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Effects of baclofen on different dopaminergic neuronal systems
GABA Nrrrrr,rrun.\mi.\sion Bruin Rewarch BuMir~,
Vol. 5,
Suppl.
2, pp. 531-535.
Printed
in the
U.S.A
Effects of Baclofen on Different Dopaminergic Neuronal Systems’ K. E. MOORE Department
of Pharmacology
AND K. T. DEMAREST’
and Toxicology,
Michigan
State
UniLversity,
East Lrrnsing,
MI 48824
MOORE, K. E. AND K. T. DEMAREST. Ejj&rs of’hr~clqfin o,z d#c>rrr~t doprrminergic nclrrroncrl .sy.st~m.s. BRAIN RES. BULL. 5: Suppl. 2, 531-53.5, 1980.--Systemic administration of d/ or I-baclofen causes a dose-related increase in the concentration of dopamine (DA), but not of norepinephrine, in the forebrains of mice and rats. Microinjections of baclofen or GABA into substantia nigra increase DA in striatum. Since only the effect of GABA is reversed by picrotoxin, it would appear that baclofen does not act through a GABAergic mechanism. Baclofen-induced changes in DA concentrations are believed due to the depression of neuronal impulse flow which results in a decreased release of DA and thus a concomitant decrease in putative autoreceptor activation. Activation of DA autoreceptors appears to inhibit the synthesis and release of DA. Baclofen increases the concentration and the rate of synthesis of DA (accumulation of DOPA after inhibition of decarboxylase with NSD 1015) in the striatum, olfactory tubercle and posterior pituitary, but not in the median eminence. Apomorphine reverses the baclofen-induced increases in the concentration and synthesis of DA in all brain regions except median eminence, presumably by activating DA autoreceptors. Since baclofen depresses the a-methyltyrosine-induced decline of DA in all brain regions it would appear that tuberoinfundibular nerves, which terminate in the median eminence, differ from other DA nerves in that they are not regulated by autoreceptors. Baclofen
Dopamine
Norepinephrine
Picrotoxin
BACLOFEN depresses the excitability of a number of different neurons in the central nervous system by a mechanism that is currently not completely understood [IS], although numerous in vivo studies suggest that the drug does not act through a GABAergic mechanism [7, 13, 17, 201. Regardless of the mechanism of action, the ability of baclofen to depress impulse traffic in dopamine (DA) neurons [ 1,2, 6, 10, 11, 231 provides the experimenter with a useful pharmacological tool with which to study the properties of these nerves. The present report describes the results of experiments which attempt to characterize putative autoreceptors [4] on different DA neurons in the rat brain.
Distrihutiorl of DA Nrrvcs in Rut Brcrin Several different DA neuronal systems have been identified in the mammalian brain (Fig. 1). These nerves subserve different functions, and, in turn, are regulated by different mechanisms. The largest group of DA nerves, those comprising the “mesotelencephalic” system [3], have cell bodies in the central tegmentum (areas A8, 9, 10 in Fig. 1) and axons which project to various regions in the forebrain. This system is generally divided into the nigrostriatal system, comprised of nerves which have cell bodies in pars compacta of substantia nigra and axons which terminate in the striatum (caudate nucleus and putamen), and the mesolimbicmesocortical system which is comprised of nerves which have cell bodies in substantia nigra and the ventromedial tegmentum and axons which project to limbic forebrain regions (e.g., olfactory tubercle) and to the cingulate, frontal
Autoreceptors
and entorhinal cortices. Another group of DA neurons have cell bodies located in the mediobasal hypothalamus (A12) and axons which terminate in the median eminence or in the posterior pituitary; these neurons belong to the tuberoinfundibular and the tuberohypophyseal systems, respectively.
The properties of nigrostriatal nerves have been studied more thoroughly than DA nerves in other systems because the former system is the largest, and thus technically the easiest to work with. Regulation of these nerves is complex, involving various neuronal inputs to substantia nigra and the striatum, neuronal feedback loops, and short loop feedback mechanisms regulated by putative “autoreceptors” [4, 16, 181. This latter mechanism is depicted schematically in Fig. 2. Under normal conditions (Fig. 2A) DA is released into the synaptic cleft from nigrostriatal nerve terminals in response to the arrival of action potentials. This DA is free to interact with both post- and presynaptic receptors. Activation of the latter autoreceptors inhibits the release and synthesis of DA. If impulse flow in the nigrostriatal nerves is disrupted (Fig. 2B) less DA is released and thus autoreceptor activation is diminished. Consequently, the autoreceptor-mediated brake on DA synthesis is removed and the rate of synthesis of the amine increases. If DA is not released yet its synthesis is increased the concentration of DA in the nerve terminal must
increase.
IThis work was supported by USPHS grants NS-09174 and MH-13174. Some of the studies reported were performed by Drs. G. Gianutsos, P. H. Kelly and S. M. Wuerthele. ‘NIH Postdoctoral Fellow.
Copyright
Co 1980 ANKHO
International
Inc.-0361-9230/80/080531-05$01.00/O
532
MOORE AND DEMAREST
A
NORMAL IMPULSE FLOW WITH AUTORECEPTOR ACTIVATION
8. REDUCED IMPULSE FLOW WITH REDUCED AUTORECEPTOR ACTIVATION
?gix PUTATIVE 0 AUTORECEPTOR
C. REDUCED IMPULSE FLOW WITH AUTORECEPTOR ACTIYATI~
FIG. 1. Sag&al view of dopaminergic nerve projections in the rat brain. The bottom figure is an enlargement of the mediobasal hypothalamus and the pituitary gland showing the tuberoinfundibular and tuberohypophyseal DA nerves. Shaded areas represent regions of DA nerve Lerminals. ap-area postrema; AP-anterior pituitary; ar-arcuate nucleus; c~caudate-pu~en (st~aturn}; ILintermediate lobe; ip-interpeduncular nucleus; me-median eminence; mp-posterior mammilary nucleus; na-nucleus accumbens; NIL-neurointermediate lobe; N&neural lobe; oc-optic chiasm; pr-paraventricular nucleus; o&-olfactory tubercle; scsuprachiasmatic nucleus.
FIG. 2, Schematic representation of possible autoreceptor regulation of dopamine release and synthesis in mesotelenceph~ic nerve terminals. The size of “I)” represents the relative amount of dopamine within the nerve and in the synaptic cleft. The size of the schematic nerve action potential represents the amount of impulse traffic in the neuron. See text for details.
.6 -1
-4
c
0
O
iP
IO
20
40 DOSE
80 I60 (mglkg)
320
640
FIG. 3. Effects of baclofen and GBL on concentrations of catecholamines in the mouse forebrain. The dopamine (DA) and norepinephrine (NE) concentrations were determined in brains of untreated mice (Cl), in mice injected with baclofen (0) 1 hr before sacrifice, or in mice injected with GBL (A) 30 min before sacrifice. Solid symbols represent values that are significantly different @<0.05) from those in untreated controls (N=4-8). Reprinted from Gianutsos G. et nf. [ill.
Eflect ofSystemic Administration Mesotelencephalic DA Nerves
of Baclofen
on
impulse ilow in nigrostriatal DA nerves can be disrupted by surgically sectioning the nerves or by ad~nist~~ng depressant drugs such as y-butyrolactone (GEL; [24]). Baclofen appears to depress the activity of nigrostriatal DA nerves
D. REDUCED IMPULSE FLOW WITH AUTOWCEPTOR BLOCKADE
30 I 2 .25 APOMCRPHINE (mg/ kg)
FIG. 4. Reverse1 by apomo~hine of the baclofen-induced increase in the mouse forebrain concentration of dopamine. Various doses of apomorphine were injected SC 15 min prior to baclofen (40 mg/kg, IP), and mice were sacrificed 1 hr after baclofen. The horizontal line and cross-hatched area represents the mean DA concentration + SE in the forebrains of vehicle-treated (N=6-8). Reprinted from Gianutsos, G. et al. [ll]. in a manner similar to that of GBL. As depcited in Fig. 3, the systemic administration of GBL and baciafen (unless specified references are to the racemic mixture of baclofen) causes a dose-related increase in the concentration of DA in the mouse forebrain, with baclofen being approximately 8 times more potent than GBL. Neither drug altered the forebrain concentration of norepinephrine (NE). Systemic administ~tion of baclofen also increases the DA concentration in limbic forebrain regions (e.g., olfactory tubercle; [14]). The ability of baclofen to increase DA concentrations in forebrain regions resides in the l-isomer of this compound Ull. If baclofen increases DA concentrations in forebrain regions as a result of its ability to depress impulse flow in mes~elenceph~ic nerves, and thus to reduce activation of
presynaptic
DA receptors,
it should be possible to reverse
BACLOFEN
ON DIFFERENT
DOPAMINERGIC
533
NERVES TABLE
1
AND DOPAC IN THE STRIATUM OF RATS FOLLOWING SYSTEMIC ADMINISTRATION OF HALOPERIDOL AND MICROINJECTIONS OF BACLOFEN INTO SUBSTANTIA NIGRA CONCENTRATIONS
OF DOPAMINE
Intranigral injections Treatments
Dopamine (ngimg protein) Saline
Systemic injections 100 2 4 Vehicle 103 ? 3 Haloperidol
901,
.06
’
.I6
*
.32
’
.63
’ ’ 1.25 2.5
* ” 5 ’ ’
*
63
* ’ ’ ’ I25 250 500 0
DOSE (pg)
FIG. 5. Dopamine concentrations in rat hemiforebrains 30 min after injection of baclofen (A) or GABA (0) into substantia nigra. In all experiments 1 ~1 of drug solution was infused (1 WLVmin)into the substantia nigra on one side of the brain while the same volume of saline was concurrently infused into the substantia nigra on the other side. Solid symbols indicate those values from the drug-injected side which are significantly different (pcO.05) from the saline-injected side. Reprinted from Kelly and Moore [141.
such as apomorphine, which has presynaptic DA receptor agonist properties (Fig. 2C) [5]). As depicted in Fig. 4, apomorphine dose-dependently antagonized the baclofen-induced increase in the forebrain content of DA. This effect of apomorphine is blocked when animals are pretreated with a DA receptor antagonist, such as haloperidol (Fig. 2D; [ll]). the effects
Effects
of baclofen
of Intrunigral
by administering
Injections
a drug,
of Baclofen
In order to determine the site at which baclofen acts to exert its effect of mesotelencephalic DA nerves, the drug was injected directly into the region containing the cell bodies of these nerves. The results depicted in Fig. 5 reveal that microinjections of baclofen and GABA into substantia nigra caused a dose-related increase in the DA concentration in the forebrain (see also [5]). Baclofen is clearly more potent than GABA. Subsequent studies revealed that this activity resided in the l-isomer of baclofen [14]. In the same study it was found that microinjections of picrotoxin into substantia nigra did not alter DA concentrations per se, but did block the ability of GABA, but not of baclofen, to increase forebrain concentrations of DA. These results suggest that GABA and baclofen do not act in the same way, and that baclofen does not appear to exert its effects of mesotelencephalic DA nerves through a GABAergic mechanism. The concentration of DOPAC in the striatum has been used as a biochemical index of nigrostriatal nerve activity, increased activity being associated with increased concentrations of DOPAC [21]. Neuroleptics, such as haloperidol, increase nigrostriatal nerve activity and striatal DOPAC concentrations. The results of the experiment depicted in Table 1 reveal that intranigral injections of baclofen prevent the haloperidol-induced increased striatal concentration of DOPAC.
DOPAC (ngimg protein)
Baclofen
Saline
Baclofen
163 t 5* 181 + 9*
10.3 2 1.6 28.5 i- 2.0
11.6 ? 1.6 14.9 -c 3.2*
Male rats received concurrent microinjections of baclofen (0.1 pg) or saline (2.0 ~1) into the right and left substantia nigra, respectively. Immediately afterwards the rats received an IP injection of haloperidol (0.1 mgikg) or its vehicle (0.3% tartaric acid) and were sacrificed 1 hr later. Dopamine and DOPAC concentrations were determined in the striatum by a radioenzymatic assay [221. Values represent mean t 1 SE of at least 11 separate determinations. * Values in striatum on the baclofen-treated side are significantly different (pcO.05) from those on the saline-treated side (adapted from J. Nc,rrrol. Truns. 45: 117, 1979).
TABLE 2 EFFECT OF APOMORPHINE ON BACLOFEN-INDUCED INCREASE OF DOPAMINE CONCENTRATIONS IN VARIOUS BRAIN REGIONS Drug treatment Vehicle Baclofen Baclofen + apomorphine
Olfactory tubercle
Posterior pituitary
Median eminence
5 6*
43 2 3 70 i- 5*
8.7 2 0.6 10.5 -c 0.3*
99 i- 7 97 ‘- 6
98 2 10t
44 2 6+
7.0 Z?0.5t
90 + 7
Striatum 962 166 ?
Baclofen (40 mg/kg, IP) and apomorphine (2 mgikg, SC) were administered 60 min prior to sacrifice. Values represent dopamine concentrations (npimg protein, mean 2 I SE) as determined from 8 animals using a radioenzymatic assay [22]. *Significantly different from vehicle @<0.05). tsignificantly different from baclofen alone @<0.05).
Ejfects of Baclofen Tuheroinfundihular
on Tuherohypoph~sc,crl DA Nerves
end
To see if the effects of baclofen on mesotelencephalic DA nerves also extended to tuberohypophyseal and tuberoinfundibular DA nerves, the effect of this drug was determined on the DA content of the posterior pituitary and the median eminence. The results summarized in Table 2 reveal that systemic administration of baclofen significantly increased the concentration of DA in the striatum, olfactory tubercle and posterior pituitary, but not in the median eminence. The effect of baclofen in the former regions was reversed by apomorphine. The administration of DA agonists inhibits the synthesis of DA in terminals of mesotelencephalic DA nerves [ 16,181. The results depicted in Table 3 reveal that apomorphine, a Potent DA receptor agonist, also reduces the rate of DA synthesis in the terminals of tuberohypophyseal but not of tuberoinfundibular DA nerves. That is, apomorphinc de-
534
MOORE AND DEMAREST TABLE 3
TABLE 4 EFFECT OF BACLOFEN ON u-IWETHYLTYROSINE-INUUCED DECLINE OF ~PAMINE 1N THE MEDIAN EMJNENCE
EFFECT OF A~~O~Hl~E ON BACLOFEN-INDUCED INCREASE OF DOPA A~CUMULA~ON IN VARIOUS BRAIN REGIONS
Drug treatment Vehicle Apomorphine Baclofen Baclofen + apomorphine Apomorphine
Striatum 10.0 + 0.3 2.3 +: O.l* 15.3 _t 0.8* 4.1 ? 0.33. (2 m&g,
Olfactory tubercle
Posterior pituitary
Il.5 i: 0.6 0.96 -+ 0.07 1.6 t 0.7* 0.73 i- 0.06” 16.3 2 I.O* 1.37 _f O.IS* 4.8 i 0.2:’
1.02 ? 0.08t
Median eminence 7.9 + 0.5 8.1 k I .2 6.8 L 0.3 8.4 i 0.7
SC), baclofen (20 mgikg, IP) and NSD
Saline (control) a-Methyltyrosine C of control
N
Vehicle
Baclofen
8 8
103.1 i 4.3 68.6 i 3.x* 66.5
100.8 + 7.4 89.2 rt 8.3 88.5
Baclofen (40 mg/kg IP) or vehicle and a-methyityrosine (250 mgikg, IP) or saline were injected 60 min prior to sacrifice. Dopamine concentrations are expressed as ndmg protein. *Significantly different from saline control. ~<0.05.
1015 (105 mgikg, IP) were administered 40, 35 and 30 min prior to sacrifice, respectively. Values represent DOPA accumulation (ngimg protein/30 min; mean t t SE) as determined from 8 animals using a radioenzymatic assay f8]. *Significantly different from vehicle (u
pressed the rate of accumulation of DOPA after administration of a DOPA decarboxylase inhibitor (NSD 1015) in the striatum, olfactory tubercle and posterior pituitary, but not in the median eminence. The effect of apomorphine on DA synthesis in the former regions could result from activation of postsynaptic receptors and thereby of an inhibitory neuronal feedback loop, or it could result from activation on DA autoreceptors. However, apomo~hine also depressed the enhanced DOPA accumulation in the striatum and olfactory tubercle after the administration of baclofen which by acting at the level of the ceil bodies of these neurons would effectively disrupt the neuronal feedback loop. These results suggest, therefore, that apomorphine reverses the effect of baclofen on DA synthesis by activating autoreceptors. The lack of effect of baclofen on the rate of DOPA accumulation in median eminence suggests that the drug either does not inhibit impulse flow in tuberoinfundibu~ar DA nerves, or that these nerves are not regulated by an autoreceptor mechanism. It has been previously reported [ 121 that the baclofen-induced depression of activity in mesotelencephalic DA nerves is expressed by a reduction in the rate of decline of DA in the striatum and olfactory tubercle following the ~ministration of the synthesis inhibitor, cY-methyltyrosine, The results summarized in Table 4 indicate that baclofen also depresses the cu-methyltyrosineinduced decline of DA in the median eminence. This suggests, therefore, that baclofen does depress the activity of tuberoinfundibular DA nerves, just as it does other DA nerves, Nevertheless, tuberoi~undibular nerves respond differently to this depression, possibly because they lack autoreceptors on their terminals, which in other DA nerves exert a regulatory influence on DA synthesis.
A comparison of some of the properties of mesoteiencephalic and tuberoinfundibular DA nerves is depicted schematically in Fig. 6. DA is released from mesotelencephalit nerve terminals, depicted at the top, into a snyaptic cleft where it is free to activate post- or presynaptic receptars. Activity of the latter receptors appears to inhibit the synthesis and release of this amine. DA is normally removed from the synaptic cleft, and thus from the regions of pre- and
DOFW
a.
3MT
----xi&\ ,-----------. -FY ;“*,
t
COMT
‘.
TYROWE: $+” DOPA ----?b)u/LcD
b.
FIG. 6. Schematic diagram of (a) mesotelencephalic and (b) tuberoinfundib~lar DA nerve terminals. COMT-catechok)methyltransferase; DOPAC-3.4-dihydroxyphenylacetic acid; HVA-homovanillic acid; MAO-monoamine oxidase: 3MT-3methoxytyramine.
postsynaptic receptors by an active mechanism which transports the amine back into the nerve terminal where it is oxidatively deaminated by intraneuronal monoamine oxidase to form DOPAC. Disruption of impulse traffic along mesotelencephalic nerves with baclofen reduces the release of DA so that the autoreceptor-induced inhibition of DA synthesis is no longer operative, and DA synthesis, measured in viva by accumulation of DOPA after inhibition of decarboxylase, is increased. The terminal of a tuberoinfundibu~ar DA nerve, depicted at the bottom of Fig. 6, differs from the mesotelencephalic nerve terminal in several respects. First, DA is not released into a synaptic cleft, but into perivascular spaces from where it is tra~s~~~ in the hypophyse~ portal blood to the anterior pituitary. There DA acts on receptors to inhibit the release of prolactin. Secondly, tuberoi~undibular DA nerves appear to lack a high affinity mechanism for transporting DA back into the nerve terminal [71; this results in a low concentration of DOPAC in the median eminence 1221. Finally, as suggested by the results of the experiments with baclofen, tuberoinfundibular nerve terminals appear to lack DA autoreceptors, which in other DA nerves play some role in regulating DA synthesis and release.
BACLOFEN
ON DIFFERENT
DOPAMINERGIC
NERVES
535
REFERENCES 1. And&, N.-E. and H. Wachtel. Biochemical effects of baclofen (/3-parachlorphenyl-GABA) on the dopamine and the noradrenaline in the rat brain. Actct Phurtnac. Toxicol. 40: 31&320, 1977. 2. Bernard, P., S. J. Edwards, S. Fielding, R. D. Robson, J. K. Saelens, J. P. Simpke and J. Welch. Baclofen: An unusual CNS-active agent. Pharmacologist 17: 256, 1975. 3. Bjiirklund, A. and 0. Lindvall. The meso-telencephalic dopamine neuron system: A review of its anatomy. In: Limbic Mechanisms, edited by K. E. Livingston and 0. Hornykiewicz. New York: Plenum Press, 1978, pp. 307-331. 4. Carlsson, A. Receptor-mediated control of dopamine metabolism. In: Pre- rend Postsynaptic Receptors, edited by E. Usdin and W. E. Bunney, Jr. New York: Marcel Dekker, Inc., 1975, pp. 4%63. 5. Carlsson, A., B. Biswas and M. Lindqvist. Influence of GABA and GABA-like drugs on monoaminergic mechanisms. Adv. Biochem.
Psychopharmuc.
16: 471-75,
1977.
6. Da Prada, M. and H. H. Keller. Baclofen and hydroxybutyrate: Similar effects on cerebral dopamine neurones. Lifr Sci. 19: 1253-1264, 1976. 7. Davies, J. and J. C. Watkins. The action of P-phenyl-GABA derivatives on neurons of cat cerebral cortex. Brctin RES. 70: 501-505,
1974.
8. Demarest, K. T. and K. E. Moore. Accumulation of dihydroxyphenylalanine in the median eminence: An index of tuberoinfundibular dopaminergic nerve activity. Endocrinolog.v 106: 463468, 1980. 9. Demarest, K. T. and K. E. Moore. Lack of a high affinity transport system for dopamine in the median eminence and posterior pituitary. Brain Rcs. 171: 545-551, 1979. 10. Fuxe, K., T. H&felt, A. Ljungdahl, L. Agnati and 0. Johansson. Evidence for an inhibitory GABAergic control of the mesolimbic dopamine neurons: Possibility of improving treatment of schizophrenia by combined treatment with neuroleptics and GABAergic drugs. Med. Bid. 53: 177-183, 1975. 11. Gianutsos, G. and K. E. Moore. Increase in mouse brain dopamine content by baclofen: Effects of apomorphine and neuroleptics. Psyc,hopharmuco/oRy 52: 217-221, 1977. 12. Gianutsos, G. and K. E. Moore. Tolerance to the effects of baclofen and y-butyrolactone on locomotor activity and dopaminergic neurons in the mouse. J. Phrtrmcrc. exp. Thu. 207: 859-869,
1978.
13. Henry, J. L. and Y. Ben-Ari. Actions of the p-chlorphenyl derivative of GABA, Lioresal, on nociceptive and non-nociceptive units in the spinal cord of the cat. Brain Res. 117: 540-544, 1976. 14. Kelly, P. H. and K. E. Moore. Dopamine concentrations in the rat brain following injections into substantia nigra of baclofen, y-aminobutyric acid, y-hydroxybutyric acid, apomorphine and amphetamine. Nc~uropharmucoloR~ 17: 169-174, 1978. 15. Koella, W. P. The pharmacology of baclofen. In: Bacl&tz: Spa.stki/y and Cerebral Parhology, edited by A. M. Jukes. Northampton: Cambridge Med. Publication, 1978, pp. 7-15. 16. Moore, K. E. and S. M. Wuerthele. Regulation of nigrostriatal and tuberoinfundibular-hypophyseal dopaminergic neurons. In: Progress in Neurobiology, Vol. 13, edited by G. A. Kerkut and J. W. Phillis. 1979, pp. 325-358. 17. Nai. S. R., A. Guidotti and E. Costa. Central GABA receptor agonists: Comparison of muscimol and baclofen. Nerrrophtrrmctcology 15: 479-484, 1976. 18. Nowycky, M. C. and R. H. Roth. Dopaminergic
neurons: Role of presynaptic receptors in the regulation of transmitter biosynthesis. frog. it1 Neurops~c~hophtrrn~~t~, 2: 13%160, 1978. 19. Olpe, H. R., W. P. Koella, P. Wolf and H. L. Haas. The action of baclofen on neurons of the substantia nigra and of the ventral tegmental area. Bruin Res. 134: 577-580, 1977. 20. Phillis, J. W. Is P-(4-chlorophenyl)-GABA a specific antagonist of substance P on cerebral cortical neurons? E.uperientirr 32: 593-594, 1976. 21. Roth, R. H.,
L. C. Murrin and J. R. Walters. Central dopaminergic neurons: Effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Eur. J. Phurmrlc. 36: 163-171, 1976. 22. Umezu, K. and K. E. Moore. Effects of drugs on regional brain concentrations of dopamine and dihydroxyphenylacetic acid. J. Phrtrmrtc. c’xp, Ther. 208: 49-56, 1979. 23. Waldmeier. P. C. and L. Maitre. Effects
of baclofen on dopamine metabolism and interaction with neuroleptic effects.
Eur. J. Phormac. 47: 191-200, 1978. 24. Walters, J. R., R. H. Roth and G. K. Aghajanian. Dopaminergic
neurons: Similar biochemical and histochemical effects of y-hydroxybutyrate and acute lesions of the nigro-neostriatal pathway. .I. Phnrmctc. exp. Ther. 186: 630-639. 1973.
Vol. 5,
Suppl.
2, pp. 531-535.
Printed
in the
U.S.A
Effects of Baclofen on Different Dopaminergic Neuronal Systems’ K. E. MOORE Department
of Pharmacology
AND K. T. DEMAREST’
and Toxicology,
Michigan
State
UniLversity,
East Lrrnsing,
MI 48824
MOORE, K. E. AND K. T. DEMAREST. Ejj&rs of’hr~clqfin o,z d#c>rrr~t doprrminergic nclrrroncrl .sy.st~m.s. BRAIN RES. BULL. 5: Suppl. 2, 531-53.5, 1980.--Systemic administration of d/ or I-baclofen causes a dose-related increase in the concentration of dopamine (DA), but not of norepinephrine, in the forebrains of mice and rats. Microinjections of baclofen or GABA into substantia nigra increase DA in striatum. Since only the effect of GABA is reversed by picrotoxin, it would appear that baclofen does not act through a GABAergic mechanism. Baclofen-induced changes in DA concentrations are believed due to the depression of neuronal impulse flow which results in a decreased release of DA and thus a concomitant decrease in putative autoreceptor activation. Activation of DA autoreceptors appears to inhibit the synthesis and release of DA. Baclofen increases the concentration and the rate of synthesis of DA (accumulation of DOPA after inhibition of decarboxylase with NSD 1015) in the striatum, olfactory tubercle and posterior pituitary, but not in the median eminence. Apomorphine reverses the baclofen-induced increases in the concentration and synthesis of DA in all brain regions except median eminence, presumably by activating DA autoreceptors. Since baclofen depresses the a-methyltyrosine-induced decline of DA in all brain regions it would appear that tuberoinfundibular nerves, which terminate in the median eminence, differ from other DA nerves in that they are not regulated by autoreceptors. Baclofen
Dopamine
Norepinephrine
Picrotoxin
BACLOFEN depresses the excitability of a number of different neurons in the central nervous system by a mechanism that is currently not completely understood [IS], although numerous in vivo studies suggest that the drug does not act through a GABAergic mechanism [7, 13, 17, 201. Regardless of the mechanism of action, the ability of baclofen to depress impulse traffic in dopamine (DA) neurons [ 1,2, 6, 10, 11, 231 provides the experimenter with a useful pharmacological tool with which to study the properties of these nerves. The present report describes the results of experiments which attempt to characterize putative autoreceptors [4] on different DA neurons in the rat brain.
Distrihutiorl of DA Nrrvcs in Rut Brcrin Several different DA neuronal systems have been identified in the mammalian brain (Fig. 1). These nerves subserve different functions, and, in turn, are regulated by different mechanisms. The largest group of DA nerves, those comprising the “mesotelencephalic” system [3], have cell bodies in the central tegmentum (areas A8, 9, 10 in Fig. 1) and axons which project to various regions in the forebrain. This system is generally divided into the nigrostriatal system, comprised of nerves which have cell bodies in pars compacta of substantia nigra and axons which terminate in the striatum (caudate nucleus and putamen), and the mesolimbicmesocortical system which is comprised of nerves which have cell bodies in substantia nigra and the ventromedial tegmentum and axons which project to limbic forebrain regions (e.g., olfactory tubercle) and to the cingulate, frontal
Autoreceptors
and entorhinal cortices. Another group of DA neurons have cell bodies located in the mediobasal hypothalamus (A12) and axons which terminate in the median eminence or in the posterior pituitary; these neurons belong to the tuberoinfundibular and the tuberohypophyseal systems, respectively.
The properties of nigrostriatal nerves have been studied more thoroughly than DA nerves in other systems because the former system is the largest, and thus technically the easiest to work with. Regulation of these nerves is complex, involving various neuronal inputs to substantia nigra and the striatum, neuronal feedback loops, and short loop feedback mechanisms regulated by putative “autoreceptors” [4, 16, 181. This latter mechanism is depicted schematically in Fig. 2. Under normal conditions (Fig. 2A) DA is released into the synaptic cleft from nigrostriatal nerve terminals in response to the arrival of action potentials. This DA is free to interact with both post- and presynaptic receptors. Activation of the latter autoreceptors inhibits the release and synthesis of DA. If impulse flow in the nigrostriatal nerves is disrupted (Fig. 2B) less DA is released and thus autoreceptor activation is diminished. Consequently, the autoreceptor-mediated brake on DA synthesis is removed and the rate of synthesis of the amine increases. If DA is not released yet its synthesis is increased the concentration of DA in the nerve terminal must
increase.
IThis work was supported by USPHS grants NS-09174 and MH-13174. Some of the studies reported were performed by Drs. G. Gianutsos, P. H. Kelly and S. M. Wuerthele. ‘NIH Postdoctoral Fellow.
Copyright
Co 1980 ANKHO
International
Inc.-0361-9230/80/080531-05$01.00/O
532
MOORE AND DEMAREST
A
NORMAL IMPULSE FLOW WITH AUTORECEPTOR ACTIVATION
8. REDUCED IMPULSE FLOW WITH REDUCED AUTORECEPTOR ACTIVATION
?gix PUTATIVE 0 AUTORECEPTOR
C. REDUCED IMPULSE FLOW WITH AUTORECEPTOR ACTIYATI~
FIG. 1. Sag&al view of dopaminergic nerve projections in the rat brain. The bottom figure is an enlargement of the mediobasal hypothalamus and the pituitary gland showing the tuberoinfundibular and tuberohypophyseal DA nerves. Shaded areas represent regions of DA nerve Lerminals. ap-area postrema; AP-anterior pituitary; ar-arcuate nucleus; c~caudate-pu~en (st~aturn}; ILintermediate lobe; ip-interpeduncular nucleus; me-median eminence; mp-posterior mammilary nucleus; na-nucleus accumbens; NIL-neurointermediate lobe; N&neural lobe; oc-optic chiasm; pr-paraventricular nucleus; o&-olfactory tubercle; scsuprachiasmatic nucleus.
FIG. 2, Schematic representation of possible autoreceptor regulation of dopamine release and synthesis in mesotelenceph~ic nerve terminals. The size of “I)” represents the relative amount of dopamine within the nerve and in the synaptic cleft. The size of the schematic nerve action potential represents the amount of impulse traffic in the neuron. See text for details.
.6 -1
-4
c
0
O
iP
IO
20
40 DOSE
80 I60 (mglkg)
320
640
FIG. 3. Effects of baclofen and GBL on concentrations of catecholamines in the mouse forebrain. The dopamine (DA) and norepinephrine (NE) concentrations were determined in brains of untreated mice (Cl), in mice injected with baclofen (0) 1 hr before sacrifice, or in mice injected with GBL (A) 30 min before sacrifice. Solid symbols represent values that are significantly different @<0.05) from those in untreated controls (N=4-8). Reprinted from Gianutsos G. et nf. [ill.
Eflect ofSystemic Administration Mesotelencephalic DA Nerves
of Baclofen
on
impulse ilow in nigrostriatal DA nerves can be disrupted by surgically sectioning the nerves or by ad~nist~~ng depressant drugs such as y-butyrolactone (GEL; [24]). Baclofen appears to depress the activity of nigrostriatal DA nerves
D. REDUCED IMPULSE FLOW WITH AUTOWCEPTOR BLOCKADE
30 I 2 .25 APOMCRPHINE (mg/ kg)
FIG. 4. Reverse1 by apomo~hine of the baclofen-induced increase in the mouse forebrain concentration of dopamine. Various doses of apomorphine were injected SC 15 min prior to baclofen (40 mg/kg, IP), and mice were sacrificed 1 hr after baclofen. The horizontal line and cross-hatched area represents the mean DA concentration + SE in the forebrains of vehicle-treated (N=6-8). Reprinted from Gianutsos, G. et al. [ll]. in a manner similar to that of GBL. As depcited in Fig. 3, the systemic administration of GBL and baciafen (unless specified references are to the racemic mixture of baclofen) causes a dose-related increase in the concentration of DA in the mouse forebrain, with baclofen being approximately 8 times more potent than GBL. Neither drug altered the forebrain concentration of norepinephrine (NE). Systemic administ~tion of baclofen also increases the DA concentration in limbic forebrain regions (e.g., olfactory tubercle; [14]). The ability of baclofen to increase DA concentrations in forebrain regions resides in the l-isomer of this compound Ull. If baclofen increases DA concentrations in forebrain regions as a result of its ability to depress impulse flow in mes~elenceph~ic nerves, and thus to reduce activation of
presynaptic
DA receptors,
it should be possible to reverse
BACLOFEN
ON DIFFERENT
DOPAMINERGIC
533
NERVES TABLE
1
AND DOPAC IN THE STRIATUM OF RATS FOLLOWING SYSTEMIC ADMINISTRATION OF HALOPERIDOL AND MICROINJECTIONS OF BACLOFEN INTO SUBSTANTIA NIGRA CONCENTRATIONS
OF DOPAMINE
Intranigral injections Treatments
Dopamine (ngimg protein) Saline
Systemic injections 100 2 4 Vehicle 103 ? 3 Haloperidol
901,
.06
’
.I6
*
.32
’
.63
’ ’ 1.25 2.5
* ” 5 ’ ’
*
63
* ’ ’ ’ I25 250 500 0
DOSE (pg)
FIG. 5. Dopamine concentrations in rat hemiforebrains 30 min after injection of baclofen (A) or GABA (0) into substantia nigra. In all experiments 1 ~1 of drug solution was infused (1 WLVmin)into the substantia nigra on one side of the brain while the same volume of saline was concurrently infused into the substantia nigra on the other side. Solid symbols indicate those values from the drug-injected side which are significantly different (pcO.05) from the saline-injected side. Reprinted from Kelly and Moore [141.
such as apomorphine, which has presynaptic DA receptor agonist properties (Fig. 2C) [5]). As depicted in Fig. 4, apomorphine dose-dependently antagonized the baclofen-induced increase in the forebrain content of DA. This effect of apomorphine is blocked when animals are pretreated with a DA receptor antagonist, such as haloperidol (Fig. 2D; [ll]). the effects
Effects
of baclofen
of Intrunigral
by administering
Injections
a drug,
of Baclofen
In order to determine the site at which baclofen acts to exert its effect of mesotelencephalic DA nerves, the drug was injected directly into the region containing the cell bodies of these nerves. The results depicted in Fig. 5 reveal that microinjections of baclofen and GABA into substantia nigra caused a dose-related increase in the DA concentration in the forebrain (see also [5]). Baclofen is clearly more potent than GABA. Subsequent studies revealed that this activity resided in the l-isomer of baclofen [14]. In the same study it was found that microinjections of picrotoxin into substantia nigra did not alter DA concentrations per se, but did block the ability of GABA, but not of baclofen, to increase forebrain concentrations of DA. These results suggest that GABA and baclofen do not act in the same way, and that baclofen does not appear to exert its effects of mesotelencephalic DA nerves through a GABAergic mechanism. The concentration of DOPAC in the striatum has been used as a biochemical index of nigrostriatal nerve activity, increased activity being associated with increased concentrations of DOPAC [21]. Neuroleptics, such as haloperidol, increase nigrostriatal nerve activity and striatal DOPAC concentrations. The results of the experiment depicted in Table 1 reveal that intranigral injections of baclofen prevent the haloperidol-induced increased striatal concentration of DOPAC.
DOPAC (ngimg protein)
Baclofen
Saline
Baclofen
163 t 5* 181 + 9*
10.3 2 1.6 28.5 i- 2.0
11.6 ? 1.6 14.9 -c 3.2*
Male rats received concurrent microinjections of baclofen (0.1 pg) or saline (2.0 ~1) into the right and left substantia nigra, respectively. Immediately afterwards the rats received an IP injection of haloperidol (0.1 mgikg) or its vehicle (0.3% tartaric acid) and were sacrificed 1 hr later. Dopamine and DOPAC concentrations were determined in the striatum by a radioenzymatic assay [221. Values represent mean t 1 SE of at least 11 separate determinations. * Values in striatum on the baclofen-treated side are significantly different (pcO.05) from those on the saline-treated side (adapted from J. Nc,rrrol. Truns. 45: 117, 1979).
TABLE 2 EFFECT OF APOMORPHINE ON BACLOFEN-INDUCED INCREASE OF DOPAMINE CONCENTRATIONS IN VARIOUS BRAIN REGIONS Drug treatment Vehicle Baclofen Baclofen + apomorphine
Olfactory tubercle
Posterior pituitary
Median eminence
5 6*
43 2 3 70 i- 5*
8.7 2 0.6 10.5 -c 0.3*
99 i- 7 97 ‘- 6
98 2 10t
44 2 6+
7.0 Z?0.5t
90 + 7
Striatum 962 166 ?
Baclofen (40 mg/kg, IP) and apomorphine (2 mgikg, SC) were administered 60 min prior to sacrifice. Values represent dopamine concentrations (npimg protein, mean 2 I SE) as determined from 8 animals using a radioenzymatic assay [22]. *Significantly different from vehicle @<0.05). tsignificantly different from baclofen alone @<0.05).
Ejfects of Baclofen Tuheroinfundihular
on Tuherohypoph~sc,crl DA Nerves
end
To see if the effects of baclofen on mesotelencephalic DA nerves also extended to tuberohypophyseal and tuberoinfundibular DA nerves, the effect of this drug was determined on the DA content of the posterior pituitary and the median eminence. The results summarized in Table 2 reveal that systemic administration of baclofen significantly increased the concentration of DA in the striatum, olfactory tubercle and posterior pituitary, but not in the median eminence. The effect of baclofen in the former regions was reversed by apomorphine. The administration of DA agonists inhibits the synthesis of DA in terminals of mesotelencephalic DA nerves [ 16,181. The results depicted in Table 3 reveal that apomorphine, a Potent DA receptor agonist, also reduces the rate of DA synthesis in the terminals of tuberohypophyseal but not of tuberoinfundibular DA nerves. That is, apomorphinc de-
534
MOORE AND DEMAREST TABLE 3
TABLE 4 EFFECT OF BACLOFEN ON u-IWETHYLTYROSINE-INUUCED DECLINE OF ~PAMINE 1N THE MEDIAN EMJNENCE
EFFECT OF A~~O~Hl~E ON BACLOFEN-INDUCED INCREASE OF DOPA A~CUMULA~ON IN VARIOUS BRAIN REGIONS
Drug treatment Vehicle Apomorphine Baclofen Baclofen + apomorphine Apomorphine
Striatum 10.0 + 0.3 2.3 +: O.l* 15.3 _t 0.8* 4.1 ? 0.33. (2 m&g,
Olfactory tubercle
Posterior pituitary
Il.5 i: 0.6 0.96 -+ 0.07 1.6 t 0.7* 0.73 i- 0.06” 16.3 2 I.O* 1.37 _f O.IS* 4.8 i 0.2:’
1.02 ? 0.08t
Median eminence 7.9 + 0.5 8.1 k I .2 6.8 L 0.3 8.4 i 0.7
SC), baclofen (20 mgikg, IP) and NSD
Saline (control) a-Methyltyrosine C of control
N
Vehicle
Baclofen
8 8
103.1 i 4.3 68.6 i 3.x* 66.5
100.8 + 7.4 89.2 rt 8.3 88.5
Baclofen (40 mg/kg IP) or vehicle and a-methyityrosine (250 mgikg, IP) or saline were injected 60 min prior to sacrifice. Dopamine concentrations are expressed as ndmg protein. *Significantly different from saline control. ~<0.05.
1015 (105 mgikg, IP) were administered 40, 35 and 30 min prior to sacrifice, respectively. Values represent DOPA accumulation (ngimg protein/30 min; mean t t SE) as determined from 8 animals using a radioenzymatic assay f8]. *Significantly different from vehicle (u
pressed the rate of accumulation of DOPA after administration of a DOPA decarboxylase inhibitor (NSD 1015) in the striatum, olfactory tubercle and posterior pituitary, but not in the median eminence. The effect of apomorphine on DA synthesis in the former regions could result from activation of postsynaptic receptors and thereby of an inhibitory neuronal feedback loop, or it could result from activation on DA autoreceptors. However, apomo~hine also depressed the enhanced DOPA accumulation in the striatum and olfactory tubercle after the administration of baclofen which by acting at the level of the ceil bodies of these neurons would effectively disrupt the neuronal feedback loop. These results suggest, therefore, that apomorphine reverses the effect of baclofen on DA synthesis by activating autoreceptors. The lack of effect of baclofen on the rate of DOPA accumulation in median eminence suggests that the drug either does not inhibit impulse flow in tuberoinfundibu~ar DA nerves, or that these nerves are not regulated by an autoreceptor mechanism. It has been previously reported [ 121 that the baclofen-induced depression of activity in mesotelencephalic DA nerves is expressed by a reduction in the rate of decline of DA in the striatum and olfactory tubercle following the ~ministration of the synthesis inhibitor, cY-methyltyrosine, The results summarized in Table 4 indicate that baclofen also depresses the cu-methyltyrosineinduced decline of DA in the median eminence. This suggests, therefore, that baclofen does depress the activity of tuberoinfundibular DA nerves, just as it does other DA nerves, Nevertheless, tuberoi~undibular nerves respond differently to this depression, possibly because they lack autoreceptors on their terminals, which in other DA nerves exert a regulatory influence on DA synthesis.
A comparison of some of the properties of mesoteiencephalic and tuberoinfundibular DA nerves is depicted schematically in Fig. 6. DA is released from mesotelencephalit nerve terminals, depicted at the top, into a snyaptic cleft where it is free to activate post- or presynaptic receptars. Activity of the latter receptors appears to inhibit the synthesis and release of this amine. DA is normally removed from the synaptic cleft, and thus from the regions of pre- and
DOFW
a.
3MT
----xi&\ ,-----------. -FY ;“*,
t
COMT
‘.
TYROWE: $+” DOPA ----?b)u/LcD
b.
FIG. 6. Schematic diagram of (a) mesotelencephalic and (b) tuberoinfundib~lar DA nerve terminals. COMT-catechok)methyltransferase; DOPAC-3.4-dihydroxyphenylacetic acid; HVA-homovanillic acid; MAO-monoamine oxidase: 3MT-3methoxytyramine.
postsynaptic receptors by an active mechanism which transports the amine back into the nerve terminal where it is oxidatively deaminated by intraneuronal monoamine oxidase to form DOPAC. Disruption of impulse traffic along mesotelencephalic nerves with baclofen reduces the release of DA so that the autoreceptor-induced inhibition of DA synthesis is no longer operative, and DA synthesis, measured in viva by accumulation of DOPA after inhibition of decarboxylase, is increased. The terminal of a tuberoinfundibu~ar DA nerve, depicted at the bottom of Fig. 6, differs from the mesotelencephalic nerve terminal in several respects. First, DA is not released into a synaptic cleft, but into perivascular spaces from where it is tra~s~~~ in the hypophyse~ portal blood to the anterior pituitary. There DA acts on receptors to inhibit the release of prolactin. Secondly, tuberoi~undibular DA nerves appear to lack a high affinity mechanism for transporting DA back into the nerve terminal [71; this results in a low concentration of DOPAC in the median eminence 1221. Finally, as suggested by the results of the experiments with baclofen, tuberoinfundibular nerve terminals appear to lack DA autoreceptors, which in other DA nerves play some role in regulating DA synthesis and release.
BACLOFEN
ON DIFFERENT
DOPAMINERGIC
NERVES
535
REFERENCES 1. And&, N.-E. and H. Wachtel. Biochemical effects of baclofen (/3-parachlorphenyl-GABA) on the dopamine and the noradrenaline in the rat brain. Actct Phurtnac. Toxicol. 40: 31&320, 1977. 2. Bernard, P., S. J. Edwards, S. Fielding, R. D. Robson, J. K. Saelens, J. P. Simpke and J. Welch. Baclofen: An unusual CNS-active agent. Pharmacologist 17: 256, 1975. 3. Bjiirklund, A. and 0. Lindvall. The meso-telencephalic dopamine neuron system: A review of its anatomy. In: Limbic Mechanisms, edited by K. E. Livingston and 0. Hornykiewicz. New York: Plenum Press, 1978, pp. 307-331. 4. Carlsson, A. Receptor-mediated control of dopamine metabolism. In: Pre- rend Postsynaptic Receptors, edited by E. Usdin and W. E. Bunney, Jr. New York: Marcel Dekker, Inc., 1975, pp. 4%63. 5. Carlsson, A., B. Biswas and M. Lindqvist. Influence of GABA and GABA-like drugs on monoaminergic mechanisms. Adv. Biochem.
Psychopharmuc.
16: 471-75,
1977.
6. Da Prada, M. and H. H. Keller. Baclofen and hydroxybutyrate: Similar effects on cerebral dopamine neurones. Lifr Sci. 19: 1253-1264, 1976. 7. Davies, J. and J. C. Watkins. The action of P-phenyl-GABA derivatives on neurons of cat cerebral cortex. Brctin RES. 70: 501-505,
1974.
8. Demarest, K. T. and K. E. Moore. Accumulation of dihydroxyphenylalanine in the median eminence: An index of tuberoinfundibular dopaminergic nerve activity. Endocrinolog.v 106: 463468, 1980. 9. Demarest, K. T. and K. E. Moore. Lack of a high affinity transport system for dopamine in the median eminence and posterior pituitary. Brain Rcs. 171: 545-551, 1979. 10. Fuxe, K., T. H&felt, A. Ljungdahl, L. Agnati and 0. Johansson. Evidence for an inhibitory GABAergic control of the mesolimbic dopamine neurons: Possibility of improving treatment of schizophrenia by combined treatment with neuroleptics and GABAergic drugs. Med. Bid. 53: 177-183, 1975. 11. Gianutsos, G. and K. E. Moore. Increase in mouse brain dopamine content by baclofen: Effects of apomorphine and neuroleptics. Psyc,hopharmuco/oRy 52: 217-221, 1977. 12. Gianutsos, G. and K. E. Moore. Tolerance to the effects of baclofen and y-butyrolactone on locomotor activity and dopaminergic neurons in the mouse. J. Phrtrmcrc. exp. Thu. 207: 859-869,
1978.
13. Henry, J. L. and Y. Ben-Ari. Actions of the p-chlorphenyl derivative of GABA, Lioresal, on nociceptive and non-nociceptive units in the spinal cord of the cat. Brain Res. 117: 540-544, 1976. 14. Kelly, P. H. and K. E. Moore. Dopamine concentrations in the rat brain following injections into substantia nigra of baclofen, y-aminobutyric acid, y-hydroxybutyric acid, apomorphine and amphetamine. Nc~uropharmucoloR~ 17: 169-174, 1978. 15. Koella, W. P. The pharmacology of baclofen. In: Bacl&tz: Spa.stki/y and Cerebral Parhology, edited by A. M. Jukes. Northampton: Cambridge Med. Publication, 1978, pp. 7-15. 16. Moore, K. E. and S. M. Wuerthele. Regulation of nigrostriatal and tuberoinfundibular-hypophyseal dopaminergic neurons. In: Progress in Neurobiology, Vol. 13, edited by G. A. Kerkut and J. W. Phillis. 1979, pp. 325-358. 17. Nai. S. R., A. Guidotti and E. Costa. Central GABA receptor agonists: Comparison of muscimol and baclofen. Nerrrophtrrmctcology 15: 479-484, 1976. 18. Nowycky, M. C. and R. H. Roth. Dopaminergic
neurons: Role of presynaptic receptors in the regulation of transmitter biosynthesis. frog. it1 Neurops~c~hophtrrn~~t~, 2: 13%160, 1978. 19. Olpe, H. R., W. P. Koella, P. Wolf and H. L. Haas. The action of baclofen on neurons of the substantia nigra and of the ventral tegmental area. Bruin Res. 134: 577-580, 1977. 20. Phillis, J. W. Is P-(4-chlorophenyl)-GABA a specific antagonist of substance P on cerebral cortical neurons? E.uperientirr 32: 593-594, 1976. 21. Roth, R. H.,
L. C. Murrin and J. R. Walters. Central dopaminergic neurons: Effects of alterations in impulse flow on the accumulation of dihydroxyphenylacetic acid. Eur. J. Phurmrlc. 36: 163-171, 1976. 22. Umezu, K. and K. E. Moore. Effects of drugs on regional brain concentrations of dopamine and dihydroxyphenylacetic acid. J. Phrtrmrtc. c’xp, Ther. 208: 49-56, 1979. 23. Waldmeier. P. C. and L. Maitre. Effects
of baclofen on dopamine metabolism and interaction with neuroleptic effects.
Eur. J. Phormac. 47: 191-200, 1978. 24. Walters, J. R., R. H. Roth and G. K. Aghajanian. Dopaminergic
neurons: Similar biochemical and histochemical effects of y-hydroxybutyrate and acute lesions of the nigro-neostriatal pathway. .I. Phnrmctc. exp. Ther. 186: 630-639. 1973.