.%*n. 1st. .Snpei.. Slii;ii
Behavioral and electroencephalographic effects of chronic aclministration of diazepam in rats and rabbits
STEFAYOSAGRATELLA, . FRANCESCA. ALOISI and MARINOhlASSOTTI Laboratogv of Pharmacology, Istituto S19eriore di Sanità, Rorna, Ita9
Summary. - In order to stirdJ>fhe electroenc~phalographic mate prescriptions and achieved a widespread use in (EEG) and behavioral effects of chronic administration of the clinic as anxiolitic, sedative, anticonvulsant 2nd benxodia?epines, rats luere treated dai& for 10 dayr witb miorelaxant drugs. 10 tnglkg i.v. of diaxepam; robbits rvere treated daib for Although it is known that benzodiazepines have 72 dgs &h 20 mg/kg i.u. The sedative effect disappeared few side effects, there are clinica1 and experimental between the 3rd day in rats and the 10th day in rabbits of reports which indicate that administration of high treatment, rid~en a yndron~e characterized &y slight moto? doses of these drugs, over a prolonged period of time, rxcitation, compnlsi~x gnawing and eating ernerged. This might produce adverse effects (Tab. 1). Under benzo- pdrome was accompanied by the appearance cf 20-30 c/s diazepine treatment, tolerance to their sedative and anti- waves (beta-li& acfivi9) in the EEG recordings, rtaching the convulsant [l-41 actions was reported. plateau at the 6th day of treatment in rats and at the 9th Withdrawal signs upon abrupt discontinuation of the day of treatment in rabbitr. drug were also described [l-51 as well as the ,, Floppy This pattern war stili present @on dircontinnation o/ the infant syndrome " (Tab. l), reported in neurborns of drng and lasied up to the 15th day affer fhe end of treatmetrt. mothers who took large doses of benzodiazepines during Bnring this withdrawai period, the adnrinistration of a dose pregnancy [6-81. of 0.7 mglkg i.u. of diaxepam eiicited an excitatoy y- dronrc accompanied by increase of the beta-li& actiuity in fhe EEG. This wus b/ocked by administration o/ naloxone Table 1. - Adverse efects dne fa the chronic administrotion (5 mg/kg iu.). oj benr(od+epines
Sommario. - Uno strtdio ektfroencefalogrnfrro e comnpor- famentale è stato equito dopo sonministraxione r$et,rta di Sedative een I181 1) Tolerance to ...... { dia~epamnei ratti (10 mglkg i.v. per 10 giorni) e nei conigli Anticonvulsivant effect 121 (20 mglkg ;.v. per l2giorni). L'effetto sedatizao de/diaxepam scompare dopo i/ terxo giorno di trattanzento nei ratti e dopo 2) Withdrawal signs ...... il decimo nei conigi, allorché subito dopo i'ass~~qionedel « Rebound Iniomnia D 151 farmaco insorge una sindrome raratterixxata da /ma leggera Muscular Hyporhonia mifaZione, masticaxione ed assnn?ione di cibo. Qwesta sin- 3) ,. Floqpy lnfant synd- Sucking Dificultirs drome i accompagnata, dal pnnto di vista EEGrafco, dalla rorne 16-81 ...... Hypotherrnia comparsa di onde a ba~souoltaggio di 20-30 c/s (ntfività beta- II Atracks of Cynnosis simile). Tale andamento EEGrafco raggiunge la massima dnrata ai resto giorno di trattamento nei ratti ed al nono nei ronigii, ed è presente dopo sospensione del traitumento per altri qilindici giorni. Dnrante qnesto periodo di ostinensa, la The discovery of benzodiazepine specific recognition somministra.yione di 0.7 mglkg ;.v. di diaxepam proroca nei sites [9], functionally linked to the GABA rccep- ratfi la ricomparsa di tale sindronre ~ccitatoria,acconipqnata tors [lo-131 in animal and human brain opened a neu. dalla presenxa di attil&ì b~ta-sindesnl tracciato EEGrajco. avenue particularly far the study of the tolerance and Tale attiuità beta-sinzih viene bloccata dalla somministra- withdrawal effects due to the chronic administration of ?ione di naloxone (5mg/kg i.~.). benzodiazepines. Experimental studies shoused no significant modifications of the binding chnracteristics of hoth GABA and benzodiazepines during chronic treatment. I-lowever, in newborn rats of dams which received high doses of diazepam throughout pregnancy hen~odiaze~inederivatives belong to the class of the capability of muscimol in stimulating 31-I-diazepam « minor tranquillizer »; due to their weaker toxicity in membrane preparation frorn cortex wm reduced up these drugs superseded the barbiturate and meprobra- to the 14th day iifter birth [l4]. ControL Fr. ~~~~-~~~~~~.~.~-~i.~~~,~~.~~~~~~1i,~~~:~wi.*~~~~~~,i~:,.~~~*u,~~.i~,cu.-iilri-i Por, '~,~~~,~~~,~.,\~.~,.;~~'.Ìc:.~-~~t~~,-~~*in~~{~~A~,'~~~ww::,~~\w~~~'~~~~~~i~~:~~~~,~yi&~~%~~~-.v\~
,~~~!I/*,*i.~:;~~~~i2irc1i...i.:~~,f.(,.~~~~irc..rrr)~.sh.~~iy~~~~~~t~,,:~~~..~:~~~~,~.I.y*:,~, t I, . kb;:~:.~;zc**r)r,~~~ I ecc, Mp],bi;),\%. j. . , ,,, i F-P d. ;v.d+*~#,,~,+r\~;..:;i+.:;+. ~~~~:~~,~~~~,~~.,~.)~lrn~~~,,.w.~:,~~~.r-r,~~t~~~~,p~\.i~ ..4,-. ./urr,-
! 10 min after diazepam
t A. R. I sedated head drop 60 min
FG 1. - EEG :C;ects ot acute administration of diazepam (20 rng/kg i.".) in rahbits. The anirnalr were subjected to the surgical procedure in ordcr 10 place t. dcctrodes, undcr local ancsthesia, just before thc recording. Few seconds after adminisrration of the dmg slow waves aPP=r in the cortlcal leads lasting appmxin.arely 10 min; the hippoampus record is dismpted wich a decrcase of voltagc; at the red nuclcus kvel tkrecord shows an inucase of voltage and a dccrcase of frequency. The animal is sedatcd with head drop and the reaction t0 cxternal stlmuli is absent. Sixty min Iatcr at thc cortieal Leads che record shoms thc presence of trains of spindes and heta-like sct'vity. Tbc hippocampus and rcd nuclcus patterns tend to nomal. The animal is sedated and the rcaction to cxtemal stimuli is present l+=antuior smsorimotor (frontal) cortcx; Par.=postcrior sensorimotor @arietal) cortcx; Occ. =optic (occipital) cortu; Hipp=hippo- campus; N.R. s.=lcft red nuclcus; N.R. d.=right mi nucleus. N.R. d.,
10 min af ter diazepam
sedated
alert, gwawing +-I 200p V i ! 2 sec l
. ~~ ~ , ..... FIG. 2. - EEG cffccts of chronic administration of diazepam (20 mg/kg i.7.) in rabbits at thc 9th day of treatmenr. The animals wcrc subjected to the rurgical procedure in ordcr to placc the eiectrodes undcr local anesthesia just before the rccording. Bcfom the adminis- tration of the dmg at the cortical leadr the record is synchronized; the hippocampal theta mavcs arc dismpted. Few min after injection, 20-30 c/s wavcs appcar in the conica1 leads. The hippocampal recording shows a reduction of voltage: at the red nudcus Icvd an increase of voltagc and a reduction of frequency are obscrvcd. Sixty min aftcr drug administration the EEG svidenccs thc prcs- ence of beta-like activity at thc cortical level: thc hippocampus is still dismptcd, whilc in the red nucleus thc record is normal. a Fr, Par., Occ., Hipp., NR as described in the Fig. 1. In the present study the electroencephalogfaphic tions obsemed in the hippocampal and red nucleus leads (EEG) and behavioral effects of chronic administration tended to disappeare 40-60 min after the injection. of diazepam have been considered in view of defining In the following days of treatment, the administration the modifications connected to the development of of diazepam elicited a short-lasting synchronization tolerance. followed by a longer periods of beta-Like activity (Fig. 2). The hippocampal record was disrupted. The red nucleus record showed the same modifications described after the first administration. The arousal EEG reaction to external (vibroacustical) stimuli was absent in the first 10-20 min after drug injection up Rabbit. In order to evaluate the EEG correlates of to the third-fourth day of treatment, while it was present the chronic administration of diazepam, 48 male rabbits in the following days. received 20 mg/kg i.v. of the drug for 12 days through a cannula chronically implanted in the ear vein. The In Fig. 3 are reported the ratios of the occurrence recordings were carried out in different animals at the of beta-like activity over the occurrence of the trains lst, 3rd, %h, 7th, 9th and 12th day of treatment. On of spindles. The values were calculated within one the day of recording under local anaestheria (2% hour after diazepam injection, at various days of treat- xylocaine) six screw electrodes were placed on the ment. The value increased progressively, reaching the plateau at the 9th day of treatment and lasting up to the 15th day after the end of treatment. In order to better evaluate the EEG signs of tolerance, starting from the 2nd day after discontinuation o€ diazepam, obsemation of the EEG effects after increa- sing doses of diazepam (0.1-10 mg/kg, i.v.) was carri- ed out. Doses of 0,7 mg/kg, which in the naive rabbits induced the appearance of slow waves and spindles, in the tolerant animal elicited long periods of beta- iike activity (Fig. 4). With higher doses of diazepam (2.5 mg/kg) only the beta-like activity was present in the 6rst hour recording. fit. Eorty three male albino rats of Wistar strain, chronically implanted with cortical electrodes, were injected i.v. for 10 days with 10 mg/kg of diazepam
l 2 3 A 5 6 7 8 9 10 11 12 Days ot Olazcpam through a cannula cronically inserted into the jugular I"jecti0" vein. The recording was performed in the freely mov- IN PARENTHESIS TUE NUMBER OF EXPERIMENTS ing animals at the Ist, 3rd, 5th, 7th and 10th day of treatment. Pro. 3. - Ratio bcta-like letivity spindlm in thc cortical EEG At the iirst day of treatment slow waves appeared throughout thc chronic matment with diazcpam (20 mg/kg i.v.) in fahbin. Thc figure shows thc mio nlucs of thc duration of in the record and the EEG reaction to external stimuli thc 20-34 c/s wavcs (ben-likc octivity) aver thc uains of spin- was absent. Fifteen-twenty min later, continuous trains dlcs moisured within one hour after edadministration of dia- of spindles appeared in the record; in the following repm .t vatious days of trePmunt. The wimd werc subjected 20-30 min the trains of spindles alternated with 20-30 to the surgicpl procedure in ordcr to place the elecuodcs, unda 1-1 mestheda. just befmc th~recording. Ths ratio values in- c/s waves @eta-like activity) were observed. crease aftcr thc 5th day of treauncnt. rraching thc maximal values In the following days of treatment the period of at thc 10th day of trutment. In parcnthesis i8 reponcd thc nurnber slow waves, which followed the dmg injection, became of erpcriments done far ach point. shorter and an increase of the beta-like activity was observed. After the third-fourth day of treatment, 24 hours after the injection the beta-iike activity spread sensorimotor and optic cortices. Concentric deep also to the optic cortex and the ratio beta-iike activity/ elearodes were placed at leve1 of dorsal hippocampus spindles, measured within one hour after dmg adminis- and red nucleus. Histological examinations port-tnortem tration increased under treatment, reaching the plateau coniirmcd the location of deep electrodes. The record- at the 5th day of injection'(Fig. 5). During the with- ing was pcrformed in the restrained non-curarized drawal perioU the recording evidenced the presence animals. of beta-iike ak+iyity which disappeared within 3-5 On the fmt day (Fig. l), the administration of diaze- days in the optic &tux and within the 15th day in pam induced the appearance, within 10 min. o€ a record the sensorimotor conex. -.-L-% .. r. rich in slow (24 clsec) high voltage waves in the cor- In ordei to better evaluate the tolerance signs, from tical leads; the hippocampal recording showed a the second day of withdrawal a dose-response curve reduction of the voltage with disappearance of the theta with diazepam (0.1-10 mg/kg i.v.) was performed. rhythm; in the red nucleus, an increase of voltage A strong increase in beta-like activity was observed at together with a ~eductionof frequency was observed. the dose of 0.7 mg/kg or higher. After the 4th day Fotty min after diazepam long trains of spindles were of withdrawal, this acuvity tended to disappear from present, together with 20-30 c/s waves which resemble the optic cortex, while in the sensorimotor cortex those observed in humans after administration of barbi- was present up to the 15th day after the end of treat- turates (ha-like activity). Conversely, tbe modifica- ment. Naive rabbits
sedated ,, 200;lv 2 sec
alert, gwawing
FIG.4. - EEG effects of acute administration of diazepam (0.7 mg/kg i.".) in tolerant rabbits. The figure shows the EEG cffects observed 10 min after acute administration of diazepam in nsive and tolerant rabbits at thc 3rd day after the end of treatmcnt with diazeparn (20 mg/kg i.v. far 12 days). In the cortical leads only beta-like activity is present in tolerant rats; the hippocampal pattern is more activnted, whilc no significant differenccs cm bc observcd betwesn the two animals in the red nuckus pattern. 7he animals were subjected to the surgical procedure in order to place the clectrodes, undel local anesthcsia, just before the recordig. Leads as reported in thcFig. 1
appropriate study the EEG effects of opiate agonist and antagonist in rats during the withdrawal period It has been reported that a short-lasting phase of after administration of diazepam. stimulation is present after administration of both Morphine and naloxone, at doses ranging from 0.5 diazepam and GABAergic drugs in rats and rabbits [15]. to 10 mg/kg i.v. did not a&ct significantly the EEG In our experimental conditions the daily administration modifications obsemed during the withdrawal period. of diazepam induced in both rats and rabbits in the However, naloxone (5 mg/kg i.v.), but not morphine, first day of treatment an immediate sedation accompani- reversed the EEG and bebavioral effects induced by ed by myorelaxation and impairment of the righting administntion of diazepam (0.7 mg/kg i+.) in tolerant reflex. Staning from the 2nd-3rd day of treatment rats. As shown in fig. 6, the ratio betwecn the occur- sniffing, hyperirritability, twitching of ears, slight motor rence of beta-like activity and of spindles, measured excitation, compulsive gnawing and, in 70% of the within one hour after administration of 0.7 mg/kg animals, compulsive eating appeared. Upon treatment i.v. of diazepam, was 0.53 in naive rats, but reached and during the withdrawal period no significant changes the value of 2.12 and 2.41 in tolerant rats at the 2nd of water and food consumptions were observed nor and 4th day of uithdramal period, respectively. In a significant modification of body weight. these experimental conditions, the administration of naloxone strongly reduced the occurrence of the beta- like activity and the ratio decreased to 0.31 (Fig. 6). In addition, tbe behavioral stirnulatory effect was hlocked. It is now aenerallv accepted that the benzodiazepine derivatives e& thei;pharkacological effects by ptenti- atine0 the GABA svnaotic, L transmission. In addition. it has been recently demonstrated that opiates can affects seberal pharmacological actions of benzodiazepines by Controversial data exist in the literature on the onset interfering with the transynaptic phenomena of the of tolerance to the various effects of diazepam. The GABA transmission (see for ref. 3). It seemed therefore present experiments have demonstrated that tolerance
5. KALES,A,, SCHARP,M.B. & KALES,J.D. 1978. Rcbound insomnia: A ncw clinical syndromc. Scicm. 201: 1039-1041 6. CREE,J.F., MAYER,J. & HALEI,D.M. 1973, Diazepam in labour its metabolism and c&ct on thc clinical condition and thcrmo- gcnesis on the neaiborn. Br. Med. J. 4: 251-255.
7. REMFNTBRIA, J.L. & BHAZT,K. 1977. Withdrawal symptoms in neonates from intniutcrinc exposurc to diazcpam. J. P&h. 90: 123-126. 8. SPEIGRT,A.N.P. 1977. Floppy-infant syndrome and materna1 diazcpam and/or nitrazepam. Lanrd. 878. 9. MOHLER,H. & OKADA,T, 1977. Benzodiazepinc-reccptor: Demonstration in tbc centra1 nervous system. Srio!rc. 198: 849-851. 10. KAEOBATH,M. & SPERK,G. 1979. Stimulation of benzodiazepine receptor binding by yaminobutyric acid. Pror. No!. Amd. Sci. USA. 76: 1004-1086. . .-- 11. MARTIN,I.L. & CANDY,J.M. 1978: Forilitation of benzodiazepine binding by sodium chloride and GABA. Ncr 15. Scorrr DE CAROLIS,A. & MASSOT~,M. 1978. Electroencephalographic and behavioral investigations on ii Gabacrgicn drugs: Musùmol, Baclofcn and Sodium y-Hydroxybutyratc. Implications on human epileptic studics. Pro,. Ncuro-P~yrhoph1mi1cd 2: 431-442. 16. Scorrr DE CAROLIS.A,, FWRIO. V. & LONGO,V.G. 1978. Furthcr analysis of tbc cffectr of hamine on the elccttical activity of the rabbit brain. Newopbarmmol. 17: 295-298. 17. DINGLBD~N~,R., IVEK?P.N, L.L. & BREUIER, E. 1978. Naloxonc as a GABA antagonist: svidence from iontophoretic, rcccptor binding 2nd convulsant studics. hop. J. Pbarmocol. 47: 19-27. 18. DUKA,T., WUSTER,M. & HERZ,A. 1979. Rapid changes in enkephalins Icvcls in rat stristum and hypothahmus. Nmyn Srbniird. Arrb. Pborn~col. 309: 1-5. hd. 1st. A'ttper, .S'l!,i/à Vd i8. N. I (19823, pp. 83-86 Poster session Spectra of psychotropic activity and neurochemical mechanisms of action of bi- and tetracyclic antidepressants Sunimary. - The aiiiidrprisiiue uffwt of IIW drqs ivas a 5-point scale was :ipplicd to the evnluation of be- iiiidiud ami compar NORMAL I RESERPINE 3 2 1 o 1 2 3 4 5 6 7 8 9 l01112131415 Behavioral classes 3 negative emotion 10-13 somatomotor cornponents . . 1 2 3 C 5 6 7 8 9 10 11 12131415 Behaviorol cLos,sec FIG.1. - Effect of bcfuraline on reserpine-induced dcpres~iosin cats. Co7ipmmfr a/ tlx neyoliut emo1;oiiol rt&: 1) acrive-dcfcnsivc re- actions; 2) passive-defcnsive reactionr; 3) ncgativism. Con~popoiie~~ o,i thporitiue motiorio1 ifofr: 4) curiosity; 5) iood behavior; 6) con- tentmcnt; 7) play; 8) huntiog; 9) erousal reaction. Sorm~onrotorronipor>riitr: 10) spontaneous loomotor activity; 11) non-cxprcssiveness of tbc cmotional reacrions; 12) depressive posture; 13) catalcpsy. I~rgptorind;d,~rIm~~ir~r: 14) mpsis; 15) relaiation of the nictitating membrane. ... -~ ~ . .~- . Potcntinring cfkm oi O~enheld Anlwonirm - D R U G rith Picroloxin 5 - HTP tcrrrbcnalinc ED.. mmphccamini HA VA PH GR tqxiiphan and picrotoxin convulsions. The duration havioral depression due to reserpine. Chlorimipraniine, ~f the amphetamine stereotypies did not change. The a serotonin uptake inhibitor [6], was someahat less zicrivity of the animals in the open held aas slightly potent in preventing the development o€ behavioral eiilianced. According to Boksay et al. (31, the effect o€ depression mmifestations and did not eliminate vege- bcfuraline is due to activation o€ tlie central adrencrgic tative disturhances. In low concentration, in which structures which is in conformity aith our findings. the specific eifect o€ the drugs develops, only des- So effect o€ befuraline on dopamine uptake by brain methylimipramine (DMI), like traiodone, produccd a synaptosomes was found. marked potentiation o€ 3H-serotonin uptake (67-79 "h Far pyrazidol a reversible inhihition of MA0 h and inhibition) and slighly affected the uptake o€ =H-GABA ari inhibition of noradrenaline uptake has been report- (Tah. 2). In higher concentration (500 p M) it inhihited cd [4]. Also this drug prevented the development o€ WGABA reuptake like the other drugs. It is evident reserpine-induced behavioral depression. The antide- that when antidepressants are used in high doses their pessant effect of pyrazidol consisted of a recovery o€ individua] differences are not observed in either be- tlie reactions to positive test-stimuli and o€ a decrease havioral or neurochemical study. The inhibition o€ <>fthe negative emotional state. Neurochemical data 3H-G.4BA uptake after high doses of the drugs indicate that pyrazidol is more active than the other is rclated to their non-s~edfic membrane-tro~ic drugs in potentiating picrotoxin convulsions and is action. iinly slightly different from trazodone in its effect on Unlike the drugs mentioned above, nialamide, a scn~toninergicmechanisms. Pyrazidol potentiates am- MA0 inhibitor, prevented the development o€ reser- phetamine sterotypies, decreases tetrahenazine ptosis pine-induced neurovegetative disturbances but Failed :)od hypotermia (Tab. 1). These results are in agree- to prevent the depression o€ the emotional-motiva- riient with the findings o€ Mashkovsky and Andreyeva tional sphere, which remained despite repeated nial- 1.51 and suggest the involvement o€ the serotoninergic amide administration. svstem in the antidepressant action as the enhancement The prolonged reserpine effects on the central and <>€the spectrum of positive emotions. peripheral monoaminergic neurons have been attrihuted Neither hefuraline nor pyrazidol iniluenced the soma- to the irreversible binding o€ small quantities of the tnmotor and the vegetative components o€ the he- alkaloid in specific sites o€ the granular men~hraneand to the inhibition o€ ATI' and Mg++ -dependent up- take in the granules [6, 71. Small quantities o€ reser- Table 2. - Tbc inj%,enre of onfidepressantr on the 3H- pine were found in the brain o€ rats and mice up t» GAAA ad3H-rerotonin ipake by rri~deynuptoso!ìzal 5 days after treatment [S, 91. Prohably there is a bal- preparation from rat broin. ance hetween lipid (large nnnspecific) 2nd granular (small specific) pools o€ reserpine, tlie lipid pool may be the source o€ reserpine for prolonged inhibition of the granular uptake leading to the depletion o€ mono- aminergic systems [IO]. Dahlstrom 2nd Haggendal [Il] found out that noradrenaline recovery in peripheral nerves developed only on the 7th day after single re- scrpine injection. Conrrol ...... 100&9 100'9 100i10 100510 The imipramine-like drugs do not modify the initial DMI ...... 21+3 77~9 19c2 1212 concentrations o€ reserpine in the rat brain [l2]. It is Befuraline ...... 80*10 121515 30=4 2555 possible that the antidepressants prevent irreversible Tramdone ...... 33*4 96~10 22*3 91510 binding o€ small quantities o€ reserpine in reccptor Pyra;idol ...... 71+8 99515 26+3 31'4 sites o€ the brain, moreover that there was no cor- ...... relation between monoamine content and rcserpine . , ~ ~ ---- ~--.~ ~ binding in the brain[l3]. This correlation was found Footnore: 10 nM of .:'H-serotonin and 3.5 @Mof :'H-GABA per I me of prorein crude synoptosoiiml fraction wirhin 20 in the peripheral terminals in accordance with the inin ar 37°C were considered ac 100 ",,. density o€ noradrenergic innervation [l4]. REFERENCES 1. KOZLOVSKAYA,M.M. 1975. The study of psychophysiological stnicture of the cmotional bchavior under the conditions ot group inter nctions. In: Plycbop6mm~cologjof fbe cmotionirl ~lresrimd ~oororijil;nfrrorfion~. A. Valdman. Ed. Pavlov 1st Medica1 'Institute, Lcningrnd, pp. 98-107. 2. RAYEVSKY,K.S. & Mairov, N.I. 1975. The influente of ncuroleptics and antidtpressants on the W-GABA uptake by the isolated nersc endings from rat arain. Bull. Biol. Med. Exp. URSS. 6: 63-65 (Russ.). 3. Bo~s~r,L, POPENDIKER, K.,:WEBER, R. & SODER,A. 1979. Synthesis and phnrmacological activity of befuraline (N-bcnzo [b] furan- 2-ylcarbonyl-N'-benzylpipernzine) a nca antidepressant compound. Aaneim.-Forsch. 29: 193-204, 4. MASHKOVSIY,M.D., GORKIN,V.Z. & ANDREYEYA,N.I. 1975. The invcstigation of the mechanism of action of pyranidol - a neu. antidepressant dmg. Form&l. Tokrikol. 5: 531-536 (Russ.). 5. MasIi~ovsxr,M.D. & ANDREYEVA,N. 1975. Expcrirncntal study of psychotropic action of pyrazidol. finnokol. Tokri&l. 1; 5-9 (Russ.). 6. CARLS~ON,A., HILLARP,N. & WALDECK,B. A Mg2+ ATP dcpendent storagc mechanism in the amine granules of thc adrenal medulh. Mcd. Exp. 6: 47-53, 1962. 7. STITZEL,R. 1976. Thc biologica1 fatc of reserpine. Pbmm. Ree. 28 (3): 179-205. 8. MAGGIOLO,C. & Ha~rr,T. 1964. Brain eoncentration of reserpine-HJ and its mctabolites in the mouse. Pror. .So<, &p. Biol. Mcd, 116: 149-151. 9. SHEPPARD.H., TSIEN,W. et al. 1958. Brain ceserpine Itvels following large and small doses of reserpinc-H'. Pmr. Soc. &p. Biol. Mcd. 97: 717-721. 10. WAGNER,L. & STITZEL.R. 1972. The relation betmcen thc sub cellular distribution of H3-rcserpine and its proposed sitc of nction. 1. Pbann. Pbmmrol. 24: 394-402, 11. DAHLSTROM.A. & HAGGENDAL,J. 1969. Reeovcry of noradrenaline in adrencrgic axon of rat sciatic ncrves after reserpinc treat- mcnt. j. Pb~rm.Pmrmrlcol. 21: 63L538. 12. MANARA,L. & GARATIINI,S. 1967. Failure of desipraminepretreatmcnt to modify the initial concentration of rcserpine in rat tisoues. Europ. j. Pbnrmarol. 2: 142-143. 13. ENNA,S. & SHORE,P. 1971. Regional distribution of persistently hound reserpine in rat brain. Biorbm. Pl,ormr?<-01.20: 2910- 2912. 14. NORN,S. & SHORE,P. 1971. Failure to affect tissue resupine concentration b" alteration of adrenergic ncrvc snivity. Biorliem. Pbarm~ml. 20: 2133-2135. Ann. Iri. Super. Sdnità Vol. 18, N. 1 (1982), pp. 87-90 Thyrotropin content in serum of rats motivated and non-motivated to alcohol S.A. BORISENKO (a) and P.T. LIANNISTO (6) (a) Laboratory of R~search/or Drzg addiction Preuention at~d Treatment, Inrtitz Summary. - It has been sbown that alcohol-motivated non-motivated to ethanol. 'Various stimulation tests Kistar frmale rats ajter 10 dayr qf repeated etì~anolad- \vere performed both during the state of pliysical nrinistratio haue a decreared cold-or TRH-stimulated TSH dependence and during the state of abstinence. Ieiiel in the blood ierrim under phyrical dependence and abrti- nence. There were no changes of TSH secretion in animals non-n~otiiiated to alcofd. Antaldehyde inhibited sjgnifi- rantb the TSH cold-rerponse 6ut did not infi~ence TRH- rnduced TSH secretion. Animals. 156 female Wistar rats, weighing 150-220 g It ir su~gested that repeated ethanol adn~inistrationrauses were used. The rats were kept 3-5 in each cage in o hypojìinction of both hypothalamir TRH nerwons and a room artificially illuminated from 7 a.m. to 7 p.m. anterior pituitary tfyrotropic celh. Partial4 it nuy de- They were given standard laboratory pellets (iodine pend on ncetnldehide inhibitory action un I?ypothalon~icleuel content 0.5-1 mg/kg) and tap water ad libitum. o/ regulation of tfyroid gland funrtioning. Experimental designs. Riassunto. - Ratti Wirtar dipendenti dail'alcool hanno I) TSH serretion during efhanol dependence or abstinence. bassi livelli di TSH in risposta alla stimoiqione con TRH Previous studies [5] have revealed a reverse correlation o dopo esposi@te al freddo. L'acetaldeide it~ibiscela risposta hetween the sleeping-time (induced by a narcotic al freddo ma non inlpuenza quella al TRH. dose of 25 ethanol, 4.5 ml/kg i+.) and the volume of the ethanol consumed when the animals had a free choice between ethanol (15 T!,) and water. In our experiments, the rats with sleeping-times of 60 min or less were classified as short-sleepers (SS) or moti- vated to ethanol and those sleeping 120 min or more Studies on motivation and non-motivation to ethanol as long-sleepers (LS) or non-motivated to ethanol. are one of the main aspects of the research o€ drugs LS rats consume only 0-3 ml of 15 46 ethanol per day for the prevention and treatment of alcoholism. It under a free-choice situation, whereas SS rats consume was previously reported that ethanol causes aggression 1040m1 per day 151. One week after the sleeping in non-motivated animals and has a calming effect test, the chronic treatment was started according to the in motivated animals [l]. Ethanol activates positive modification of the method used by Gothoni and reinforcement in motivated animals but does not Ahtee [lo]. In brief, 10 $L (V/V) ethanol in water work in the animals which reject ethanol [Z]. The was given 3 times daily (cvery 8 hours) by a gastric rats motivated to ethanol have been found to have tube in increasing volumes: on the 1st-3rd day 8 ml/kg, an increased activity in the hypothalamic tyrosine on the 4th-6th day 15 ml/kg and on the 7th-10th day hydroxylase[3], an increased 5-HT content in some 20 ml/kg. Contro1 animals were given equa1 volumes brain structures [4] and an increased activity of ethanol of water. The experiments were performed on the metabolizing enzymes [5]. 10th day either 4-6 h after the last ethanol dose (physi- There are some data about the difierence in the cal dependence) or 16-18 h after the last dose (absti- effects of ethanol on the hypothalamus-hypophysis nence). The following tests were performed. 1) Basal neurosecretory system in the animals which prcfer serum TSH leve1 were measured. 2) The TRH-induced or reject ethanol [6]. These effects seem to depend TSH secretion was studied by giving 50ng/100 g on the duration of the treatment and on the severity of body weight of TRH i.p. and killing the rats 30 min o€ the ethanol intoxication [7-91. later. Serum TSH levels were measured. This test The present work was devoted to study the effect monitors the function of the anterior pituitary thyro- of repeated ethanol administration on the thyrotropin trophic cells. 3) The cold-induced (the rats kept at concentration in serum of rats either motivated or a temperature of + 4 OC for 30 min) TSH secretion 88 S erum TSHng /m1 PHYSICAL I CONTROL DEPENDENCE ABSTINENCE ( L -6 hours ) (16-18 hours ) T Inclined ( short - sleepers) B TRH 4°C 8 TRH C°C B TRH C°C Non inclined (long- sleepers) T l O00 800 600 LO0 200 B TRH 4°C B TRH L°C FIG. 1. - Thyrotropin (TSM) content in blood SeNni of rats inclined and non-inclined ro alcohol in the starc of physical dependence and absrinence. Ordinates: serum content in ng/ml. Ahscissa: Basal (n), TRII-stimulnted (TRH) and cold-induced (4 'C) TSH levels. Figures in che columns indicare the number of erperiments. is a measure o€ the activity of the hypothnlamic TRH Table 1. - The effrct o/ acefaidtbyde on the cold-indwed neurons. TSH secrdon. ~ ...... 11) Effect of acetaldehp'e on TSH rerretioi~. The rats -...... -~--p--...... p-p-.. Seium TSH (both SS and LS) u-ere given either 0.9<:: saline or TREATMENT P 100, 200 or 3OOmg/kg of acetaldehyde i.p. Then inslmli the rats were transferred to + 4 "C and killed hy decapitation after 30 min. The hlood of the whole 0.9 saline, ... 30°C ...... 920172 4 0.001 trunk was collected and serum TSH assayed. Some rats were left at a room temperature. In another study, 0.9 "" saline, i 4°C ...... 3,085*579 6 -- the rats were given either 0.9 saline or 200, 300 or Acetaldehyde 100 mgikg t- 4°C 3.1561397 6 NS 450 mg/kg of acetaldehyde i.p. together with 50 ng/ 100 g or 150 ng/100 g of TRH at the same time. After Acetaldehyde 200 mg/kg -i 4°C 1,526&139 7 0.05 30 min the rats were decapitated and serum TSH Acetaldehyde 300 nigjkg + 4°C 1,3101313 6 0.05 assayed. - ~ 111) TSH radioin,rnwoassay. Serum TSH was mea- Mean t SEM. n = number oi animals. sured using a radioimmunoassay kit which was a gift frorn NIAMDD, the Rat l'ituitary Program, Bethesda, Maryland. It contained TSH for radioio- inhibited. Again, the non-motivated rats had normal dination, antithyrotropin antiserum and a rat TSH responses (Fig. 1). standard (biol. potency 0.22 USI' bovine units/mg 3) Effect o/ acefaldebyde on TSH ieuels. Acetaldehyde in the McKenzie assay). The results are expressed (200 and 300 mg/kg) inhibited significantly the TSH in ng/ml of this standard (TSH-RP 1). cold-response (Tah. l), but only 450 mg/kg inhibited IV) Stafistics. Arithmetic means, SEMs and SDs the TRH-induced TSH secretion (Tab. 2). vere calculated. Student's t-test was used for compa- rison of two means. In case of three or more means an one-way analysis of variance was first applied. The data ohtained suggest that the ethanol-moti- RESULTS. vated rats (SS) have a normal hypothalamusanterior pituitary a& However, repeated ethanol administration 1) Behauiow. After 5 days on ethanol, the rats had seems to cause a slight hypofunction of both the hypo- behavioural changes which resembled withdrawal thalamic TRH neurons and the anterior pituitary syndrome (stiffness and tremor of the body and tail, thyrotrophic cells. In the state o€ abstinence TRH- moderate rigidity, aggression). These symptoms were response is absent suggesting an impairment of the most pronounced 16-18 h after the last ethanol dose. anterior pituitary gland functions. This rnay be related Aggression was the most typical sign. to an increased dopaminergic activity during absti- 2) TSH secrefion during p&u/ dependence and absti- nence [Il, 121, because dopaminergic drugs have nence. Without any ethanol treatment, the ethanol- been shown to inhibit TSH secretion, although not at motivated rats had a greater TRH-response than the anterior pituitary level [l3]. In the ethanol non- the non-motivated rats, hut there was no differente motivated rats (LS) only basal TSH levels were signi- in the basal or cold-stimulated TSH levels. Under ficantly decreased in the state of abstinence, but both physical dependence, in the rnotivated (SS) rats both the TRH- and cold-stimulation tests gave normal the TRH- and cold-induced TSH secretions were results. A partial inbibition of the cold-response and somewhat decreased. This was not seen in the non- especially the inhibition o€ the TRH-response by motivated (LS) rats. In the state of abstinence, the high doses of acetaldehyde are evidently unspecific motivated rats did not have any TRH response at al1 and related to the effect o€ various anaesthetics as and also the response to cold exposure was significantly was shown hy Mannisto et al. [14]. Table 2. - The heffect o/ acefaldehy&~or2 tha TRH-induced TSH secreiion. .. -~...... - ---~ ..~ ~ ~. - ~ ...... -p--p- ..... ----p~-~-~ ...... ---p--. ~.~-- ...... Dox o< TRH Doae a< TRH Dose of TRH O 150 ng,ioog IO n4,10o~ DOSE OF ACETALDEHYDE -p-p.- SEI~~TSN ngiml 0 ...... 5191126 (6) 1.423i373 (6) 1,622&211 (6) 200 mgjkg 1,8321303 (7) 2,043i529 (7) 300 n~gjka ...... -. 1,9971470 (7) 2,501&354 (7) 450 ingjkg ("*l ...... - 542+ 182Y4) 495;2,438 (2) 1 .-- ...... -- . .. ~~ ~- ~ - ~ p--~-~-~.~-~-p~ .-.--.---p - Mean * SEM. Number oi nnimals in parenrhesis. (") LDaoof acetaldehyde. (*) P < 0.05 vs. the corresponding TRH- saline control. 1 REFERENCLS 1. BORISENKO,S.A. & KA~IPO~---POLLYOY,hiBi 1980. Individua1 features of thc emotional reactiviry of animals as a prerequisitc'for alcohol rnotivation. %h. 1,~yrsh.ATevii. De~rlf.3: 513-516 (Russian). 2. Bu~ov,Yu.V. & Bonrir.~~~,S.A. 1979. Thc influente of ethanul and aceta1deh.de on the structures of positive reinforcenient in rats. I'armakol. l~okrikol. 3: 291-293 (Russian). 3. MINETEVA,M.F., KUVRIN,VS. & B.~Iov, A.I. 1980. The role of brain tyrosine hydroiylase in the formarion of alcohol motiva- tion. Proc. All-Union Symposium a Biochernistry of Alcoholism n (Abstr.), Grodnn, p. 94 (Russian). 1. Anroe, L. 1975. Brain monoamines in alcuhol sclectiun and depeiidence. Proi. Iiit. Cnopr. Pliormniol. Helsinki, Finland. 3: 41-50. . BVROV,YuV. 1980. Pharmacalogicnl study of thc mechanisms of ethanol morivarion and dependrnce and search for thc drugs for alcohul trcatmcnt. Self-abstract of duct. diss. hlorcow (Russian). . Y~vonsrr,A.N. & Lru~rnrov,E.I. 1980. The studg of the reactivity «f hypothalarno-hypophysial and ncurusecremr" rysrem in rats prefcrring and rejccting alcohol. Bull Bio/. Al&. Ex$ 6: 658460. . GOLDBERG,M. 1960. The occurrence and trcatment of hypothvroidism among alcoholics. I. Cliu. Eiido Pharmacological and electr~physiolo~icaldifferences between ;i-hydroxybutfric acid and GABA- mimetic drugs R.U. OSTROVSKAYA . . Imtitufe of Pburmacolog~,Arademy of Medicai Sciences o/ tbe USSR, Moscow Summary. - In this paper ore prerented pbarmaco- B) GHBA and hypoxic condifionr. lo@l und neurophysio/ogical dutu rupporting the conceppl thd GHBA is not a GABA-mimetic drug and tbaf its It has been described that GHBA may increase the dctions ore similar to those aj ruccinic semi-uldebyde. survival time in animals exposed to hypoxic condi- tions [li, 121. In Table 3 the effects of GHBA, SSA, valproic acid and AOAA in two different models eiassunto. Sono presentati dati farmocologici e neuro- - of hypoxia are reported. fisi~logici che dimostrano che i'acido y-idrossibutirrico non The results suggest that GHBA, SSA and valproic n gaba-mimctico ". ma piuttosto ricalca gli effetti del- acid have a profile of action completely di&rent from I'd ezde emrsucrinrca. .; AOAA. At the dose used, AOAA increased brain GABA content by 270 %. These data suggest that the increase of the survival rate and of the survival time caused by SSA, GHBA and valproic acid [l3441 in hypoxic conditions is not due to their effects on It is widely accepted that y-hydroxybutyric acid brain GABA levels. (GHBA) may be considered a GABA-mimetic ageqt[l4]. Moreover it has been suggested that GABA is metabolized into GHBA and viceversa [5, 61. Coniroversial reports are available on the pharmaco- logidal actions of GHBA and on its possible modifi- C) The compariron of tbe effect of GABA-mimetic drrgs catio~sof the brain GABA content [7-91. In this paper and GHBA on e/ec~ophysioIogicu/ indiccs of cortical are presented pharmacological and neurophysiological inhibition. data supporting the concept that GHBA is not a GABA- mimqtic drug and that its actions are similar to those The recovery cycle of the evoked potentials is known of succinic semi-aldehyde (SSA). to be one of rhe most informative indices of the degree of inhibitory processes. Using this test, we have dif- ferentiated the effects of GABA-mimetic dmgs and GHBA. The recovery cycle of the primary cortical ~'HARMACOLOGICAL STUDIES. response evoked by the stimulation of the tooth pulp in the rabbit is characterized by a non-constant phase i) The antagonism o/ bicucuiihe und qf AOAA induced of postexcitatory facilitation which is most marked ronvui'sions. when a 5 msec intervd between conditioning and testing impulses is followed by the phase of postexci- Bic+xlline is a GABA-antagonist and may cause tatory depression lasting up to 30 msec. CE-GABA convulsions when administered in suthciently high in doses of 5-10 mg/kg caused a marked increase in doses. GABA-mimetic agents antagonize this effect. postexcitatory depression (Fig. lA, B). In the recovery Table 1 shows that GHBA does not antagonize this cycle of corticocortical response recorded in the motor f convulsions, while other GABA-mimeucs cortex during the stimulation of the sensory area which re effettive. Another differente between GHBA normally has a more marked and longer phase of agents was evidentiated using postexcitatory facilitation (20-100 msec) CCGABA the an~a~onismto convulsions induced by amino completely eliminated the facilitation phase. A simi- oxyaceqic acid (AOAA). lar effect was produced hy intraventricular GABA Tablq 2 sbows that this type of convulsions are as well as by the eartier studied benzodiazepine deriva- nntagd+zed by SSA, by valproic acid and by GHBA, tives (clonazepam, lorazepam, diazepam, chlordia- but not by GABA cetyl-ester, a GABA-mimetic zepoxide)[l4, 151. These effects were competitively molecul'! originally described in our laboratory (101. antagonized by bicuculline. The actions of GHBH are different and dependent upon the dose. When nonselective potentiation of the inhibitory processa 30-500 mg/kg were adrninistered, GHBA decreased coupled with the reduction of the excitatory ones the threshold of the evoked potentials and increased makes the effects of high doses of GHBA similar ti> the degree of the facilitation to the testing response. that of barhiturates. Thus, in a wide dose rangc~ At high doses (1500-2000 mgkg) it increased the GHBA was not characterized hy the ability to'imitatc. threshold of the condiuoning responses and sornewhat the effect of GABA-ergic compounds on the inhibi- increased postexcitatory depression of the testing tory processes but by the ability to decrease GABA- primary response (Fig. lB, C, D). These changes of ergic inhibitor. It can be supposed that the abilit? excitability are associated to EEG modifications. Such of GHBA to cause characteristic spike-like discharges FIG. 1. - Effect of CE-GAB (A, B) and GHBA-Na (C, D, E) on the recovery & O,, Convulsing animals ...... 98 50 50 500 100 Degree of convulrionr ...... 60-80 22-80 20-80 28-80 75-80 Death rate (76) ...... 40 10 5 15 20 Bicuculline (3 i,pJ was administered 180 min after AOAA and 30 min after murcimol, GABA CE or GHBA. The degree of che convulsior* Fas- eviluated on arbitrary basis C80 = max). 20 animals for each group were used. Table 2. - Antagonirm fo AOAA-induced ronuulrionr. - .- -p------P---- ~ p--.--.-~- SSA GHBA V1a. CE-OABA Contro1 iSOOms/kgi (5OOrnsIkg) (300mg1kci UOm8lka 7; Convulring animals ...... 95 10 25 15 95 MomslirY ...... 30 O O O 20 Latency (min) ...... lL+O.2 4012 3511.7 3711.5 1212 -. p-- --~--~------p-.----p------AOAA (150 mg/kg) was injected i.p. Twenty rats for each group were used. Values are percentage or mean + SEM. in the cerebral cortex 1161 is due to a competition of exogenously administeied GHBA with endogenous GABA for the GABA receotors. This antawo- The metabolites of the so called « GABA shunt » a have severa1 pharmacological properties not related nism mav, explain the GHBA induced mvoclonic ierks. to their transformation into GABA. GHBA in low GHBA and GABA competition for GABA-ergic doses has a tranquilizing effect and, in high doses, receptors may be the.reason for a decrease of GABA a narcotic one; SSA resembles the neuroleptics. Both inhibitory effect observed in the experiments with a compounds have a marked protective activity in simultaneous microiontophoretic application of both hypoxic states. Neurophysiological data indicate that substances [3]. they do not potentiate the degree of GABA-ergic inhibition in the cerebral coaex and that in certain conditions they antagonize it. None of them has a protective effect against bicuculline-induced convul- Table 3. Tbe effect of GHBA, SSA and AOAA - sions. Intraventricular administration of GABA and on the ruruiuaI time and on the rwviurll rate o/ mices expored of GABA-mimetics specifically increases GABA- to b~poxicrondifionr. ergic inhibition and manifests antibicuculline action. ---p The effects of GHBA and SSA are not dependent A B Survivd Survivd upon their conversion into GABA. In fact AOAA timc (mi") rate O,I inhibits their transformation into GABA but does not modify their aaions. Moreover, the electrophysiological experiments re- iontrol ...... 20i1.1 15 ported here suggest that GHBA and SSA are able SHBA (300 mg/kg) ...... 49f 2.2 68 of antagonizing the action of GABA. The different pharmacological and electrophysiological profile of REFERENCES 1. DEFEUDIS, F. & COLLIER,B. 1970. Amino acids oi brain and y-hydroxyhutyrate-induced depressiun. .+C/>. In/. l'hormomd~n. Thw, 187: 30-36. 2. GIARMAN,N.J. & SC~~YT.K.F. 1963. Some ~ieurochemicalnspects oi rhr depressant action 6. MITOMA,CH. & NEUBAUEK,S.E. 1968. Gamma-hydroxybutyric acid and slecp. Exprientia. 24: 12-13. 1 l 17. WOLLEMANN,M. & DEVENY,T, 1963. Thc mctabolism of 4-OH-butyric acid. A~c11010gie 4: 593-598. l 18. PxxTn*, G., ILI.I~~.Io,G., CAPANO,V. & RAVA,K. 1966. In vivo conversion of y-hydroxybutyrate. Arofure. 210: 733-735. 9. BALAI harmacological effects of nicotinamide. robable endogenous ligand of benzodiazepine receptors T A. VORONINA 11I titute of Pharmacolo~,,Aiadeny of Medical Sciences of the USSR, Moscoiu results are reported rod); far a detailed description of methods, see [6, 71. eficts of nicotinamide All the substances were injected intraperitoneally, sus- between NAM, inosine pended in Tween-80, 30 min before the experiment. Because of the poor penetration of NAM into tbe brain [8] only high doses (250-2000 mg/kg) were used. effects ond affects Riassunto. - Sono sfati studiati gli efetti della nicotina- m'de (NAM) su/ sistema nervoso fentra/e, comparando /o s azione con qziel/a dell'inosina e delle benzodiaxepine. I NAM in doses of 500-1000 mg/kg caused alterations ri ultati indicano che la NAM possiede effetti sedatiui, mio- of the orientation reaction, potentiated barbiturate sleep riassunti ed antironuulsiuanti e injuenza il turnouer dei neuro- and had antiaggressive activity. At higher doses, anti- f smettitori in modo analo~oa/ dia~epam. convulsive effects and disturbance of movements were 1 ohserved (Tab. 1). These effects resemble those o€ the benzodiazepines [9]; however, different potencies in the I TRODUCTION. different tests were evident: the benzodiazepines had iP greatest activity in antagonizing convulsions, while the 1 After the discovery of the benzodiazepine receptors, antiae~ressive-- and sedative activities o€ NAM were aqtempts have been made to identify their endogenous more evident. li@ands: Presently, four groups of substances capable NAM in doses of 250-500 meike-, ', was active in con- o specific binding with benzodiazepine receptors have flict situations, eliminating the action of the punishing been proposed for this role: purines (inosine, hypoxan- factor as shovin by the increase in number of drinkings tdine), p-carbolines, proteins and nicotinamide (NAM). (Tah. 2). A subthreshold dose of NAM (125 mg/kg) Available experimental findings indicate that the latter was able to potentiate the anxiolytic effect of phena- has sedative, myorelaxant and anticonvulsive effects zepam (l) or calcium valproate. apd affects neurotransmitter turnover as does diazepam On the other hand, bicuculline, a GABA-ergic re- In the present paper the results are reported of ceptor blocker, reduced NAM potency. This data of the neuropharmacological effects of NAM, provide evidence for an involvement of the GABA- tolerance hetween NAM, inosine ergic system in the anxiolytic effect of NAM, as is the case of benzodiaze~ines. As Enna and Snyder [lo] have shown, NAM is not capable of binding with GABli-receptor. To extend our knowledge on the sirnilarity of NAM 2nd inosine to benzodiazepines, cross-tolerance was used. It is known that in &n and animals tolerance The study \vas carried out on albino mice (inales, to the myorelaxant effect of benzodiazepines develops 1 -24 g) and rats (males, 150-250 g). Tranquillizing rapidly [Il]. The lack of correlation between blood e ect was estirnated using the method of conflict situ- concentration of benzodiazepines or its metabolites and a ion in rats, tbe latter resulting from clectric stimula- development of tolerance to myorelaxant effect in mice ti n during the performance of a drinking reflcx[5]. [l21 indicates that the mechanism of this tolerance is he following effects were studied in mice: antiaggres- not rclated to pharmacokinetic factors. Drawing an 1e (fight betu.een two animals on an electrified analogy with tolerance to morphine, it can be sug- sedative (disturbed orientation and potentiation sleep), anticonvulsive (mtagonism to and maximal electroshock seizures), (1) Phenazcpam: 7-bromo-~~hlorphenyI)-1,2-dihydro-3H (motor incoordination in the rota -1,4-benzodiazepine-2-0ne. Original Soviet tranquilizcr. Table 1. - ~Veuro~hurr,laco/o~~icaiacfiuig of IVAM in mice. ~~ ~-~~ ~ -. ~ ~ -- -~~- ~ - ~--~ METHOD. Antagonism to pentylentetrazoi ...... ED,, mg/ke Antsgonism to maximal &i-troshock ...... EDno mg!kp Amiaggressive effect ...... ED,,, mglkg Disturbsnce of orienration reactions ...... ED*,, mg:kg Disturbante of mouement ruordination ...... EDio mg!kg Potentiation uf hexobarbital sleep ...... Sleep duratioii (niin) gested that the tolerance to benzodiazepines is under- and after the fourth one the effect cotnpletely lied by an alteration of the interaction between the disappeared. In mice tolerant to phenazepam (day 6), drug and the receptor. lorazepam or diazepam had no myorelaxant effect; Phenazepam in a sin& high dose (40 mg/kg) after tofizopam myorelaxant etTect occurred in only 20:,, caused a marked myorelaxant effect (loss of motor of animals. In contrast to the benzodiazepines, chlor- coordination) in 100 of animals. After a second promazine, trifluoperazine, ethanol, phenobarbital, me- dose, given 24 hr later, the effect of phenazepam prohamate, hydroxyzine, or the GABA-ergic receptor remained in 20 :," of mice, after the third one in 5 l:,,, agonist muscimol given during tolerance to phena- Table 2. - Effect~o/ Nirotinamidu ond ofher driigr on conpict behnvior in roti Control ...... Nicotinamide ...... Phenazepam ...... Valproate Ca ...... Diarepam ...... Nicotinamide ...... -t Phenazepnm ...... (Nicotinamide ...... l + Valproate Ca ...... I Nicotinamide ...... 1 l t~ 1Bicuculline ...... mate, hydroxyzine, chlorpromazine, or phenobarbital revealed a differentiation o€ the effects o€ the drugs: each compound acted independently, with no connec- tion with each other. Myorelaxant effect o€ phena- zepam under alternating administration disappeared al- ready at day 2-3 o€ the administration, whereas the effects o€ chlorpromazine, meprobamate and hydroxy- zine remained stable in 100 % o€ animals even at day 10 (Fig. 2). In contrast, mhen phenazepam was alter- nated with diazepam or lorazepam, the action o€ the compounds was interrelated and the picture o€ toler- ance was the same as under Dhenaze~amalone. NAM and calcium valproate (the latter at the initial stages o€ tolerance development) had partial cross-tolerance with phenazepam duiing alternate administration. These hndings suggest specihcity o€ cross tolerance to benzodiazepines. The compounds which have suffi- cient affinity €or binding with benzodiazepine recep- tors are those capable o€ replacing benzodiazepines (nicotioamide, inosine) [2, 13, 141. The compounds Fidi 1. - C~SStolerance between bemodiazepines and other 1)rugj. Ordinate: percent nf thc effect; absdssa: day of treat- iment. Upper drawing. Myorelaaant efiects of different sub- sraneqs in animals tolerant to phsnazcpam: 1-0- phenazepam 40 rrig/kg; 2-a- meprobarnate 200 mglkg; hydronyzinc 100 loc-pam 50 mg/kg; 9 -ediazepam 40 mg/kg. Lower are che same as in the upper drawing. zepafn caused a marked myorelaxation in al1 the animals (Fig. l). Lonetyl(2), calcium valproate, sodium hydro- xybjtyrate, inosine and NAM proved capahle o€ par- tial phenazepam substitution and produced weak my- orelqxant effects. or phenobarbital-tolerant animals it was follqwed by myorelaxation in 100 0/, o€ mice. series o€ experiments, phenazepam was ated with other drugs. The administration of to the same animal in turn with meproba- Fic. 2. - Devclopnient of tolcrnncc to phenazepam under :alternate administratioo with orher psychrirropic drugs. Or- dinatc: percent of thc effcct; abscirsa: day of treatmenr. Sym- bols: --a- phenazepam; l - diazepnm (-O-); or lorazepam (-X-); 2 - ualproate Ca(-O-); 3 - KAbI (-O-);4 - meprobarnate(-O-); 5 .. chlorpromazine (-O-). Doses areas in Fig. 1. Dotted lines iiirlicate the changes in the activity of the compounds (substi- tutes) during their daily administration to miee in experiments wirhout che alternation with phenaepam. unable to bind (neuroleptics, phenobarbital, non-ben- cross-tolerance o€ NAM and inosine with the benzo- zodiazepine tranquilizers, muscimol) [15, 161, do not diazepines, suggest their resemblance with benzodia- show cross-tolerance. zepines and supports the hypothesis o€ the probable Thus, NAM is one of the few endogenous substances role o€ these compounds endogenous ligands. The capable of specific binding with benzodiazepine recep- development o€ tolerance to benzodiazepines does not tors and it is similar to benzodiazepines with respect seem to involve the GABA receptor system, since to various manifestations o€ anxiolytic, hypnotic, and muscimol, a direct GABA-receptor agonist, does not myorelaxant effects. These data, as well as the partial interfere with the process o€ tolerance development. REFERENCES l. BEATON,J.M., PEGRAM,G.V., SMYTHILS,J.R. & BRADL~Y,R.J. 1974. The eKects of nicotinamide on mouse slecp. fizpcrientza 30: 926-927. 2. MOHLER,H,, POLC,P., CUMIN,R., PIERI,L. & KETTLER,R. 1979. Nicotinamide is a brain constituent with bemodiazcpinc-like auions. Nolm. 278: 563-565. 3. SCHERBR,R. & KRAMER,W. 1972. Influcnce of Ncotinamidr administration on brain 5-HT and a posrible mode of action. I.$ Sci. 11: 189-195. 4. KENNEDI, B. & LEONADD,B. 1980. Similarity between thc action of nicotinamide and diazcpam on ncurotransmittcr metablism in the %t. Riocbcm. Soc. Tram 8: 5940. 5. KLYGUL,T.A. & KRIYOP.+LOV,V.A. 1966. A devicc with automatic recording of rat bchavior fnr cipcrimental evaluation of the donof minor tranquilizers. Formokol. Tokoikol. 2: 241-244 (Russ.). 6. Virri~ua~av,YL-.L, KLYGUL,T.A., BOGATBX~,A.V. & ANDRONATI,S.A. 1971. I,&bemodiarepines and their derivativcs. IV. On the relationships between chemica1 stnicture 2nd phamacological activity in the series of subrtituted derivatives uf 1,4-benwdia- repine. In: Pby1ioIo~ici1llyorliue rompotmdr. P.S. Pelkis (Ed.). Kiev, Naukova dumka. 3: 265-279 (Russ). 7. TURNER,A. 1965. Dspressants of the central ncrvous system. In: Srrerning mr/hodi in plornrocolo&y. Acad. Press, Ncw York, London, pp. 69-99. 8. DEGUCHI,T., ICHIYAMA,A. & NISHJZIIKA.Y. 1968. Studies on thc biosynthesis of nicotinamide - adcnine dinucleotidc in the hnin. Biorbrm. Biophy~.Arts. 158: 382-393. 9. VIKHLYAYEV,Yu.1, & VORONIN,T.A. 1979. The spectrum of phamacological actiuity of phenazepam. In: Minor h(~qdi~in fba hrotmrnt md rrbabili/atiot~ ofp1ycboitcurologiii1l potientr. E.A. Rabayan (Ed.). Leningrad, pp. 5-14 (Russ.). 10. ENNA,S. & SNIDER,S. 1975. Properties of yaminobutyric acid (GABA) rcceptor binding in rat brain synaptic membrane fractions. Bmin Ru. 100: 81-87. 11. VIK~L~AYEV,Yu.~., DZHKATSPANIAN, I.A. & KLYGUL,T.A. 1972. Peculiarities of the devdopmcnt of tolerance to benzodiazcpinc tmquilizers with respect to sedative and myorclarant eKccts. Formokol. Tokdol. 4: 421-425 (Russ.). 12. CHRISTENSEN,J, 1973. Tolcrance dcvelopment with chlordiazepoxide in the plasma leve1 of thc parcnt compound and itr mnin mctabolites in mice. Arto Pbarmmol. Toxirol. 33: 262-272. 13. BRAESZRUP,C. & NIELSEN,1%. 1980. Bemodiazepine receptors. Aq!.-Forrrh. 30: 852-857. 14. SKOLNIK,PH., MARANGOE,P,, STIPIN, P,, GOODWIN,F. & PAUL,S. 1979. CNS bemodiazepine receptors: physiological studici and putative cndogcnous ligands. Pbormorol. Biorbm. Beh. 10: 815-823. 15. MOHLER.H. & OXADA,T. 1977. Benrodiazepine receptor: demonstration in the centra1 riervous system. Sciorre. 198: 849-851. 16. SQU~KCS,R. & Bnmsr~u~,A. 1977. Bemodiazepine receptors in rat brain. Nofwe. 266: 732-734. Ann. 1st. Super. Sanird Vol. 18, N. 1 (1982), pp. 99-102 Influente of cholino-and adrenotropic drugs on immunogenesis .. . . ' . E.A. KORNEVA, G.V. GUSCWN .and G.M. POLTAVCHENKO Institute of Experimenta/ Medicine, Academy of Medica/ Sciences, Leningrad, USSR . .. Summary. - The present resu/ts show the important cyte microenvironment resulting from the activation rob of mediators, phyed at diferent /eue/s of the wwohu- or inhibition of the corresponding neuro vegetative mora/ maintenance of immune processes. Tbe data on the centers may partiupate in the regulation of immuno- participation of cbolin* and adrenorcactive rystemr in the cyte functions. In order to analyse the mechanisms modu/ation of thc intensi5 of immune rcactiofi of tbc organism modulating the intensity of the immune reaction, we point to tbc inportance of drugs in the imm~nogenesis. have studied the changes of notadrenaline content in the spleen after antigen administration, the inliuence Riassunto. - I risultati delle presenti esperien~e indi- o€ adrenergic agents affecting presynaptic (amphetamine cano i'importante ruolo dei mediatori chimiri, giocato a and cocaine) or postsynaptic membranes on the level pari &ve//; de/h regolqione dei processi immunologici. I of 3,5 c-AMP in spleen cells, and the influence of &ti su/h partecipqione dei sistemi colinergici e udrenergici M- and N-cholinergic drugs on the intensity of the ne//a modu/a+me ddi'intensiti della rea+mc immnno/ogica rosette-forming cell proliferation. The experiments indicano i'importan~a dei farmaci neli'immnnogenesi. have been carried out io Wistar rats and CBA mice. A significant increase of noradrenaline level occurs in the spleen 2 min after the inrravenous administration The centra1 nervous system influences immuno- o€ sheep etitrocytes, this increase is not observed genic processes through hypophysial hormones and after 10-30-60 min. Noradrenaline level was found the autonomic nervous system [l, 21. increased 24 h later. The noradrenaline level is not The presence of cholino and adrenoreceptors on increased in the heart after anugen administration the lymphocyte membrane and the possibiiity of me- and in the spleen after injection of autologous eri- diator-like substances to influence the functional trocytes (Fig. 1). activity of lymphoid cells has been demonstrated [3-51. The changes in noradrenaiine level aie accompanied The change in the neuromediator levels in the lympho- by a raise in 33c-AMP in the spleen occurring 10 min 2 min 10 min 30 min 1-h 24-h ' FIG. 1. - Contcnts of noradrenaline after sheep red blood ccUs injection in spleen tirsue (I), heart tissue (111), and after autologous red blood cells injection in splccn tissus (U). Ordinate = "/, increase of noiadrendine; Abscissa = timc; = significnnt diiicrence (P <0,05) as rompamd with control. FIG. 2. - Influcnce of adrencrgic dmgs on 3,s c-AMP Icsel in splcen cells. Ordinate = conccntration of cvclic AhfP (picomolcs/lOfl cells); Abscissa = I: control (saline); 11: amphetamine; 111: cocaine; IV: phen~x~bcnzamine;V: pmpranolol;~* = significmt difference !P <0.05) as compared with ontrol. FIG.3. - Changes inducrd hy injection of cholinergic drugs in the number of roserre forming cells (RFC) at 4 OC(a) and at 37 OC (h) in thc splccn of CBA mice on the 6th day after immunization with sheep er>-rhrocytes. Ordinate = Ig RFC per IOS spleen cells; whiic dots:,values of each animal treated with drugs; black dats: values of control mice in the same erpcriments (injection of salinc). 1 = nlcot'nc (0,5,,mg/kg); 2 = ~ediphcn (I,l-diphenyl-6-diethylamin~hcx-l-cn-4-in dihydrochloridc) (25 mg/kg); 3 = benrohexaminc (Hcsamcthnnil bcnzosulfonas) (3 mgjkg); 4 = arccoline (12.5 mgjkg); 5 = ~ilocaroinc(5 mplkg). * = signilicant difference as compared with corresponding contro1 in Wilcoxon test. after the antigen administration and by changes of influence the population of rosette-forming cells the functional state of severa1 hypothalamic structures revealed at 4 OC (Fig. 3). evidenced by shifts in DC potentials [o, 71. The increase of noradrenaline levels and of the The intraperitoneal injection of amphetamine (7,s intracellular 3,s c-AMP in the spleen following antigen and 2 mg/kg) and cocaine (10 and 1 mg/kg) increases administration suggests that the shifts obsemed result the content of 3,5 c-AMP in spleen cells. Alpha- from the activation of adrenergic mechanism. These adrenoblocker phenoxybenzamine (10 and 1 mg/kg) effects can hardly be associated with processes develop- has an opposite effect while the beta-adrenoblocker ing within the immunocytes, responding specifically ppanolol (10 and 1 mgjkg) is inactive (Fig. 2). to the antigen. They can be attributed to nonspecific The paaicipation of "the chafinergic system in the components of the reaction to antigens arising in maintenance of immune processes has been studied parallel with specific processes; changes in hypotha- administenng cholinergic drugs to mice immunized lamic functions are probably one of this components. with sheep eritrocytes repeatedly. N-cholinomimetic The obtained data show the important role of me- drugs lead to an increase in the population of rosette- diators, played at different levels of the neurohumoral forming cells, revealed in the spleen at the 6th day maintenance of immune processes. The data on the after immunization by the rosette formation method participation of cholino- and adrenoreactive systems at 4oC. The injection of hexamethonium benzosul- in the modulation of the intensity of immune reaction fonate (3 mg/kg) and of 1,l-diphenyl-6-diethylami- of the organism point to the importance o€ drugs nohex-l-en+in, dihydrochloride (25 mg/kg) (N-cho- in the immunogenesis. The influence of the autonomic linolytic drugs) leads to a decreased cell population. vegetative system is directed to the processes of for- On the other hand, the administration of M-cholino- mation of the immunocyte population after antigen mimetic drugs arecoline (12,5 mg/kg) and pilocarpine stimulation, the character of reconstruction in lymphoid (5 mg/kg) increases the population of rosette-forming tissue being greatly dependent on a change in bal- cells, revealed by the method at 370C, and does not ance between sympathetic 2nd parasympathetic system. REFERENCES 1. KOKNEVA,E.A., SHCHINEK,E.K. & KLIMENKO,V.M. 1978. Neuro-humoral ccgulation of thc immune homsostasis (Russ.). Mcdi- Una. I.cningd. 2. Bmz~ovs~r,H. & SORKIN,E. Nemozk of immune-neuroendocrinc intcroctions. Clin. EIp. Immtmol. 27: 1-22. 3. WEINSTEIN.Y. & MELMON,K.L. 1976. Contro1 of immune responces by cyclic AMP and lymphocytes that adhere to histamine columns. Immmol. Commun. 5: 401416. 4. GORDON,M.A., COHGN,J.J. & WILSON,J.B. 1978. Muscacinic cholinergic rcccptors in murine lymphocytes: demonstratian of dircct binding. Fed. Proc. 37: 609-613. 5. DELESPESSE,G., POCHET, R., GOSSART.B. & VANDBNBROECK.J. 1980. Correlation bctwcen the numbcr of betaadrenergic receptors and c-AMP synthesis induced by isoproterenol in thymocytes and spleen cdls. In: Abrtrd 4-th Intemafiono1Congrm o/ Immunolqu o/ thr Intcnrdionol Union of Imm1molo~iri1lSorirliu (IUIS). J.I. Prcud'Hommc & V.A.L. Hawken (Eds.). Parir, Frane, abstr. N 7.4.05. 6. G~rconmv,V.A. & POLTAVCHENIO,G.M. 1981. Changcs in the hinctional state of immunocompetent cclls and the centra1 nervous system at various timn in thc coarse of immune rcsponsc dcvclopmcnt (Russ.). Vtn. Aksd. M4 hhauk SSSR. 5: 33-36. 7. Gnrconrrv, V.A. 1981. Steady potentials shifrs in the rabbit hypothalamic stmcturcs during early pst-iods of immune response (Russ.). Pbiriologii6crkj gmml SSSR. 67: 463-467.