d so t h a t each dose doubled the previously a d m i n i s t e r e d c u m u l a t i v e dose (see text). Doses of flurazepam were .125, .125, .25, .50, 1.0, 2.0 and t~.0 mg/kg; (total dose, 8.0 mg/kg). Doses of diazepam were .031, .031, .063, .125, .25, .50, 1.0, 2.0, and to.0 mg/kg; (total dose g.0 mg/kg).
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Benzodiazepines and SN Pars Reticulata
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110 100 90
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Figure 2 Cumulative log dose-response curves of the effects of increasing i.v. doses of FLUR (O) n--16) and DIAZ (O, n=17) on the f i r i n g rates of substantia nigra pars reticulata neurons. Points represent the mean firing rates in the 2 minute intervals following each dose. Vertical lines represent the standard errors of the means.
Diazepam had a more potent inhibitory effect) although there was still considerable variability in the sensitivity of individual neurons (Figs. 192). When this drug was administered i.v. in increasing increments to a cumulative dose of g.0 mg/kg, 87% of cells were inhibited by at least 20%. Twenty-seven percent of cells were inhibited by 80% or more. The average degree of inhibition following a cumulative dose of 0.5 mg/kg was 45% (n=17); following a cumulative dose of 8.0 mg/kg) cells were inhibited by an average of 53% (n=12). A f t e r a single 0.5 mg/kg dose, cells were inhibited by an average of 49% (n=18); their f i r i n g rates did not return to baseline until 30-50 minutes had elapsed (n=5). When administered alone, the diazepam vehicle produced either no change or a modest excitation (n= 12). In contrast to its ability to inhibit pars reticulata neurons, diazepam (doses up to 8.0 mg/kg) i.v.) did not consistently alter the firing of pars compacta dopamine neurons. Of the 10 cells tested) 5 cells were moderately but significantly excited, 3 cells were unaffected, and 2 cells were inhibited. Two blockers of GABAergic transmission, bicuculline and picrotoxin, two methylxanthines, caffeine and theophylline, and Ro 15-1788 could all reverse the inhibition produced by a single 0.5 mg/kg i.v. dose of diazepam (Figs. 3,4). Firing was restored to near baseline rates by bicuculline) at doses of 0.04-1.0 mg/kg, Lv. (n=5). Reversals by picrotoxin occurred at doses of 5-7 mg/kg i.v. (n=5). Caffeine)
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1030
Benzodiazepines
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SN Pars R e t i c u l a t a
Vol.
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1982
administered i.v. to a cumulative dose of 80 mg/kg, reversed the diazepam-induced inhibition in each of the 10 neurons studied. Theophylline, administered i.v. to a cumulative dose of 100 mg/kg, less consistently reversed the inhibition produced by diazepam (6 of 9 neurons tested). The specific benzodiazepine antagonist Ro I5-1788, administered as a single 0.53 mg/kg dose, consistently reversed the inhibitory effect of diazepam (n=$). On a tJmol/kg basis, this dose of Ro 15-178g was equivalent to the dose of diazepam previously given (Fig. 4). The Tween 80 vehicle, administered alone in control studies, failed to reverse the inhibition (n= 4).
DIAZ (1.76 ~mol/kg)
~>
Ro 15-1788 (1.76 ~mol/kg)
200
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Ro 15-1788 (cumulative doses to 8 5 m g / k g )
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0
Figure t~ Up: Action of i.v. Ro 15-1788 in reversing the inhibition of a substantia nigra pars reticulata neuron produced by an equimelar i.v. dose of DIAZ (0.5 mg/kg). Down: Effect of i.v. Ro 15-1788 on the firing rate of a substantia nigra pars reticulata neuron. The drug was administered so that each dose doubled the previously administered cumulative dose. Doses were equivalent, on a molar basis, to the doses described previously for diazepam (see Fig. l).
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Benzodiazepines and SN Pars Reticulata
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In a separate group of experiments, we also studied the e f f e c t s of caffeine and Ro 15-175g, administered alone, on r e t i c u l a t a cell firing. Caffeine, given to a cumulative dose of 80 mg/kg, excited all neurons tested (n=$). However, Ro 15-1755, suspended in an aqueous solution of Tween 80 and given to a cumulative dose of g.52 mg/kg (equivalent on a ]amol/kg basis to the total dose of diazepam administered above), significantly excited only 1796 of neurons studied. This drug had little e f f e c t on the remaining cells tested (total n=l$; Fig.0). When the Tween gO aqueous solution was administered alone in control studies, it was found that this vehicle had an e f f e c t comparable to that of the Ro 15-17gg - Tween 80 combination; i.e. 14% of the cells were excited, and the remainder were u n a f f e c t e d (n--7).
Discussion Our findings demonstrate that the systemic administration of either of two benzodiazepines is able to inhibit the firing of neurons of the SN pars reticulata. While flurazepam and diazepam were both effective, the latter was more potent. This difference in potency, which may reflect differences in lipid solubility and kinetics of distribution, remained when the dosages were compared on a lamol/kg basis. One possible explanation for the difference is diazepam's greater a f f i n i t y for the benzodiazepine receptor, but i t seems unlikely that the disparity between the two K. values obtained in binding studies (9 nM vs 16 nM) (7) is sufficiently great to account fo~ the inequivalent physiological effects found here. In any case, the different potencies observed in the rat parallel the relative clinical potencies of these two drugs in man (21). In contrast to its inhibition of SN pars reticulata neurons, an equivalent i.v. dose of diazepam did not consistently alter the firing rates of SN pars compacta dopamine neurons. While most dopamine cells monitored were unaffected or moderately stimulated by the drug, a result similar to that observed after i.v. doses of the GABA agonist muscimoI (1%22,23), a few do,amine neurons were inhibited by i.v. diazepam. During the 5 rain interval following the systemic administration of a single 0.5 mg/kg dose of diazepam, the average effect on reticulata cell firing was inhibition by ~9%. This is of a similar magnitude to the previously reported inhibitory effect of diazepam on neurons of the locus coeruleus recorded in the anesthetized rat (2#). In that study, no changes in blood pressure were observed after the systemic administration of doses of diazepam up to 5 mg/kg, and a hemodynamic explanation of the decrease in unit a c t i v i t y was therefore considered unlikely. In the cat, i t has been shown that a large part of the hypotensive response to an i.v. infusion of diazepam (2 mg/kg) can be attributed to the propylene glycol vehicle; a slow infusion of diazepam (0.5 mg/kg/min) produced only a small drop in blood pressure which rapidly reverted to normal (25). No effect of the diazepam vehicle, injected alone, was demonstrated in this study. It therefore seems unlikely that either a change in blood pressure or some direct effect of the vehicle can explain the inhibition of reticulata cell a c t i v i t y which was observed. It is possible that the effects of the benzodiazepines may not be mediated entirely through a GABAergic mechanism. For example9 i t has been argued that the benzodiazepines block the reuptake of adenosine, thereby potentiating the depressant action of this endogenous inhibitory substance (35). However, there is substantial evidence that the physiological effects of the benzodiazepines are mediated through a modulation of GABAergic transmission and that the respective receptor mechanisms are tightly coupled (3,%5). In considering the possibility that the benzodiazepines produced their effect on reticulata cell firing at least in part through a GABAergic mechanism, we studied the actions of both bicuculline and picrotoxin in reversing the diazepam-mediated inhibition. Both drugs, a GABA receptor antagonist and a blocker of the GABA-associated Cl-ionophore, respectively, antagonized the effect of a single 0.5 mg/kg dose of diazepam. These findings are compatible with, but not specific proof
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of, a common locus of action of the benzodiazepines and GABA on r e t i c u l a t a neurons. Also supportive of this hypothesis is the finding t h a t p r e t r e a t m e n t with a low, 50 g g / k g i.v. dose of d i a z e p a m , which only minimally a l t e r s neuronal a c t i v i t y , significantly p o t e n t i a t e s the inhibitory e f f e c t of Lv. muscimol on r e t i c u l a t a cell firing (Waszczak, unpublished observations). Our results a r e consistent with other observations concerning the e f f e c t s of the benzodiazepines in the substantia nigra. Although they did not s p e c i f i c a l l y l o c a t e their recording sites to the pars r e t i c u l a t a , Wolf e t al. also r e p o r t e d t h a t cells in the SN a r e inhibited by s y s t e m i c a l l y a d m i n i s t e r e d benzodiazepines (26). In evoked potential studies in the c a t , Haefely e t al. found t h a t a nigral response to s t r i a t a l stimulation, which was inhibited by bicuculIine, was r e s t o r e d by diazepam (5). There are numerous examples from other electrophysiological studies of this presumed i n t e r a c t i o n between the benzodiazepines and GABA (27,25,29,30,31,32,33). For example, Gallager (34) r e p o r t e d t h a t neurons of the rat dorsal raphe nucleus were inhibited by s y s t e m i c a l l y a d m i n i s t e r e d diazepam a f t e r p r e t r e a t m e n t with the GABAt r a n s a m i n a s e inhibitor AOAA; iontophoretically applied chlordiazepoxide and f l u r a z e p a m themselves had no inhibitory e f f e c t , but they did p o t e n t i a t e the inhibition produced by GABA. Geller e t al. (8) found t h a t diazepam enhanced the e l e c t r i c a l l y evoked, G A B A - m e d i a t e d inhibition of cultured tuberal hypothalamic neurons at doses which failed to e x e r t a d i r e c t depressant e f f e c t ; f u r t h e r m o r e , both bicuculline and picrotoxin antagonized the inhibition which had been produced by the benzodiazepines. Although pars r e t i c u l a t a cells were variably a f f e c t e d by the benzodiazepines, neurons which were minimally inhibited by f l u r a z e p a m were still inhibited by muscimol 3.2-6.~ mg/kg (n=4). Waszczak e t al. (14) found t h a t all r e t i c u l a t a neurons t e s t e d could be t o t a l l y inhibited by the i.v. administration of muscimol (12.8 mg/kg or lower). This implies t h a t some r e t i c u l a t a neurons a r e highly sensitive to GABA and considerably less sensitive to the benzodiazepines, and t h e r e b y raises the possibility t h a t GABA r e c e p t o r s on r e t i c u l a t a neurons may not always be linked to benzodiazepine r e c e p t o r s . This i n t e r p r e t a t i o n ~. consistent with the findings in a r e c e n t autoradiographic study in the r a t t h a t : l) H - f l u n i t r a z e p a m binding is uniformly s t i m u l a t e d by exogenous GABA, suggesting t h a t most or all benzodiazepine r e c e p t o r s a r e coupled to a form of the GABA r e c e p t o r ; and 2) not all GABA r e c e p t o r s , perhaps only a low a f f i n i t y subset, are linked to benzodiazepine sites (36). There a r e , in f a c t , known to be regional differences, within the SN in the density of benzodiazepine binding, with the pars l a t e r a I i s exhibiting a p a r t i c u l a r l y high level c o m p a r e d to the medial pars r e t i c u l a t a , where most of the cells r e c o r d e d in t h e s e studies were l o c a t e d (9). Caffeine and theophylline were able to r e v e r s e the diazepam-induced inhibition of firing. These. methylxanthlnes, which a r e m e t h y l a t e d purines, a c t as c o m p e t i t i v e inhibitors of H-diazepam binding (18). Because they have stimulating p r o p e r t i e s and, in high doses, cause convulsions (21), it has been p o s t u l a t e d t h a t they might e x e r t their pharmacologic action a t the site of the benzodiazepine r e c e p t o r , perhaps by blocking an endogenous Iigand. C o m p a t i b l e with this idea are: I) clinical observations t h a t the benzodiazepines have potent anxiolytic and anticonvulsant properties; and 2) the suggestion t h a t several purine nucleosides might function as natural Iigands for the benzodiazepine r e c e p t o r (18). Our finding t h a t two methylxanthines, c a f f e i n e and theophyUine, reversed the inhibitory e f f e c t of diazepam on r e t i c u l a t a cell firing is consistent with the hypothesis t h a t all of these drugs might a c t a t a common site. While r e l a t i v e l y high doses of c a f f e i n e (up to 80 mg/kg) were required to r e v e r s e the d i a z e p a m - i n d u c e d inhibition, it has been pointed out t h a t , in various m a m m a l i a n systems, the antagonism of benzodiazepine-induced e f f e c t s also occurs a t high doses, ranging up to 180 mg/kg, p.o. (37). Similarly, binding studies d e m o n s t r a t e a low a f f i n i t y of the methylxanthines for the benzodiazepine r e c e p t o r (18). However, while t h e methylxanthines may function as benzodiazepine r e c e p t o r blockers, these compounds a r e also known to have a wide range of e f f e c t s a t the cellular level which a r e not r e l a t e d to their ability to c o m p e t i t i v e l y inhibit benzodiazepine binding. These other actions, which include phosphodiesterase inhibition, mobilization of intraceUular calcium, and antagonism of a d e n o s i n e - m e d i a t e d neurotransmission (21), could
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conceivably have played some role in the ability of these drugs to increase the a c t i v i t y of pars reticulata neurons alter the administration of diazepam. However, since theophylline is a considerably more potent phosphodiesterase inhibitor than caffeine, its lesser potency in antagonizing the inhibitory effect of diazepam on reticulata ceils argues against a mechanism involving cyclic nucIeotide accumulation (35). Analogous reasoning suggests that the blockade of adenosine receptors is not involved (39). The relative potencies of caffeine and theophyHine in inhibiting benzodiazepine binding do, however, parallel their potencies in restoring reticulata cell firing (18). This suggests that an action of the methylxanthines at the benzodiazepine receptor might be involved in their ability to reverse the diazepam-induced inhibition of neuronal firing. If such were the case, the observation that caffeine, administered alone, excited reticulata neurons would raise the possibility that an endogenous ligand for the benzodiazepine receptor were being displaced. Our studies with Ro 15-1788, discussed below, provided additional insight into this possibility. Various approaches have been used in investigating whether there might exist a naturally occurring compound which functions as a physiological ligand for the benzodiazepine receptor. In binding studies, several putative agonists have been identified through their abilities to serve as competitive inhibitors (t0). In an electrophysiological study, the effects of several of these substances on cultured spinal neurons have been observed, and the findings have been compared with those obtained from the application of a benzodiazepine (01). In the present study, the effects of systemically administered Ro 15-1788 on reticulata cell a c t i v i t y were examined in order to gain some insight into whether these benzodiazepine-sensitive cells are tonically influenced by an endogenous benzodiazepine-like ~ompound. Ro 15-1788 is an imidazodiazepine with a potent inhibitory effect on H-diazepam binding in brain (16). It acts as a specific antagonist of many behavioral, electrophysiological, and biochemical effects of the typical benzodiazepines and by itself generally lacks the tranquilizing properties of these benzodiazepines (17). Consistent with these reports, Ro 15-178g readily reversed the inhibitory effect of diazepam on reticulata cell firing. Given alone, Ro 15-I788 generally had l i t t l e excitatory effect. While the few instances of stimulation of cell firing by Ro 15-1758 may suggest a minor role for an endogenous benzodiazepine-like inhibitor, i t seems quite likely that these cases reflected a response of cells to the aqueous Tween gO vehicle in which the Ro 15-178g was suspended. Consequently, it would appear that reticulata neurons are not subject to a significant tonic inhibitory control mediated by an endogenous ligand for the benzodiazepine receptor. These findings then also imply that the excitation produced by caffeine, the much less specific benzodiazepine antagonist, most likely reflects a mechanism of action unrelated to the blockade of benzodiazepine sites. This is consistent with other evidence suggesting that the opposite actions of the benzodiazepines and the methylxanthines do not necessarily reflect an interaction at benzodiazepine receptors (K¢2,~3). It is of interest to speculate about possible correlations between the electrophysiological effects of the benzodiazepines on the SN and certain documented behavioral effects of these drugs. Recently i t has been reported that both muscimol and gamma-vinyl GABA (an irreversible GABA-transaminase inhibitor), injected bilaterally into the SN, protected against bicuculline-induced seizures. On this basis, i t has been suggested that GABAergic systems in the SN might be involved in limiting the propagation of seizure a c t i v i t y ( ~ ) . The possibility exists that the anticonvulsant action of the benzodiazepines involves a facilitation of GABAergic transmission within the SN and a reduction in a c t i v i t y in an efferent pathway from the pars reticulata. Other workers have reported that oral diazepam significantly affects saccadic e y e movements in humans (45,~(;). Since two important efferent projection sites of the SN pars reticulata are the superior colliculus and the reticular formation, both areas presumably involved in the generation of saccadic eye movements, i t might be hypothesized that the benzodiazepines influence saccades through an action on nigral pars reticulata neurons which project to either or both of these regions.
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Acknowledgements We wish to thank William E. Scott, Ph.D. (Hoffmann-La Roche, Inc., Nutley, N.J.) tor supplying the benzodiazepines and Ro 17-1788 used. We also acknowledge Steven M. Paul, M.D. and Frederick K. Goodwin, M.D. for helpful discussions during the course of these studies.
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