- Email: [email protected]
The relative significance of norepinephrine, dopamine and 5-hydroxytryptamine in electroshock seizure in the rat
THE RELATIVE SIGNIFICANCE OF NOREPINEPHRINE, DOPAMINE AND 5-HYDROXYTRYPTAMINE IN ELECTROSHOCK SEIZURE IN .THE RAT* P. C. JOBE?, R. E. STCLL$ and P. F. GEIGER Division
of Pharmacology and Toxicology. College of Pharmacy and Allied Health Northeast Louisiana University, Monroe, Louisiana 71201 (AcWpted
Professions,
IO May 1974)
Summary-The effects,of various treatments on brain norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) were compared to the effects of these same treatments on electroshock seizure (ES) intensity. The data indicate that depletion of all three biogenic amines by reserpine or the benzoquinolizme Ro 4-1284, enhances ES intensity and that treatments which protect against depletion of NE and/or DA antagonize ES enhancement. However, protection of 5-HT stores against depletion does not protect against ES enhancement, Additional studies showed that, following depletion of all three amines by Ro 4-1284, subsequent repletion of NE and DA singly or in combination is sufficient to antagonize Ro 4-1284-induced ES enhancement. However, repletion of DA stores alone did not antagonize Ro 4-1284 seizure enhancement in animals treated with pimozide, a central DA receptor blocking agent. Thus. it appears that both NE and DA exert central nervous system inhibitory effects which limit the spread of the electroshock seizure discharge and consequently regulate intensity of ES. The data indicate that 5-HT does not play a significant role in the modulation process.
Recently, JOBE. PICCHIOYI and CHIN (1973a,b,c) reported that, following various combination treatments which alter the synthesis, storage, and release of catecholamines and/or 5-hydroxytryptamine (5-HT) in brain tissue, there is a consistent correlation in rats between increased audiogenic seizure severity and drastically reduced levels of norepinephrine (NE) and 5-HT, but not of dopamine (DA). Based on the data presented, it was proposed that endogenous NE and 5-HT play an attenuating role in audiogenic seizure. The data strongly indicated that of the three biogenic amines studied, NE and 5-HT are more important than DA in modulating audiogenic seizure intensity in rats. Other investigators have indicated that, in the rat, NE. DA and/or 5-HT may also subserve a similar modulating function in electroshock seizure (PROCKOP, SHORE and BRODIE, 1959; RUDZIK and JOHNSON, 1970; KILIAN and FREY, 1973; STULL, JOBE, GEIGER and FERGUSON, 1973). However, from these studies it is not clear which of the amines are most directly involved in electroshock seizure processes. Furthermore, certain evidence fails to support a role for any of these biogenic amines (PICCHIONI, CHIN and BREITNER, 1962; KOSLOW and ROTH, 1971). The present study was conducted to obtain additional evidence concerning the possible functional significance of endogenous NE, DA and 5-HT in electroshock seizure. Various * The work was presented in part at the 58th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 1974. t Present address: Department of Pharmacology and Therapeutics, School of Medicine in Shreveport, Louisiana State University Medical Center, Shreveport, Louisiana 71101. : Present address: Department of Pharmacology. School of Medicine, University of California, San Francisco, California 94143. 961
962
P. C.
JOBE. R. E. STULL
and
P. F. GEIGER
procedures were employed to modify brain catecholamines and .S-HT and the effects of these treatments on seizure intensity were observed. Since some of these procedures selectively modified the brain concentrations of either the catecholamines or 5-HT or selectively blocked dopamine receptors, it was possible to study the relative significance of all three biogenic amines as modulators of electroshock seizure activity. Brain concentrations of the biogenic amines, determined by spectrophotofluorometric analysis, served as indications of neuronal availability of these amines. METHODS
Experimental animals and treatments Female Sprague-Dawley rats (I 5&200 g) were used throughout this investigation. All drugs were administered as aqueous solutions with the exception of L,-3,4-dihydroxyphenylalanine (L-DOPA), 5-hydroxy-D,L-tryptophan (%HTP) and pimozide which were injected intraperitoneally as suspensions in normal saline. Ro 4-1284 (2-ethyl-1,2,3,4,6,7-hexahydro-2-hydroxy-3-isobutyl-9, IO-dimethoxy-1 lbH-benzoquinolizine hydrochloride) was injected subcutaneously; Ro 4-4602 [IN-(DL-seryl-N’-2,3,4_trihydroxybenzyl) hydrazine hydrochloride], reserpine phosphate, iproniazid phosphate and diethyldithiocarbamate sodium (DEDTC) were injected intraperitoneally. Sources of drugs were as follows: L-DOPA, Regis Chemical Co. ; 5-HTP, Sigma Chemical Corp.; Pimozide, Janssen Pharmaceutics; Ro 4-1284, Ro 4-4602, Iproniazid, Roche Laboratories; DEDTC, Fisher Scientific Co. The dosage regimens, test times and sacrifice times used in the various experiments are indicated in the tables. In each set of experiments, test animals which were treated with a single drug were administered supplementary injections of saline by appropriate routes and time schedules such that they were handled and treated in the same manner as corresponding test animals which were administered a combination of drugs. Similarly, control animals were injected with saline and manipulated in a manner comparable to the corresponding test animals which received a combination of drugs. The number of animals used in the calculation of reported test values is indicated in the tables. Electroshock procedure Elecktroshock seizures were induced by stimulation of each animal with 26 mA for O-2 set through cornea1 electrodes. This procedure routinely produced clonic seizures in control (saline-treated) animals. The electroshock seizure pattern (either clonic or tonic) in rats was used to test the effects of selected drugs. Since the tonic pattern is more intense than the clonic pattern (WOODBLTRY and ESPLIN, 19.59; SWINYARD,1963), drugs which increase the incidence of tonus obviously enhance seizure intensity, whereas drugs which decrease the incidence of tonus, diminish seizure intensity. Similar methods of assessing seizure intensity have been used by PROCKOPet al. (I 959) and DE LA TORRE,KAWANAGAand M~~LLAN(1970). Spectrophotq~uoPo,net~ir analysis Catecholamines and 5-HT were extracted from whole brain by the method of WIEGAN~ and PERRY (1961). 5-Hydroxytryptamine was assayed by the method of BOGDANSKI, PLETSCHER,BRODIEand UDENFRIEND(1956); NE and DA were assayed by the methods of SHOREand OLIN ( 1958) and CARLSSON and WALDECK(1958), respectively.
Brain amines and electroshock
Statistical
seizure
963
procedure
The statistical significance of the differences between per cent tonus values were determined by the Chi Square Test. Significant differences between mean whole brain NE, DA, and 5-HT values were determined by Student’s t test. RESULTS
effects
qf‘reserpine
in Ro 4-1284 pretreated
animals
CARLSSON and LINDQVIST (1966) have reported that tetrabenazine interferes with the amine depicting effects of reserpine. In the present investigation. we have studied the capacity of Ro 4-1284 (a benzoquinolizine closely related to tetrabenazine) to interfere with reserpine-induced biogenic amine depletion and seizure enhancement (Table 1). Reserpine alone markedly enhanced seizure intensity and strongly diminished brain NE, DA, and 5-HT levels. When Ro 4-1284 was administered 0.5 hr prior to reserpine, it protected against the reserpine-induced seizure enhancement and brain NE, DA, and 5-HT depletion. In this experiment, Ro 4- 1284 alone had no effect on seizure intensity or brain amine levels; however, this lack of effect when administered alone is not in conflict with its seizure enhancing and brain amine depleting actions which will be described subsequently in this paper and which have been previously reported (JOBE rt a/., 1973a,h; STULL et al., 1973). Table
I. Protecting effects of Ro 4-1284 pretreatment against reserpine-induced enhancement of electroshock seizure intensity and depletion of brain norepinephrine. dopamine and 5-hydroxytryptamine
Treatmenl
and times of injection*
(1) Saline, 0. 0.5 hr
23 (22):
(2) Ro J- 1284. IO mg/kg. 0 hr
9 (22)
+ Saline. 0.5 hr (3) Saline, 0 hr + Reserpine. 2.5 mgikg. 0.5 hr (4) Ro 4-1284. IO mgjkg. 0 hr + Rcscrpinc. 2.5 mgikg. 0.5 hr
Brain amine concentrationst /cg,!g i S.E. NE DA
Per cent tonic seiruret
100 (24) P < 0~0005i; 35 (20)
0.24 (8) ‘r 0.01 0.23 (12) +0.01 0.03 (8) *0,01 P < 0.001 0.15 (12) *010 P < PO25
0.75 (X) + 0.04 064 (12) f 0.06 0.17 (8) * 0.05 P < 0.001 0.52” ( 12) + wJ7 P < 0.01
5-HT 0.31 (8) + 0.02 0.32 (12) io.01 0.17 (8) f 0.02 P < 0.001 0.77 (12) * 0.02
_ * Times of injection are reported as elapsed time from beginning of experiment (0 hr) to the time of injection. i- A.lI scizurc values and brain amine levels were determined 24.5 hr after beginning of experiment (0 hr). $ Number of animals used in each determination is indicated in parentheses. 6 Each tabulated P value indicates a significant difference from saline control value. 1Significantly different from corresponding reserpine value (Treatment No. 3) (P < 0.005).
In these instances, where Ro 4-1284 causes effects on seizure and amine levels. experimental determinations were made within 0.75-4 hr postadministration, whereas, in the present experiment, determinations were made 24.5 hr postadministration, a time period when the effects of Ro 4-12X4 have completely dissipated (JOBE et al., 1973a). EfJcts
qf L-DOPA,
5-HTP
and Ro 4-4602 in reserpine
treated animals
CARLSSON (1967) and BUTCHER, RHODES and YUWILER (1972) have reported that in animals pretreated with Ro 4-4602 (in a small dose which inhibits only peripheral aromatic amino acid decarboxylase), L-DOPA, administered shortly before and after reserpine. will
964
P. C. JOBE. R. E. STULL and P. F. GEIGER
Table 2. Protecting effects of L-DOPA, 5-HTP and Ro 4-4602 against two reserpine actions: (I) depletion brain NE. DA. and 5.HT levels; and (2) the enhancement of electroshock seizure intensity
Treatment
and times of injection*
(1) Saline. 0. 0.5, 1.0, 2.5~ 4.5 hr (2) Saline, 0. 0.5. 2.5. 4.5 hr + Reserpine, 2.5 mg/kg,
Per cent tonic seizure?
0 @)I 62.5 (8) 1.0 hr
(3) Ro 4-4602, 50 mg/kg, 0 hr + L-DOPA, 473 mgjkg, 0.5 hr; 354 mg/kg, 2.5, 4.5 hr + Reserpine, 2.5 mgikg, I.0 hr (4) Ro 4-4602, 50 mg/kg, 0 hr + 5-HTP, 400 mg/kg. @5 hr; 200 mg/kg. 2.5, 4.5 hr + Reserpine, 2,5,mg/kg, I.0 hr (5) Ro 4-4602. 50 mg/kg, 0 hr + 5-HTP, 400 mg/kg, 0.5 hr; 200 mg/kg, 2.5, 4.5 hr + L-DOPA. 473 mg/kg, @5 hr; 354 mg: kg. 2.5, 4.5 hr + Reserpine, 2.5 mg/kg, 1.0 hr
P < @Ol$ 25 (12)
62.5 (8)
P < 0.01
20.0 (IO)
NE
Brain amine concentrations+ /Lg/g f S.E. DA
0.27 (8) + 0.01 0.06 (8) ‘I 0.01
P < 0.001 0.1611 (10) * 0.02 P < 0.001
0.65 (8) + 0.08 0.09 (7) * 0.05 P < 0,001 047’1 (IO) i: 0.05
5-HT
P < 0.05
036 (8) + 0.03 0.15 (8) & 0.02 P < OGOI 0.21 (IO) * 0.03 P < 0,005
0.04 (8) i_ 0.01 P < 0.001
0.14 (8) k 0.06
0.69 (8) f 0.04
P < 0.001
P < 0.001
0.12)’ (9) k 0.02
0.4411 (9) f 0.06
0.67 (9) & 005
P < 0,001
P < ~05
of
P < 0.001
* Times of injection are reported as elapsed time from beginning of experiment (0 hr) to the time of injection. t All seizure values and brain amine levels were determined 25 hr after beginning of experiment (0 hr). $ Number of animals used in each determination is shown in parentheses. 8 Each tabulated P value indicates a significant difference from corresponding salme control value. Significantly different from corresponding reserpine value (Treatment No. 2) (P < 0.01).
protect brain NE and DA stores against the depleting actions of reserpine and that 5-HTP administered in an analogous fashion will protect against depletion of 5-HT stores. In the present investigation, we have studied the capacity of L-DOPA and 5-HTP administered in combination with a small dose of Ro 4-4602 to protect against reserpine-induced biogenie amine depletion and seizure enhancement (Table 2). Brain NE, DA, and 5-HT were strongly depleted and ES was markedly intensified 24 hr after reserpine treatment. However, in animals treated with Ro 4-4602, 5-HTP plus L-DOPA given 0.5 hr prior to and 2.5 and 4.5 hr.after reserpine, protected against reserpine-induced depletion of NE, DA and 5-HT and enhancement of ES. Similarly, L-DOPA given alone rather than in combination with 5-HTP, strongly inhibited the reserpine-induced depletion of brain NE and DA as well as the enhancement of ES but afforded no protection of 5-HT stores. In contrast, 5-HTP alone protected 5-HT stores against depletion by reserpine, yet it did not protect against ES enhancement and brain NE and DA depletion. lZfects
ofpimozide
and DEDTC
on the L-DOPA
plus iproniazid-Ro
4-1284
interaction
Ro 4- 1284 markedly enhanced seizure intensity and severely depleted brain NE and DA stores (Table 3). In contrast, L-DOPA administered in combination with iproniazid completely antagonized the seizure enhancement as well as NE and DA depletion. Neither pimozide nor DEDTC diminished the L-DOPA-iproniazid antagonism of Ro 4-l 284 seizure enhancement or brain DA depletion; however, DEDTC, but not pimozide, did prevent the antagonism of NE depletion. Combined treatment with DEDTC and pimozide com-
Brain amines and eiectroshock seizure
965
Table 3. Effects of pimozide and DEDTC on the L-DOPA- and ~proniazid-induced antagonism of seizure enhancement and brain NE and DA depletion produced by Rb 4- 12SJ
Treatment and times of injection*
(1) Saline, 0, 0.25, 0’75. 1.5. 2.5 hr (2) Saline, 0, 0.25, 0.75. 25 hr + Ro 4-1284, 10 mg&g. 1.5 hr (3) Saline, 0. 0.25 hr -t Iproniazid, 40 mg/kg. 0.75 hr + Ro 4-12X4. IO mg/kg, 15 hr -t L-DOPA, 250 mg/kg. 2.5 hr (4) Saline. 0 hr + Pimozide. 10 mg:kg, 0.25 hr + Iproniazid, 40 mgikg, 075 hr -t Ro 4-1284. 10 mg/kg, 1.5 hr (51 D~~,~~~~~~~~~~i~ 25 hr
Per cent tonic seizure? 0.0 (8): s75 (8) P < @ool~ 0.0 (8)
12.5 (8)
25.0 (8) + Saline, 0.25 hr + Iproniazid, 40 mgikg, @75 hr + Ro 4-1284, 10 mgikg. I.5 hr + L-DOPA. 250 mg!‘kg, 2.5 hr (6) DEDTC, 400 mg/kg. 0 hr -t Pimozide, 10 mg/kg. 0.25 hr + Iproniazid, 40 mgikg. 0.75 hr + Ro 4-1284. 10 mg/kg. 1.5 hr + L-DOPA, 250 mg/kg, 2.5 hr
Brain amine concentrationst ug:g f S.E. NE ‘-DA i” -.__ ,. _ 0.29 (6) Q93 (6) & 0.02 j, 0.13 0.06 (8) @05 (8) * DO1 + OG4 P < 0~001 P < 0.001 @26 (5) 489 (8) + 0.04 + 1.60 P < 0.05
18.53(7) f 5.49
0.55 (6) *o-Ii P < 0.05
P < 0.01
0.09 (7) * 0.01
1.99 (8) + O-53
P < O-001
750 (8) P < OQOI
0.08 (7) * 0.02 P < 0.001
I .84 (7) f 0.50
* Times of injection arc reported as elapsed time from beginning of experiment (0 hr) to the time of injection. t All seizure values and brain amine levels were determined 3 hr after beginning of experiment(0 hr). $ Number of animals used in each determination is indicated in parentheses. 5 Each tabulated P value indicates a significant difference from saline control value.
pletely prevented the L-DOPA-iproniazid antagonism of Ro 4-1284 enhancement ure intensity and the antagonism of NE but not DA depletion.
of seiz-
DISCUSSION
Depletion of brain NE, DA, and 5-HT in rats is associated with increased severity of electroshock seizure. In this regard, both reserpine and Ro 4- 1284 (drugs which interfere with storage of NE, DA, and 5-HT) resulted in marked seizure intensification. In contrast, pharmacologic treatments (Ro 4-1284 administered just prior to reserpine, or 5-HTP plus L-DOPA administered just prior to and shortly after reserpine) which protect against reserpine-induced depIetion of all three biogenic amines also protect against enhancement of seizure intensity. Since these treatments caused simultaneous changes in each of the biogenie amines, it is not possible on the basis of the above data to ascribe greater importance to one or the other of these substances as a modulator of electroshock seizure. However, other observations in the present study provide evidence that NE and DA are more important than 5-HT in the modulation process. For example, administration of L-DOPA just prior to and shortly after reserpine treatment, strongly inhibited the reserpine-i~lduced depletion of brain NE and DA as well as the enhancement of seizure intensity. In contrast, the L-DOPA treatment afforded no protection of 5-HT stores against reserpine action.
966
P. C. Jam,R.E.STULL and P. F. GIXZX
This observation indicates that depletion of both NE and DA may produce seizure enhancement, whereas depletion of 5-HT alone may be of no consequence. Additional support for this concept is provided by the observation that 5-HTP administered just prior to and shortly after reserpine treatment protected 5-HT stores against the depleting action of reserpine. yet it did not protect against seizure intensification and brain NE and DA depletion. Further evidence alsb favours NE and DA as important modulators in electroshock seizure. In this regard, the administration of L-DOPA (1 hr after Ro 4-1284) and iproniazid completely antagonized the Ro 4-1284-induced seizure enhancement as well as NE and DA depletion. Neither pimozide (a DA receptor blocking agent) nor DEDTC (a DA-Phydroxylase inhibitor) diminished the L-DOPA-iproniazid antagonism of Ro 4- 1284 seizure enhancement. yet DEDTC did prevent the antagonism of NE depletion and pimozide blocks DA receptors at the dosage used in the present experiment (ANIG:N, BUTCHEK. COKRODI, FUXE and UNGERSTEDT, 1970). This failure of pimozide or DEDTC to block the antagonizing effects of L-DOPA on seizure enhancement could be viewed as cvidence against an important modulating role of both NE and DA unless each of these two biogenic amines subserves the same inhibitory function in the central nervous system. Under these conditions either selective or concomitant replenishment of DA or NE activity by L-DOPA administration would conceivably antagonize seizure enhancement. Conversely. if L-DOPA simultaneously failed to restore DA and NE activity. it would also fail to antagonize Ro 4-1284-induced seizure enhancement. This possibility was studied in animals pretreated with both pimozide and DEDTC so that NE synthesis and DA receptors were simultaneously blocked. Under these conditions. the administration of L-DOPA failed to antagonize Ro 4-1284-induced seizure enhancement. Our hypothesis regarding the inhibitory influence of NE and DA in electroshock seizure in the rat agrees with the concepts advanced by a number of investigators who used other experimental approaches and/or animal species (BILLIET, BERNARI), DELALJNOISand DE SCHAIPDRYVER, 1970a,b:SCHLESINWR, BOGCAN and GRIEK, 1968; STULL et ~1..1973). However, it is in partial disagreement with the findings of KILIAN and FREY (1973) who reported that NE, but not DA, is of importance in electroshock seizure activity in rats and mice. Their conclusions regarding rats were derived from two types of experiments. In the first type of experiment they observed that simultaneous depletion of NE and DA or that selective depletion of NE alone was associated with a decreased electroshock seizure threshold. In our view, this data only indicates that depletion of both NE and DA or depletion of NE alone will cause seizure enhancement. Whether or not depletion of DA alone will produce seizure enhancement remains unanswered by their data. In the second type of experiment. they observed that haloperidol, which causes selective blockade of DA receptors (ANDY& et al., 1970), failed to lower electroshock seizure threshold in mice. Although KILIAN and FREY (1973) interpreted these data as militating against a modulator role for DA in rats. we believe that such an interspecies extrapolation of data is questionable. Indeed, MCKENZIE and SOROKO (1972) have presented evidence which suggests that enhanced DA receptor activity modulates maximal electroshock seizure in the rat but not in the mouse. The failure of our data to support a role for 5-HT in electroshock seizure in the rat agrees with observations of RUIIZIK and JOHNSON (1970). However, our results are in contrast to those reported by KILIAN and FREY (1973). The findings of these investigators. which according to their own interpretation favoured a role for 5-HT in electroshock con-
Brain amines and electroshock
seizure
961
vulsions in rats do not, in our opinion, differentiate betwten the relative significance of this brain amine and NE. In this regard, they found that p-chlorophenylalanine administered 48 hr prior to testing, significantly decreased electroshock seizure threshold; however, rather than lowering only brain 5-HT stores, NE levels were also reduced. Consequently, the possibility remains that p-chlorophenylalanine-induced seizure enhancement could be related to the effects of this drug on NE rather than on 5-HT metabolism. In conclusion, NE and DA appear to be the amines most directly involved in regulating electroshock seizure intensity. Our data do not support the concept that 5-HT plays a significant role in this activity. We are currently studying the effects of 6-hydroxydopamine and 5,6_dihydroxytryptamine, agents which produce selective degeneration of catecholamine and 5-HT neurones, respectively, on experimentally-induced seizure. Preliminary evidence in each of these investigations supports our hypothesis. Ackrlowledyrnlr,nts~This work was supported by a Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation, U.S.A. The authors are grateful to PAUL A. J. JANSSIZNof Janssen Pharmaceutics (Beerse. Belgium) for the pimozide; to Dr. W. E. SCOTT of Roche Laboratories (Nutley, New Jersey, U.S.A.) for the Ro 4-1284. Ro 4-4602 and iproniazid; and to Dr. ALBERT J. PLUMMER of Ciba Pharmaceutical Company (Summit. New Jersey, U.S.A.) for the reserpine.
REFERENCES AND~N. N. E., BUTCHER, S. G., CORRODI, H., FUXE, K. and UNGERSTEDT, U. (1970). Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. Eur. J. Pharmac. 11: 303-3 14. BILLIET, M., BERNARD, P., DELAUNOIS, A. and DE SCHAEPDRYVER,A. (1970a). Electroshock and caudate nucleus dopamine. Archs int. Pharmacodyn. Thir. 186: 179-181. BILLIET, M., BERNARD, P., DELAUNOIS,A. and DE SCHAEPDRYVER,A. (1970b). Induced changes in caudate nucleus dopamine and electroshock threshold. Archs int. Phar?nacodyn. Thhr. 188: 39&400. BOGIIANSKI. D. F., PLETSCHER,A., BR~DIE, B. B. and UDENFRIEND, S. (1956). Identification and assay of serotonin in brain. J. Pharmac. esp. T&r. 117: 82-88. BUTCHER, L. L., RHOIIES, D. L. and YUWILER, A. (1972). Behavioral and biochemical effects of preferentially protecting monoamines in the brain against the action of reserpine. Eur. J. Pharmac. 18: 204-212. CARLSSON, A. (1967). Selective protection of 5-hydroxytryptamine stores against the action of reserpine by treatment with 5-hydroxytryptophan. J. Pharm. Pharmac. 19: 783-784. CARLSSON, A. and LINDQVIST,M. (1966). The interference of tetrabenazine, benzquinamide and prenylamine with the action of reserpine. Acta pharmac. tox. 24: 112-120. CARLSSON, A. and WALDECK, B. (1958). A fluorometric method for the determination of dopamine (3-hydroxytyramine). Acta physiol. stand. 44: 293-298. Dt LA TORRE. J. C., KAWANAGA, H. M. and MULLAN, S. (1970). Seizure susceptibility after manipulation of brain serotonin. Archs int. Pharmacodyn. T/l&. 188: 298-304. Jour. P. C., PICCHIONI, A. L. and CHIN. L. (1973a). Role of brain norepmephrine in audiogenic seizure in the rat. J. P/tarmac. cup. Thtir. 184: l-10. JOBE, P. C., PICCHIONI, A. L., and CHIN, L. (197313). Role of brain 5-hydroxytryptamine in audiogenic seizure in the rat. Life Sci. 13: I-13. JOBS, P. C., PICCHIONI, A. L. and CHIN, L. (1973~). Effect of lithium carbonate and cc-methyl-p-tyrosine (r-MPT) on audiogenic seizure intensity. J. Pharm. Pharmac. 25: 83Ck83 1, KILIAN, M. and FREY, H. H. (1973). Central monoamines and convulsive thresholds in mice and rats. Neuropharmacolog~, 12: 6X1-692. KOSLOW, S. H. and ROTH, L. J. (1971). Reserpine and acetazolamide in maximum electroshock seizure in the rat. J. Pharmac. cup. Thdr. 176: 71 l-71 7. McKruzl~, G. M. and SOROKO, F. E. (1972). The effects of apomorphine. (+)-amphetamine and L-DOPA on maximal electroshock convulsions-a comparative study in the rat and mouse. J. Pharm. Pharmac. 24: 696 701. PITCHIONI. A. L., CHIN, L. and BREITNER, C. (1962). Relationship between brain levels of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) and susceptibility to electrically-induced seizures. Fed11 Proc. Fedn Am. Sots exp. Biol. 21: 416. PROCKOP, D. J., SHORE, P. A. and BRODIE, B. B. (1959). Anticonvulsant properties of monoamine oxidase inhibitors. Anr7.N.Y Acad. Sci. 80: 643-651.
968 RUDZIK, A. D. and
P. C. Jon~, R. E. STULL and P. F. GEIGER
JOHNSON,G. A. (1970). Effect of amphetamine and amphetamine analogs on convulsive thresholds. In: Amphetamines and Related Compounds. (COSTA, E. and GARATTINI, S.. Eds.), pp. 715-728, Raven Press, New York. SCHLESINGER, K., BOGGAN, W. 0. and GRIEK, B. J. (1968). Pharmacognetic correlates of pentylenetetrarol and electroconvulsive seizure thresholds in mice. Psychophar,naco/ogiL 13: 18 l&188. SHORE, P. A. and OLIN, 1. S. (1958). Identification and chemical assay of norepinephrine in brain and other tissues. J. Pharmac. exp. ThPr. 122: 295300. STULL, R. E., JOBE. P. C., GEIGER, P. F. and FERGUSON, G. G. (1973). Effects of dopamine receptor stimulation and blockade on Ro 4-1284-induced enhancement of electroshock seizure. J. Pharm. Pharmuc. 25: 842-844. SWINYARD, E. A. (1963). Some physiological properties of audiogenic seizures in mice and their alteration by drugs. In: Psychophysiologie, Neuropharwzacologie, et Biochimie de la Crisr Audiogew. pp. 405-421. Editions du Centre National de la Recherche Scientifique, Paris. WIEGAND, R. G. and PERRY, J. E. (1961). Effect of L-DOPA and N-methyl->V-benzyl-2-propynylamine . HCI on DOPA, dopamine, norepinephrine, epinephrine, and serotonin levels in mouse brain. b’I’och~‘~xPhcr~~ac. 7: 181-186. WOOD~JRY, D. M. and ESPLIN. D. W. (1959). Neuropharmacology and neurochemistry of anticonvulsant drugs. Proc. Ass. Res. nerc. mem. Dis. 37: 24-56.
of Pharmacology and Toxicology. College of Pharmacy and Allied Health Northeast Louisiana University, Monroe, Louisiana 71201 (AcWpted
Professions,
IO May 1974)
Summary-The effects,of various treatments on brain norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) were compared to the effects of these same treatments on electroshock seizure (ES) intensity. The data indicate that depletion of all three biogenic amines by reserpine or the benzoquinolizme Ro 4-1284, enhances ES intensity and that treatments which protect against depletion of NE and/or DA antagonize ES enhancement. However, protection of 5-HT stores against depletion does not protect against ES enhancement, Additional studies showed that, following depletion of all three amines by Ro 4-1284, subsequent repletion of NE and DA singly or in combination is sufficient to antagonize Ro 4-1284-induced ES enhancement. However, repletion of DA stores alone did not antagonize Ro 4-1284 seizure enhancement in animals treated with pimozide, a central DA receptor blocking agent. Thus. it appears that both NE and DA exert central nervous system inhibitory effects which limit the spread of the electroshock seizure discharge and consequently regulate intensity of ES. The data indicate that 5-HT does not play a significant role in the modulation process.
Recently, JOBE. PICCHIOYI and CHIN (1973a,b,c) reported that, following various combination treatments which alter the synthesis, storage, and release of catecholamines and/or 5-hydroxytryptamine (5-HT) in brain tissue, there is a consistent correlation in rats between increased audiogenic seizure severity and drastically reduced levels of norepinephrine (NE) and 5-HT, but not of dopamine (DA). Based on the data presented, it was proposed that endogenous NE and 5-HT play an attenuating role in audiogenic seizure. The data strongly indicated that of the three biogenic amines studied, NE and 5-HT are more important than DA in modulating audiogenic seizure intensity in rats. Other investigators have indicated that, in the rat, NE. DA and/or 5-HT may also subserve a similar modulating function in electroshock seizure (PROCKOP, SHORE and BRODIE, 1959; RUDZIK and JOHNSON, 1970; KILIAN and FREY, 1973; STULL, JOBE, GEIGER and FERGUSON, 1973). However, from these studies it is not clear which of the amines are most directly involved in electroshock seizure processes. Furthermore, certain evidence fails to support a role for any of these biogenic amines (PICCHIONI, CHIN and BREITNER, 1962; KOSLOW and ROTH, 1971). The present study was conducted to obtain additional evidence concerning the possible functional significance of endogenous NE, DA and 5-HT in electroshock seizure. Various * The work was presented in part at the 58th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey, April 1974. t Present address: Department of Pharmacology and Therapeutics, School of Medicine in Shreveport, Louisiana State University Medical Center, Shreveport, Louisiana 71101. : Present address: Department of Pharmacology. School of Medicine, University of California, San Francisco, California 94143. 961
962
P. C.
JOBE. R. E. STULL
and
P. F. GEIGER
procedures were employed to modify brain catecholamines and .S-HT and the effects of these treatments on seizure intensity were observed. Since some of these procedures selectively modified the brain concentrations of either the catecholamines or 5-HT or selectively blocked dopamine receptors, it was possible to study the relative significance of all three biogenic amines as modulators of electroshock seizure activity. Brain concentrations of the biogenic amines, determined by spectrophotofluorometric analysis, served as indications of neuronal availability of these amines. METHODS
Experimental animals and treatments Female Sprague-Dawley rats (I 5&200 g) were used throughout this investigation. All drugs were administered as aqueous solutions with the exception of L,-3,4-dihydroxyphenylalanine (L-DOPA), 5-hydroxy-D,L-tryptophan (%HTP) and pimozide which were injected intraperitoneally as suspensions in normal saline. Ro 4-1284 (2-ethyl-1,2,3,4,6,7-hexahydro-2-hydroxy-3-isobutyl-9, IO-dimethoxy-1 lbH-benzoquinolizine hydrochloride) was injected subcutaneously; Ro 4-4602 [IN-(DL-seryl-N’-2,3,4_trihydroxybenzyl) hydrazine hydrochloride], reserpine phosphate, iproniazid phosphate and diethyldithiocarbamate sodium (DEDTC) were injected intraperitoneally. Sources of drugs were as follows: L-DOPA, Regis Chemical Co. ; 5-HTP, Sigma Chemical Corp.; Pimozide, Janssen Pharmaceutics; Ro 4-1284, Ro 4-4602, Iproniazid, Roche Laboratories; DEDTC, Fisher Scientific Co. The dosage regimens, test times and sacrifice times used in the various experiments are indicated in the tables. In each set of experiments, test animals which were treated with a single drug were administered supplementary injections of saline by appropriate routes and time schedules such that they were handled and treated in the same manner as corresponding test animals which were administered a combination of drugs. Similarly, control animals were injected with saline and manipulated in a manner comparable to the corresponding test animals which received a combination of drugs. The number of animals used in the calculation of reported test values is indicated in the tables. Electroshock procedure Elecktroshock seizures were induced by stimulation of each animal with 26 mA for O-2 set through cornea1 electrodes. This procedure routinely produced clonic seizures in control (saline-treated) animals. The electroshock seizure pattern (either clonic or tonic) in rats was used to test the effects of selected drugs. Since the tonic pattern is more intense than the clonic pattern (WOODBLTRY and ESPLIN, 19.59; SWINYARD,1963), drugs which increase the incidence of tonus obviously enhance seizure intensity, whereas drugs which decrease the incidence of tonus, diminish seizure intensity. Similar methods of assessing seizure intensity have been used by PROCKOPet al. (I 959) and DE LA TORRE,KAWANAGAand M~~LLAN(1970). Spectrophotq~uoPo,net~ir analysis Catecholamines and 5-HT were extracted from whole brain by the method of WIEGAN~ and PERRY (1961). 5-Hydroxytryptamine was assayed by the method of BOGDANSKI, PLETSCHER,BRODIEand UDENFRIEND(1956); NE and DA were assayed by the methods of SHOREand OLIN ( 1958) and CARLSSON and WALDECK(1958), respectively.
Brain amines and electroshock
Statistical
seizure
963
procedure
The statistical significance of the differences between per cent tonus values were determined by the Chi Square Test. Significant differences between mean whole brain NE, DA, and 5-HT values were determined by Student’s t test. RESULTS
effects
qf‘reserpine
in Ro 4-1284 pretreated
animals
CARLSSON and LINDQVIST (1966) have reported that tetrabenazine interferes with the amine depicting effects of reserpine. In the present investigation. we have studied the capacity of Ro 4-1284 (a benzoquinolizine closely related to tetrabenazine) to interfere with reserpine-induced biogenic amine depletion and seizure enhancement (Table 1). Reserpine alone markedly enhanced seizure intensity and strongly diminished brain NE, DA, and 5-HT levels. When Ro 4-1284 was administered 0.5 hr prior to reserpine, it protected against the reserpine-induced seizure enhancement and brain NE, DA, and 5-HT depletion. In this experiment, Ro 4- 1284 alone had no effect on seizure intensity or brain amine levels; however, this lack of effect when administered alone is not in conflict with its seizure enhancing and brain amine depleting actions which will be described subsequently in this paper and which have been previously reported (JOBE rt a/., 1973a,h; STULL et al., 1973). Table
I. Protecting effects of Ro 4-1284 pretreatment against reserpine-induced enhancement of electroshock seizure intensity and depletion of brain norepinephrine. dopamine and 5-hydroxytryptamine
Treatmenl
and times of injection*
(1) Saline, 0. 0.5 hr
23 (22):
(2) Ro J- 1284. IO mg/kg. 0 hr
9 (22)
+ Saline. 0.5 hr (3) Saline, 0 hr + Reserpine. 2.5 mgikg. 0.5 hr (4) Ro 4-1284. IO mgjkg. 0 hr + Rcscrpinc. 2.5 mgikg. 0.5 hr
Brain amine concentrationst /cg,!g i S.E. NE DA
Per cent tonic seiruret
100 (24) P < 0~0005i; 35 (20)
0.24 (8) ‘r 0.01 0.23 (12) +0.01 0.03 (8) *0,01 P < 0.001 0.15 (12) *010 P < PO25
0.75 (X) + 0.04 064 (12) f 0.06 0.17 (8) * 0.05 P < 0.001 0.52” ( 12) + wJ7 P < 0.01
5-HT 0.31 (8) + 0.02 0.32 (12) io.01 0.17 (8) f 0.02 P < 0.001 0.77 (12) * 0.02
_ * Times of injection are reported as elapsed time from beginning of experiment (0 hr) to the time of injection. i- A.lI scizurc values and brain amine levels were determined 24.5 hr after beginning of experiment (0 hr). $ Number of animals used in each determination is indicated in parentheses. 6 Each tabulated P value indicates a significant difference from saline control value. 1Significantly different from corresponding reserpine value (Treatment No. 3) (P < 0.005).
In these instances, where Ro 4-1284 causes effects on seizure and amine levels. experimental determinations were made within 0.75-4 hr postadministration, whereas, in the present experiment, determinations were made 24.5 hr postadministration, a time period when the effects of Ro 4-12X4 have completely dissipated (JOBE et al., 1973a). EfJcts
qf L-DOPA,
5-HTP
and Ro 4-4602 in reserpine
treated animals
CARLSSON (1967) and BUTCHER, RHODES and YUWILER (1972) have reported that in animals pretreated with Ro 4-4602 (in a small dose which inhibits only peripheral aromatic amino acid decarboxylase), L-DOPA, administered shortly before and after reserpine. will
964
P. C. JOBE. R. E. STULL and P. F. GEIGER
Table 2. Protecting effects of L-DOPA, 5-HTP and Ro 4-4602 against two reserpine actions: (I) depletion brain NE. DA. and 5.HT levels; and (2) the enhancement of electroshock seizure intensity
Treatment
and times of injection*
(1) Saline. 0. 0.5, 1.0, 2.5~ 4.5 hr (2) Saline, 0. 0.5. 2.5. 4.5 hr + Reserpine, 2.5 mg/kg,
Per cent tonic seizure?
0 @)I 62.5 (8) 1.0 hr
(3) Ro 4-4602, 50 mg/kg, 0 hr + L-DOPA, 473 mgjkg, 0.5 hr; 354 mg/kg, 2.5, 4.5 hr + Reserpine, 2.5 mgikg, I.0 hr (4) Ro 4-4602, 50 mg/kg, 0 hr + 5-HTP, 400 mg/kg. @5 hr; 200 mg/kg. 2.5, 4.5 hr + Reserpine, 2,5,mg/kg, I.0 hr (5) Ro 4-4602. 50 mg/kg, 0 hr + 5-HTP, 400 mg/kg, 0.5 hr; 200 mg/kg, 2.5, 4.5 hr + L-DOPA. 473 mg/kg, @5 hr; 354 mg: kg. 2.5, 4.5 hr + Reserpine, 2.5 mg/kg, 1.0 hr
P < @Ol$ 25 (12)
62.5 (8)
P < 0.01
20.0 (IO)
NE
Brain amine concentrations+ /Lg/g f S.E. DA
0.27 (8) + 0.01 0.06 (8) ‘I 0.01
P < 0.001 0.1611 (10) * 0.02 P < 0.001
0.65 (8) + 0.08 0.09 (7) * 0.05 P < 0,001 047’1 (IO) i: 0.05
5-HT
P < 0.05
036 (8) + 0.03 0.15 (8) & 0.02 P < OGOI 0.21 (IO) * 0.03 P < 0,005
0.04 (8) i_ 0.01 P < 0.001
0.14 (8) k 0.06
0.69 (8) f 0.04
P < 0.001
P < 0.001
0.12)’ (9) k 0.02
0.4411 (9) f 0.06
0.67 (9) & 005
P < 0,001
P < ~05
of
P < 0.001
* Times of injection are reported as elapsed time from beginning of experiment (0 hr) to the time of injection. t All seizure values and brain amine levels were determined 25 hr after beginning of experiment (0 hr). $ Number of animals used in each determination is shown in parentheses. 8 Each tabulated P value indicates a significant difference from corresponding salme control value. Significantly different from corresponding reserpine value (Treatment No. 2) (P < 0.01).
protect brain NE and DA stores against the depleting actions of reserpine and that 5-HTP administered in an analogous fashion will protect against depletion of 5-HT stores. In the present investigation, we have studied the capacity of L-DOPA and 5-HTP administered in combination with a small dose of Ro 4-4602 to protect against reserpine-induced biogenie amine depletion and seizure enhancement (Table 2). Brain NE, DA, and 5-HT were strongly depleted and ES was markedly intensified 24 hr after reserpine treatment. However, in animals treated with Ro 4-4602, 5-HTP plus L-DOPA given 0.5 hr prior to and 2.5 and 4.5 hr.after reserpine, protected against reserpine-induced depletion of NE, DA and 5-HT and enhancement of ES. Similarly, L-DOPA given alone rather than in combination with 5-HTP, strongly inhibited the reserpine-induced depletion of brain NE and DA as well as the enhancement of ES but afforded no protection of 5-HT stores. In contrast, 5-HTP alone protected 5-HT stores against depletion by reserpine, yet it did not protect against ES enhancement and brain NE and DA depletion. lZfects
ofpimozide
and DEDTC
on the L-DOPA
plus iproniazid-Ro
4-1284
interaction
Ro 4- 1284 markedly enhanced seizure intensity and severely depleted brain NE and DA stores (Table 3). In contrast, L-DOPA administered in combination with iproniazid completely antagonized the seizure enhancement as well as NE and DA depletion. Neither pimozide nor DEDTC diminished the L-DOPA-iproniazid antagonism of Ro 4-l 284 seizure enhancement or brain DA depletion; however, DEDTC, but not pimozide, did prevent the antagonism of NE depletion. Combined treatment with DEDTC and pimozide com-
Brain amines and eiectroshock seizure
965
Table 3. Effects of pimozide and DEDTC on the L-DOPA- and ~proniazid-induced antagonism of seizure enhancement and brain NE and DA depletion produced by Rb 4- 12SJ
Treatment and times of injection*
(1) Saline, 0, 0.25, 0’75. 1.5. 2.5 hr (2) Saline, 0, 0.25, 0.75. 25 hr + Ro 4-1284, 10 mg&g. 1.5 hr (3) Saline, 0. 0.25 hr -t Iproniazid, 40 mg/kg. 0.75 hr + Ro 4-12X4. IO mg/kg, 15 hr -t L-DOPA, 250 mg/kg. 2.5 hr (4) Saline. 0 hr + Pimozide. 10 mg:kg, 0.25 hr + Iproniazid, 40 mgikg, 075 hr -t Ro 4-1284. 10 mg/kg, 1.5 hr (51 D~~,~~~~~~~~~~i~ 25 hr
Per cent tonic seizure? 0.0 (8): s75 (8) P < @ool~ 0.0 (8)
12.5 (8)
25.0 (8) + Saline, 0.25 hr + Iproniazid, 40 mgikg, @75 hr + Ro 4-1284, 10 mgikg. I.5 hr + L-DOPA. 250 mg!‘kg, 2.5 hr (6) DEDTC, 400 mg/kg. 0 hr -t Pimozide, 10 mg/kg. 0.25 hr + Iproniazid, 40 mgikg. 0.75 hr + Ro 4-1284. 10 mg/kg. 1.5 hr + L-DOPA, 250 mg/kg, 2.5 hr
Brain amine concentrationst ug:g f S.E. NE ‘-DA i” -.__ ,. _ 0.29 (6) Q93 (6) & 0.02 j, 0.13 0.06 (8) @05 (8) * DO1 + OG4 P < 0~001 P < 0.001 @26 (5) 489 (8) + 0.04 + 1.60 P < 0.05
18.53(7) f 5.49
0.55 (6) *o-Ii P < 0.05
P < 0.01
0.09 (7) * 0.01
1.99 (8) + O-53
P < O-001
750 (8) P < OQOI
0.08 (7) * 0.02 P < 0.001
I .84 (7) f 0.50
* Times of injection arc reported as elapsed time from beginning of experiment (0 hr) to the time of injection. t All seizure values and brain amine levels were determined 3 hr after beginning of experiment(0 hr). $ Number of animals used in each determination is indicated in parentheses. 5 Each tabulated P value indicates a significant difference from saline control value.
pletely prevented the L-DOPA-iproniazid antagonism of Ro 4-1284 enhancement ure intensity and the antagonism of NE but not DA depletion.
of seiz-
DISCUSSION
Depletion of brain NE, DA, and 5-HT in rats is associated with increased severity of electroshock seizure. In this regard, both reserpine and Ro 4- 1284 (drugs which interfere with storage of NE, DA, and 5-HT) resulted in marked seizure intensification. In contrast, pharmacologic treatments (Ro 4-1284 administered just prior to reserpine, or 5-HTP plus L-DOPA administered just prior to and shortly after reserpine) which protect against reserpine-induced depIetion of all three biogenic amines also protect against enhancement of seizure intensity. Since these treatments caused simultaneous changes in each of the biogenie amines, it is not possible on the basis of the above data to ascribe greater importance to one or the other of these substances as a modulator of electroshock seizure. However, other observations in the present study provide evidence that NE and DA are more important than 5-HT in the modulation process. For example, administration of L-DOPA just prior to and shortly after reserpine treatment, strongly inhibited the reserpine-i~lduced depletion of brain NE and DA as well as the enhancement of seizure intensity. In contrast, the L-DOPA treatment afforded no protection of 5-HT stores against reserpine action.
966
P. C. Jam,R.E.STULL and P. F. GIXZX
This observation indicates that depletion of both NE and DA may produce seizure enhancement, whereas depletion of 5-HT alone may be of no consequence. Additional support for this concept is provided by the observation that 5-HTP administered just prior to and shortly after reserpine treatment protected 5-HT stores against the depleting action of reserpine. yet it did not protect against seizure intensification and brain NE and DA depletion. Further evidence alsb favours NE and DA as important modulators in electroshock seizure. In this regard, the administration of L-DOPA (1 hr after Ro 4-1284) and iproniazid completely antagonized the Ro 4-1284-induced seizure enhancement as well as NE and DA depletion. Neither pimozide (a DA receptor blocking agent) nor DEDTC (a DA-Phydroxylase inhibitor) diminished the L-DOPA-iproniazid antagonism of Ro 4- 1284 seizure enhancement. yet DEDTC did prevent the antagonism of NE depletion and pimozide blocks DA receptors at the dosage used in the present experiment (ANIG:N, BUTCHEK. COKRODI, FUXE and UNGERSTEDT, 1970). This failure of pimozide or DEDTC to block the antagonizing effects of L-DOPA on seizure enhancement could be viewed as cvidence against an important modulating role of both NE and DA unless each of these two biogenic amines subserves the same inhibitory function in the central nervous system. Under these conditions either selective or concomitant replenishment of DA or NE activity by L-DOPA administration would conceivably antagonize seizure enhancement. Conversely. if L-DOPA simultaneously failed to restore DA and NE activity. it would also fail to antagonize Ro 4-1284-induced seizure enhancement. This possibility was studied in animals pretreated with both pimozide and DEDTC so that NE synthesis and DA receptors were simultaneously blocked. Under these conditions. the administration of L-DOPA failed to antagonize Ro 4-1284-induced seizure enhancement. Our hypothesis regarding the inhibitory influence of NE and DA in electroshock seizure in the rat agrees with the concepts advanced by a number of investigators who used other experimental approaches and/or animal species (BILLIET, BERNARI), DELALJNOISand DE SCHAIPDRYVER, 1970a,b:SCHLESINWR, BOGCAN and GRIEK, 1968; STULL et ~1..1973). However, it is in partial disagreement with the findings of KILIAN and FREY (1973) who reported that NE, but not DA, is of importance in electroshock seizure activity in rats and mice. Their conclusions regarding rats were derived from two types of experiments. In the first type of experiment they observed that simultaneous depletion of NE and DA or that selective depletion of NE alone was associated with a decreased electroshock seizure threshold. In our view, this data only indicates that depletion of both NE and DA or depletion of NE alone will cause seizure enhancement. Whether or not depletion of DA alone will produce seizure enhancement remains unanswered by their data. In the second type of experiment. they observed that haloperidol, which causes selective blockade of DA receptors (ANDY& et al., 1970), failed to lower electroshock seizure threshold in mice. Although KILIAN and FREY (1973) interpreted these data as militating against a modulator role for DA in rats. we believe that such an interspecies extrapolation of data is questionable. Indeed, MCKENZIE and SOROKO (1972) have presented evidence which suggests that enhanced DA receptor activity modulates maximal electroshock seizure in the rat but not in the mouse. The failure of our data to support a role for 5-HT in electroshock seizure in the rat agrees with observations of RUIIZIK and JOHNSON (1970). However, our results are in contrast to those reported by KILIAN and FREY (1973). The findings of these investigators. which according to their own interpretation favoured a role for 5-HT in electroshock con-
Brain amines and electroshock
seizure
961
vulsions in rats do not, in our opinion, differentiate betwten the relative significance of this brain amine and NE. In this regard, they found that p-chlorophenylalanine administered 48 hr prior to testing, significantly decreased electroshock seizure threshold; however, rather than lowering only brain 5-HT stores, NE levels were also reduced. Consequently, the possibility remains that p-chlorophenylalanine-induced seizure enhancement could be related to the effects of this drug on NE rather than on 5-HT metabolism. In conclusion, NE and DA appear to be the amines most directly involved in regulating electroshock seizure intensity. Our data do not support the concept that 5-HT plays a significant role in this activity. We are currently studying the effects of 6-hydroxydopamine and 5,6_dihydroxytryptamine, agents which produce selective degeneration of catecholamine and 5-HT neurones, respectively, on experimentally-induced seizure. Preliminary evidence in each of these investigations supports our hypothesis. Ackrlowledyrnlr,nts~This work was supported by a Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation, U.S.A. The authors are grateful to PAUL A. J. JANSSIZNof Janssen Pharmaceutics (Beerse. Belgium) for the pimozide; to Dr. W. E. SCOTT of Roche Laboratories (Nutley, New Jersey, U.S.A.) for the Ro 4-1284. Ro 4-4602 and iproniazid; and to Dr. ALBERT J. PLUMMER of Ciba Pharmaceutical Company (Summit. New Jersey, U.S.A.) for the reserpine.
REFERENCES AND~N. N. E., BUTCHER, S. G., CORRODI, H., FUXE, K. and UNGERSTEDT, U. (1970). Receptor activity and turnover of dopamine and noradrenaline after neuroleptics. Eur. J. Pharmac. 11: 303-3 14. BILLIET, M., BERNARD, P., DELAUNOIS, A. and DE SCHAEPDRYVER,A. (1970a). Electroshock and caudate nucleus dopamine. Archs int. Pharmacodyn. Thir. 186: 179-181. BILLIET, M., BERNARD, P., DELAUNOIS,A. and DE SCHAEPDRYVER,A. (1970b). Induced changes in caudate nucleus dopamine and electroshock threshold. Archs int. Phar?nacodyn. Thhr. 188: 39&400. BOGIIANSKI. D. F., PLETSCHER,A., BR~DIE, B. B. and UDENFRIEND, S. (1956). Identification and assay of serotonin in brain. J. Pharmac. esp. T&r. 117: 82-88. BUTCHER, L. L., RHOIIES, D. L. and YUWILER, A. (1972). Behavioral and biochemical effects of preferentially protecting monoamines in the brain against the action of reserpine. Eur. J. Pharmac. 18: 204-212. CARLSSON, A. (1967). Selective protection of 5-hydroxytryptamine stores against the action of reserpine by treatment with 5-hydroxytryptophan. J. Pharm. Pharmac. 19: 783-784. CARLSSON, A. and LINDQVIST,M. (1966). The interference of tetrabenazine, benzquinamide and prenylamine with the action of reserpine. Acta pharmac. tox. 24: 112-120. CARLSSON, A. and WALDECK, B. (1958). A fluorometric method for the determination of dopamine (3-hydroxytyramine). Acta physiol. stand. 44: 293-298. Dt LA TORRE. J. C., KAWANAGA, H. M. and MULLAN, S. (1970). Seizure susceptibility after manipulation of brain serotonin. Archs int. Pharmacodyn. T/l&. 188: 298-304. Jour. P. C., PICCHIONI, A. L. and CHIN. L. (1973a). Role of brain norepmephrine in audiogenic seizure in the rat. J. P/tarmac. cup. Thtir. 184: l-10. JOBE, P. C., PICCHIONI, A. L., and CHIN, L. (197313). Role of brain 5-hydroxytryptamine in audiogenic seizure in the rat. Life Sci. 13: I-13. JOBS, P. C., PICCHIONI, A. L. and CHIN, L. (1973~). Effect of lithium carbonate and cc-methyl-p-tyrosine (r-MPT) on audiogenic seizure intensity. J. Pharm. Pharmac. 25: 83Ck83 1, KILIAN, M. and FREY, H. H. (1973). Central monoamines and convulsive thresholds in mice and rats. Neuropharmacolog~, 12: 6X1-692. KOSLOW, S. H. and ROTH, L. J. (1971). Reserpine and acetazolamide in maximum electroshock seizure in the rat. J. Pharmac. cup. Thdr. 176: 71 l-71 7. McKruzl~, G. M. and SOROKO, F. E. (1972). The effects of apomorphine. (+)-amphetamine and L-DOPA on maximal electroshock convulsions-a comparative study in the rat and mouse. J. Pharm. Pharmac. 24: 696 701. PITCHIONI. A. L., CHIN, L. and BREITNER, C. (1962). Relationship between brain levels of 5-hydroxytryptamine (5-HT) and norepinephrine (NE) and susceptibility to electrically-induced seizures. Fed11 Proc. Fedn Am. Sots exp. Biol. 21: 416. PROCKOP, D. J., SHORE, P. A. and BRODIE, B. B. (1959). Anticonvulsant properties of monoamine oxidase inhibitors. Anr7.N.Y Acad. Sci. 80: 643-651.
968 RUDZIK, A. D. and
P. C. Jon~, R. E. STULL and P. F. GEIGER
JOHNSON,G. A. (1970). Effect of amphetamine and amphetamine analogs on convulsive thresholds. In: Amphetamines and Related Compounds. (COSTA, E. and GARATTINI, S.. Eds.), pp. 715-728, Raven Press, New York. SCHLESINGER, K., BOGGAN, W. 0. and GRIEK, B. J. (1968). Pharmacognetic correlates of pentylenetetrarol and electroconvulsive seizure thresholds in mice. Psychophar,naco/ogiL 13: 18 l&188. SHORE, P. A. and OLIN, 1. S. (1958). Identification and chemical assay of norepinephrine in brain and other tissues. J. Pharmac. exp. ThPr. 122: 295300. STULL, R. E., JOBE. P. C., GEIGER, P. F. and FERGUSON, G. G. (1973). Effects of dopamine receptor stimulation and blockade on Ro 4-1284-induced enhancement of electroshock seizure. J. Pharm. Pharmuc. 25: 842-844. SWINYARD, E. A. (1963). Some physiological properties of audiogenic seizures in mice and their alteration by drugs. In: Psychophysiologie, Neuropharwzacologie, et Biochimie de la Crisr Audiogew. pp. 405-421. Editions du Centre National de la Recherche Scientifique, Paris. WIEGAND, R. G. and PERRY, J. E. (1961). Effect of L-DOPA and N-methyl->V-benzyl-2-propynylamine . HCI on DOPA, dopamine, norepinephrine, epinephrine, and serotonin levels in mouse brain. b’I’och~‘~xPhcr~~ac. 7: 181-186. WOOD~JRY, D. M. and ESPLIN. D. W. (1959). Neuropharmacology and neurochemistry of anticonvulsant drugs. Proc. Ass. Res. nerc. mem. Dis. 37: 24-56.