Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Neurochem. Int. Vol. 7, No. 4, pp. 675-681, 1985 Printed in Great Britain.All rights reserved

0197-0186/85$3.00+0.00 Copyright © 1985PergamonPress Ltd

MELATONIN REVERSES PINEALECTOMY-INDUCED DECREASE OF BENZODIAZEPINE BINDING IN RAT CEREBRAL CORTEX PEDRO R. LOWENSTEIN*, RUTH ROSENSTEIN* and DANIEL P. CARDIN^LIt Centro de Estudios Farmacol6gicos y de Principios Naturales (CEFAPRIN), Serrano 665, (1414)-Buenos Aires, Argentina (Received 15 May 1984; accepted 2 November 1984)

Atstract--Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [3H]flunitrazepam binding) in cerebral cortex 3-14 days after surgery without affecting thor affinity significantly. A single s.c. injection of melatonin (800/~g/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600/~g/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added/n vitro to rat cerebral cortex membranes only slightly depressed [3H]flunitrazepambinding at 100#M concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.

Current experimental evidence indicates that the pineal gland exerts a general, homeostatic influence on central nervous system (CNS) excitability (Quay, 1975; Romijn, 1978). Removal of the pineal gland in rabbits changed the electrical activity of dorsal hippocampal neurons towards a epileptiform electroencephalographic pattern (Bindoni and Rizzo, 1975). In pinealectomized (Ix) rats electrical recording of cerebral cortex exhibited intermittent paroxysmal outbursts of seizure-like discharges (Nir et al., 1969) and in parathyroidectomized rats a subsequent Px induced violent and often fatal clonic-tonic type convulsions (Reiter et al., 1973). The antiepileptic activity of the pineal gland is further supported by observations describing epileptiform behavior in Mongolian gerbils as revealed by wild running, clonus and tonus shortly after Px (Philo and Reiter, 1978). However such pineal antiepileptic properties may not be universal inasmuch as in mice IX inhibited subsequently induced convulsions (Hata and Kita, 1978). In view of the fact that one of the major pineal products, melatonin, and its brain metabolite Nacetyl-5-methoxykynurenamine were reported to in-

teract with brain benzodiazepine (BZP) binding sites in vitro (Marangos et al., 1981), and inasmuch as these BZP sites are presumably involved in the antiepileptic, anxiolytic, and sleep-promoting action of BZP in animals (Haefely et al., 1983) we considered worthwhile to assess whether pineal removal or melatonin treatment could affect BZP receptor density or affinity in rat cerebral cortex, the region of the brain that exhibits one of the highest concentation of BZP binding sites (M6hler et al., 1980). EXPERIMENTAL PROCEDURES

Adult male Wistar rats (180-220 g), kept under fight from 0700 to 2100 h daily and given access to food and water ad libitum were used. Pinealectomy or its sham-operation was performed under light ether anesthesia as described by Kuszak and Rodin (1977). Animals were active within l0 or 20 min after the operation and there was no mortality or brain damage (as determined from macroscopical examination at death) from the operation. Melatonin (Sigma Chemical Co., St. Louis, MO, U.S.A.) (800-1600/~g/kg body wt) or vehicle (saline-ethanol, 9:1, v/v) was injected s.c. at either morning (0900 h) or evening (1800 h) schedules. Three hours after the last injection groups of 3-6 rats were killed by decapitation, the brains were quickly removed rostral to the spino-occipital junction and a portion of the parietal cerebral cortex was removed and frozen at -70°C. Binding experiments were carded out on crude P2 mem*Research Fellow, Consejo Nacional de Investigaciones brane fractions (Bennet, 1978). Tissue was homogenized in cold 0.32 M sucrose and the homogenates were centrifuged Cientificas y T6enicas (CONICET), Argentina. at 900g for 20 min. The supernatant was centrifuged at tEstablished Investigator, CONICET. 675

0"7(3

PEt)RI) R. L()WI{NSTFIN dl ~1[.

30,000g for 20 min at 0 C, the pellct being resuspended in either 50mM Tris-HC1 buffer, pl-] 7.4 (at 0 ('i or 20 mM phosphate buffer pH 7.4. Similar results were obtained regardless of the buffer used. Resuspended membranes (0.8-1.0 mg protein) were incubated in triplicate at OC in a total volume of I ml buffer with [3H]flunitrazepam [FNZP] (83.6 Ci/mmol, New England Nuclear Co., Boston, Ma, U.S.A.) and in the presence (non-specific binding) or absence (total binding) of 12/tM unlabeled FNZP. Specific binding was linear up to 1.5mg of membrane protein. Incubations were carried out at optimal conditions (at (1C for 90rain). At the end of incubations the reaction was stopped by adding 3 ml of cold buffer to each tube and by filtering the samples under reduced pressure through Whatman GF/B glass fiber filters to separate free from bound ligand. Filters were rinsed thrice with 4 ml of ice-cold buffer and were transferred to counting vials. A total washing volume of 15 ml had previously been found as optimal in lowering nonspecific binding without affecting significantly specific binding. Radioactivity in the tilters was determined by liquid scintillation spectrometry. In all experiments, FNZP specific binding, i.e. the amounts of total binding displaced by excess unlabeled FNZP, was greater than 60o./o of total binding. Essentially similar results to those of FNZP were obtained when excess clonazepam (which binds almost exclusively to central type BZP receptors) was used. This observation together with the concentration of [3H]FNZP used (up to 6 nM), below the dissociation constant (K~0 of FNZP for peripheral-type BZP receptors in brain (i.e. 25 nM, Marangos et al., 1982) strongly suggest that the binding observed correspond to the central type BZP receptors. Binding data at equilibrium were obtained by two different procedures: (a) by increasing the concentration of radioactive ligand; (b) by increasing the concentration of unlabeled ligand and recalculating the specific activity of [3H]FNZP at each concentration of ligand, Equilibrium binding constants were calculated by Scatchard analysis, the line slopes and the intercepts being determined by regression analysis. Repeated estimates of specific binding capacity of brain membranes yielded coefficients of variation of 6--10%. Each saturation isotherm experiment was repeated at least thrice. When single-point assays were carried out, specific radioligand binding of individual brains was assessed at a 6 nM concentration.

~ :(It ~:.,

T h e effect of Px o n rat b r a i n B Z P receptor binding is s h o w n in Fig. 1. Pineal removal depressed Bma, b u t did n o t affect Kd significantly 3-14 days after surgery. Single p o i n t binding assays of individual brains o f normal, Px a n d s h a m Px rats are s h o w n in Fig. 2. A t every time after surgery [3H]FNZP b i n d i n g of Px rats was significantly lower t h a n t h a t of controls ( P < 0.01). A c r a n i o t o m y - r e l a t e d depression o f B Z P binding sites t o o k place in s h a m - o p e r a t e d animals, as revealed by the 61 a n d 29% decrease in [3H]FNZP binding observed 3 a n d 7 days after surgery, re-

35riM •

c~

. . . . . .

• FiFeqle2tt;rr

iC4~'nMeNe X

50 100 1.50 450k(7 days)

400 ~14, doys)

3°°F X

200t- X X 136"M

"t.%

150 300 $00 600 SH-FNZPSPEC)FICALLYBOUND(fmol/mgprotein) Fig. 1. Effect of pinealectomy or sham-operation on BZP binding sites in rat cerebral cortex. Groups of 3-4 rats subjected to surgery 3, 7 or 14 days before sacrifice were killed at noon. Data are shown as Scatchard plots; slopes and intercepts were calculated by regression analysis and analyzed statistically by an analysis of covariance ( P < 0.05 for differences in Bronx).Two other independent saturation isotherm experiments yielded essentially similar results.

spectively. B Z P binding of n o r m a l a n d shamo p e r a t e d rats killed 14 days after surgery did n o t differ significantly (Fig. 2). P e r h a p s this accounts for the convulsive - p r o n e state observed after c r a n i o t o m y in s h a m - o p e r a t e d rats. B Z P receptor c o n c e n t r a t i o n of

{SURGERY) J- -t~'~CmO400 --1 ~

RESULTS

Effect o f pineal removal and brain surgery on cerebral cortex B Z P binding

, 1:yS)

~ ~ { /t Sham" PX

= 2oc

-'""P~

# 1

0

_ I

3

~

L

}4

DAYSELAPSEDAFTERSURGERY Fig. 2. Effect of pinealectomy (Px) or sham-operation on [3H]flunitrazepam ([3H]FNZP) binding by rat cerebral cortex. Shown are the m e a n s _ SEM of individual brains (n = 5~5/group). At every time interval Px differed from sham-Px significantly ( P < 0.01, Student's t-test). An analysis of variance indicated significant differences between Px and intact control rats (circle) at all studied times, and between sham-Px and intact control rats 3, and 7 days after surgery ( P < 0.05). Rats were killed at noon.

Pineal and benzodiazepine binding 30O

4 l e l -e-x

F KOO.enM L ~122 fmollmll I~rOl

uJ uJ n-

",,,\

~

I

~ o\

I

k.

t) P,+ r Kd'°.se"" %%~11)" ~ ' i n 15C

677

\

Kd ,O.72 nM

I

,_, ,, r v , . o g 4 ~ ,', b~,... ~41~,d/.,s p,~. ~l ' , ÷ I'~o.w,,u

L Bmem'154fmM/me Pto1

Q Z

( 7 do ys ) IOO

200

O

IOO

200

SH- FNZP SPECIFICALLY BOUND (fmol/mg prof.)

Fig. 3• Effect of melatonin (800 p g/kg, 3 h earlier) on pinealectomy (Px)-induced decrease of BZP binding in rat cerebral cortex, 3 or 7 days after surgery. Data are shown as Scatchard plots and were calculated as in Fig. 1. Bm~xof Px rats differed from the other 2 groups at either time interval (P < 0•05, analysis of covariance). No statistical differences in binding affinity of the 3 experimental groups were detectable• Two other independent saturation isotherm experiments yielded essentially similar results. rat cerebral cortex similar to those found in the present work have been reported in the literature (e.g. Gallager et al., 1978; Bowdler et al., 1983; Niehoffet al., 1983; Wilkinson et al., 1983a, b; Ramanjaneyulu and Ticku, 1984).

Effect of melatonin treatment on cerebral cortex B Z P binding As shown in Fig. 3 a single injection of 800 pg/kg of melatonin at 0900 h restored 3 h later the depressed BZP receptors observed in Px rats 3 or 7 days after surgery; no changes were apparent in the / ~ value after melatonin injection• Table 1 summarizes individual single point assays of rats Px 3 or 7 days earlier and injected with melatonin 3 h earlier• In every case melatonin treatment restored the depressed brain [3H]FNZP binding of Px rats. The injection of melatonin (800-1600#g/kg) to

Table 1. Effect of pinealectomy (lax) or melatonin on [3H]FNZP binding in rat cerebral cortex membranes [3H]FNZP specifically bound (fmol/mg prot) Experimental group Sham-operation Px Px + melatonin

3 days after surgery

7 days after surgery

133 + 6 (5) 102 ± 6 (6)* 144 5=4 (6)

246 + 10 (5) 196 5= 12 (6)* 238 5= 8 (6)

Animals subjected to Px or sham-operation 3 or 7 days earlier received a single s.c. injection of melatonin (800 pg/kg body wt) at 0900 h and were killed 3 h later. Shown are the means 5= SEM of n individual brains. Single-point binding assays were carried out at a 6nM-concentration of [31-I]FNZP as described in Experimental Procedures. *Significantly different from the other two groups, P < 0.05, analysis of variance, Dunnet's t-test.

intact rats at 0900 or 1800 h did not affect BZP binding sites significantly when assessed 3 h later (Fig. 4). The smallest melatonin dose given for 5 days to intact rats at either morning or evening schedule, changes significant BZP receptor binding, i.e. melatonin treatment increased Bm~ without affecting Kd at morning hours, while it affected K d without modification of Bin,, at evening hours (Fig. 5). Thus only morning melatonin injections modified BZP specific binding in cerebral cortex membranes when assessed at single, saturating concentrations of [3H]FNZP (Table 2).

Competition of [3H]FNZP binding by melatonin and B Z P analogs In vitro competition of [3H]FNZP binding to rat cerebral cortex membranes by melatonin and several BZP agonists and antagonists is shown in Fig. 6. The Table 2. Effect of melatonin treatment on [3H]FNZP binding rat cerebral cortex membranes Experimental group

[3H]FNZP specifically bound (fmol/mg prot)

0900 h-injections Vehicle Melatonin

298 + 11 (6) 398 + 15 (6)*

180Oh.injections Vehicle Melatonin

291 + 16(5) 284 5= 19 (5)

Rats were injected for 5 days with either melatonin (800 pg/kg body wt) or vehicle s.c., and were killed 3 h after the last injection. Injections were performed at 0900 or 1800 h. Shown are the means + SEM o f n individual brains. Single-point binding assays were carried out at a 6 nM-concentration of [3H]F'NZP as described in Experimental Procedures. *Significantly different from vehicle-treated controls, P < 0.01, Student's t-test.

67g

I;H)R() R. I~OWI!NSIEIN ('/ ~z/.

700

Kd= 0 . 6 8 3 nM Bma x = 4 7 2 t m o l t m g

o Control

\

c~

z

prot

g lOO~

? ~ •

• Melotonin ( 8 0 0 ug/Kg) •

L

[ K d = O 808 nM Brno x = 499 f r'r,ol / m(J prol

Melo lonin [ KiIr~-- O. 9 0 0 tIM ~ -OOug/K~) 8rnox = 5 4 4 f m o l / m g p r o l

~

50

350

IIR0

5 -4864

c o

I (.... i n g i njecti on) 0

I

E

250

500

BOO

rKa= o ZZ6.M Bmox = 435 f m o l / m g prot.

L

0 Con,ro, U_ ~" Z

0 (30

•

•

10

87 654 -log M

° I'1~---

Melofonin

[800ug/Kg)

[- Kd= 1.03OHM 8mQX=464 fmol/mg

L

• Meiatonin ~ (1600 u g / K g )

prot

Kd = 0 . 5 8 8 nM

40o~.%~.

Bmox = 4 4 9 fmol/mq p r o t

Fig. 6. Effect of malatonin and several BZP analogs on [3H]FNZP binding to rat cerebral cortex membranes. Each point is the average of triplicate samples which varied less than I 0°4,. following order of affinity [K~; nM] (Bennet, 1978) was found: clonazepam (0.22)> Ro. 15-1788 (0.48) > F N Z P (0.95) > melatonin, Ro. 5-4864 ( > 10,000). DISCUSSION

,o,.,,oo, 0

250 3H--FNZP

SPECIFICALLY

500 BOUND

(fm01/rng

prot)

Fig. 4. Effect of morning (at 0900 h) or evening (at 1800 h) single melatonin injections (800-1600/ag/kg body wt) on BZP receptor binding in rat cerebral cortex. Data are shown as Scatchard plots and were calculated as in Fig. 1. No statistically significant differences in Bm~~ or Kd's values were detectable by an analysis of covariance. Two other independent saturation isotherm experiments yielded essentially similar results.

BZP binding sites in brain are believed to be the pharmacological receptors for the anticonvutsivant, anxiolytic, sleep-promoting and muscle relaxation properties of BZP (Haefely et al., 1983). Although some alternative mechanisms have been considered (Phillips et al., 1980) it is widely held that BZP act on brain by increasing gamma aminobutyric acid ( G A B A ) neurotransmission (Costa et al., 1978; Olsen, 1981). Current experimental evidence suggest that the BZP receptor is a part of a supramolecular complex formed also by the G A B A receptor, the chloride channel and a barbiturate recognition site,

J ( o } Control r K d = O . 6 5 n M (0) Conlrol L LB~.~QX=317 fmoi/mg p .... L BOO / e) MoOonn r ' K d = O 5 6 n M IN • -~x, L Bmox-~ 4 0 3 f . . . . . q prol 6 O 0 [ - - N X ~ " . . . . . .

'L\

u\

o_

Kd=O 4 3 n M [Bmox=279fmol/mg

prot

rKd=O 96nM Bmox = 219 fmol/mg pror

L

400 •

z

I o L~

\o" __~_

200

I

. . . . . . _~_ ~

o L ......

400

t

1

100

200

\ ~

300

3 H - F N Z P SPECIFLCALLY BOUND ( f r n o i / m g prot)

Fig. 5. Effect of morning (at 0900 h) or evening (at 1800 h) melatonin treatment for 5 days (800 #glkg body wt/day) on BZP receptor bindingin rat cerebral cortex. Data are shown as Scatchard plots and were calculated as in Fig. 1. By an analysis of covariance Bin,~ differed significantly at the morning schedule and Kd at the evening schedule (P < 0.01). Two other independent saturation isotherm experiments yielded essentially similar results.

Pineal and benzodiazepine binding its role being the regulation of affinity state of the GABA receptor or the coupling of this receptor with the ion channel (Haefely et al., 1983). A still uncharacterized brain constituent could be the endogenous ligand for BZP receptor sites (Hamon and Soubri6, 1983); alternatively the sites to which BZP are bound might be a region of regulatory or coupling protein having no physiological function (and thus no endogenous ligand) but exhibiting an altered function when occupied by an active BZP. Foregoing results indicate that Px reduces the density of BZP binding sites in rat cerebral cortex without affecting their affinity. The effect of pineal ablation is manifested as early as 3 days after surgery and persists for 2 weeks. Providing that an endogenous agonist ligand does exist for BZP in brain the convulsive prone state (Nir et al., 1969; Reiter et al., 1973) or increased paradoxical sleep (Romijn, 1978; Yamaoka, 1981) of Px rats could be interpreted as reflecting an impaired BZP receptor activity and consequently an impaired GABA neurotransmission after pineal removal. Although Px-induced decrease of Bm,x of BZP binding sites could be due to presumptive changes in GABA content brought about by gland's removal (Anton Tay, 1971), GABA is known to affect the affinity of BZP receptors (Olsen, 1981) rather than the concentration of binding sites, as observed in the experiments reported herein. Besides melatonin, other pineal preparations like unrefined aqueous extracts (Roldan and Anton-Tay, 1968) or alleged components as arginine vasotocin (Pavel, 1979) have been reported to decrease induced seizures or to promote sleep and sedation in animals; however the bulk of available information deals with melatonin-evoked changes of brain excitability. Pharmacological doses of melatonin protect against Pxinduced seizure in gerbils (Rudeen et al., 1980), ouabain-elicited convulsions in rats (Izumi et al., 1973) and pentylenetetrazole or kindled seizures in rats (Albertson et al., 1981). In mice melatonin potentiated barbiturate-induced sleep, antagonize pentylenetetrazole-, 3-mercaptopropionic acid- and electroshock-induced seizures and had analgesic activity (Sugden, 1983). It also improved electroencephalographic activity of patients with temporal lobe epilepsia (Anton-Tay et al., 1971), diminished light-induced seizures in baboons (Brailowsky, 1976) and reduced focal epileptic activity of primary sensory areas in cats (Fariello and Bubenik, 1976). Intracerebroventricular injections of antimelatonin antibody brought about transient epileptiform abnormalities and occasional convulsions in rats (Fariello et al., 1977). Sleep induction follows to melatonin

679

treatment of humans (Anton Tay et aL, 1971; Cramer et al., 1974; Vollrath et ai., 1981), rats (Yamaoka, 1981; Holmes and Sugden, 1981) or birds (Pang et al., 1976; Bermudez et al., 1983). Our present results indicate that a single injection of a pharmacological dose of melatonin restored 3 h later the depressed BZP receptor sites in Px rats. Similar or twice as high doses of melatonin injected at either morning or evening hours did not affect BZP receptor sites in normal animals. However when daily melatonin injections were given for 5 days to intact rats, changes in B~x (morning treatment) of affinity (evening treatment) were apparent. Indeed significant differences in putative melatonin receptor sites of rat or hamster brains are detectable at similar time intervals (Vacas and Cardinali, 1979). In confirmation of Marangos et al.'s results (1981) melatonin added/n vitro to cerebral cortex membranes competed for [3H]FNZP binding only at concentrations greater than 10/~M. Since such concentrations are not achieved presumably in brain at the pharmacological melatonin doses employed, the melatonin-evoked changes in brain BZP receptors seem to be independent on direct effects on the binding sites. Melatonin has been reported to increase brain GABA levels in rats (Anton-Tay, 1971) and the changes reported herein could be due to GABA-induced modifications of BZP receptor sites. However none of the changes observed after melatonin treatment in either Px or intact rats fit with the increase in BZP receptor affinity and unchanged Bma~ reported in brain membranes treated with GABA (Olsen, 1981). Further experiments are needed to assess the possible physiological meaning of the pharmacological experiments performed herein; in particular a complete dose-response curve for melatonin effect in Px rats would be useful, since the doses of melatonin employed by us are about 1000 times greater than the daily secretion rate of the pineal hormone. Several biochemical mechanisms have been proposed for melatonin action in brain (Cardinali, 1981; Cardinali et al., 1983; Waldhauser and Wurtman, 1983), and the present results suggest that changes in BZP receptor sites can be one of them. Since a lithium-sensitive diurnal cycle of BZP receptors occurs in rat brain (Kafka et al., 1982), to what extent the pineal gland and melatonin are involved in this rhythm deserves further exploration. Acknowledgements--These studies were supported by grant No 6638 from CONICET, Argentina, and by Hoecbst AG, Werk Albert, Wiesbanden, F.R.G. Clonazepam, Ro 15-1788, Ro 5-4864 and FNZP were a generous gift from Productos Roche, Buenos Aires, and Hoffmann-LaRoche, Basel, Switzerland.

680

I)t])RO R. [,()WI..NSFEIN el N].

REFERENCES Alberton T. E., Peterson S. L., Stark L. G., Lakin M. L. and Winters W. D. (1981) The anticonvulsant properties of melatonin on kindled seizures in rats. Neuropharmacology 20, 61-66. Anton-Tay F. (1971) Pineal-brain relationships. In: The Pineal Gland(Wolstenholme G. E. W. and Knight 1., eds), pp. 213 227. Churchill Livingstone, London. Bennet J. P. Jr (1978) Methods in binding studies. In: Neurotransmitter Receptor Binding (Yamamura H. I., Enna S. J. and Kuhar M. J., eds), pp. 57-90. Raven Press, New York. Bermudez F. F., Forbes J. M. and lnjidi M. H. (1983) Involvement of melatonin and thyroid hormones in the control of sleep, food intake and energy metabolism in the domestic fowl. J. Physiol. (Lond.) 337, 1927. Bindoni M. and Rizzo R. (1965) Hippocampal evoked potentials and convulsive activity after electrolytic lesions of the pineal body in chronic experiments on rabbits. Archs Sci. Biol. 49, 223-233. Bowdler J. M., Green A. R., Minchin M. C. W. and Nutt D. J. (1983) Regional GABA concentration and ~H-diazepam binding in rat brain following repeated electroconvulsive shock. J. Neural Transm. 56, 3 12. Brailowsky S. (1976) Effects of melatonin on the photosensitive epilepsy of the baboon, Papio papio. Electroenceph. Clin. Neurophysiol. 41, 314-319. Cardinali D. P. (1981) Melatonin. A mammalian pineal hormone. Endocr. Rev. 2, 324 346. Cardinali D. P., Vacas M. I., Keller Sarmiento M. 1. and Morguenstern E. (1983) Melatonin action: sites and possible mechanisms in brain. In: The Pineal Gland and Its Endocrine Role (Axelrod J., Fraschini F. and Velo G. P.. eds), pp. 277 301. Plenum Press, New York. Costa E., Guidotti A. and Toffano G. (1978) Molecular mechanisms mediating the action of diazepam on GABA receptors. Br. J. Psychiat. 133, 239 248. Cramer H., Rudolph J., Consbruch U. and Kendel K. (1974) On the effects of melatonin on sleep and behavior in man. Adv. Biochem. Psychopharmac. II, 187--191. Fariello R. and Bubenik G. (1975) Melatonin-induced changes in the sensory activation of acute epileptic foci. Neurosci. Lett. 3, 151 155. Fariello R., Bubenik G., Brown G. and Grota L. (1977) Epiteptogenic action of intraventricularly injected antimelatonin activity. Neurology 27, 567-570. Gallager D. W., Thomas J. W. and Tallman J. F. (1978) Effect of gabaergic drugs on benzodiazepine binding sensitivity in rat cerebral cortex. Biochem. Pharmac. 29, 2745 2749. Haefely W., Pole P., Pieri L., Schaffner R. and Laurent J-P. (1983) Neuropharmacology of benzodiazepines: synaptic mechanism and neural basis of action. In: The Benzodiazepines: From Molecular Biology to Clinical Practice (Costa E., ed.), pp. 21-65. Raven Press, New York. Hamon M. and Soubri6 P. (1983) Searching for endogenous ligand(s) of central benzodiazepine receptors. Neurochem. lnt. 5, 663-672. Hata T. and Kita T. (1978) A newly designed method for removal of the pineal body and depression of convulsions and enhancement of exploratory movements by pinealectomy in mice. Endocr. Jap. 25, 407-413. Holmes S. W. and Sugden D. (1982) Effects of melatonin on

sleep and neurochemistry in the rat. Br. ,I. Pharmac. 76, 95- 101. lzumi K., Donaldson J., Minnich J. and Barbeau A. t1973~ Ouabain-induced seizures in rats: modification by' melatonin and melanocyte-stimulating hormone. ('dn. J. Physiol. Pharmac. 51, 572-578. Kafka M. S., Wirz-Justice A., Naber D., Marangos P. J.. O'Dohonhue T. L. and Wehr T. A. (1982) Effect of lithium on circadian neurotransmitter receptor rhythm. Neurop.2vchobiology 8, 41-47. Kuszak J. and Rodin M. (1977) A new technique of pinealectomy of adult rats. Experientia 33, 283. Marangos P. J., Patel J., Boulenge J. P. and ClarkRosenberg R. (1982) Characterization of peripheral type benzodiazepine binding site in brain using 3H- Ro 5-4864. Molec. Pharmac. 22, 26-32. Marangos P. J., Patel J., Hirata F., Sondhein D., Paul S. M., Skolnick P. and Goodwin F. K. (1981) Inhibition of diazepam binding by tryptophan derivatives including melatonin and its brain metabolite N-acetyt-5-methoxy kynurenamine. Lift, Sci. 29, 256-267. M6hler H., Battersby M. K. and Richards J. G. (1980) Benzodiazepine receptor protein identified and visualized in brain tissue by photoaffinity label. Proc. natn. Acad. Sci., U.S.A 77, 1666-1668. Niehoff D. L., Mashal R. D. and Kuhar M. J. (1983) Benzodiazepine receptors: preferential stimulation of type 1 receptors by pentobarbital. Eur. J. Pharmac. 92, 131 134. Nir 1., Behroozi K., Assaet M., lvriani I. and Sulman F. G. (1968) Changes in the electrical activity of the brain following pinealectomy. Neuroendocrinology 4, 122-127. Olsen R. W. (1981) GABA-benzodiazepine-barbiturate receptor interactions. J. Neuroehem. 37, 1 -13. Pang S. F., Ralph C. L. and Petrozza J. A. (t976) Effect of melatonin administration and pinealectomy on the electroencephalogram of the chicken (Gallus domesticus) brain. Life Sci. 18, 961-966. Pavel S. (1979) Pineal vasotocin and sleep: involvement of serotonin-containing neurons. Brain Res. Bull. 4, 731 734. Phillis J. W., Siemmens R. K. and Wu P. H. (1980) Effects of diazepam on adenosine and acetylchotine release from rat cerebral cortex: further evidence for a purinergic mechanism in the action of diazepam. Br. J. Pharmac. 70, 341- 348. Philo R. and Reiter P. J. (1978) Characterization of pinealectomy-induced convulsions in the Mongolian gerbil ( Meriones unguiculatus). Epilepsia 19, 485-492. Quay W. B. (1975) Pineal Chemistry. Chapter 14, Charles C. Thomas, Springfield, Ill. Reiter R. J., Blask D. E., Talbot J. A. and Barnett M. P. (1973) Nature and time course of seizures associated with surgical removal of the pineal gland from parathyroidectomized rats. Expl Neurol. 38, 386-397. Ramanjaneyulu R. and Ticku M. K. (1984) Interactions of pentamethylene-tetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore. Eur. J. Pharmac. 98, 337-345. Rold~m E. and Anton-Tay F. (1968) EEG and convulsive threshold changes produced by pineal extract administration. Brain Res. !1, 238-245. Romijn H. (1978) The pineal, a tranquillizing organ? L(ti" Sci. 23, 2257-2274.

Pineal and benzodiazepine binding Rudeen P. K., Philo R. C. and Symmes S. K. (1980) Antiepileptic effects of melatonin in the pinealectomized mongolian gerbil. Epilepsia 21, 149-154. Sugden D. (1983) Psychopharmacologlcal effects of melatonin in mouse and rat. J. Pharmac. exp. Ther. 227, 587-591. Vacas M. I. and Cardinali D. P. (1979) Diurnal changes in melatonin binding sites of hamster and rat brains. Correlation with neuroendocrine responsiveness to melatonin. Neurosci. Lett. 15, 259-264.

681

Vollrath L., Semm P. and Gammel G. (1981) Sleep induction by intranasal application of melatonin. Adv. Biosci. 29, 327-329. Waldhausvr F. and Wurtman R. J. (1983) The secretion and action of melatonin. In: Biochemical Actions o f Hormones (Litwack G., ed.), Vol. 10, pp. 187-225. Academic Press, New York. Yamaoka S. (1981) The role of the pineal gland in the steroidal modification of sleep circadian rhythm. Jikeikai Med. J. 28 (Suppl. 1), 17-21.