PRELIMINARY NOTES OPIATE-RECEPTQRS TN SUBSTANTIA NIGRA: ROLE IN TAE REGULA'FION Ol? STR32iTAL TYRQSINE HYDROXYLASE ACTIVl'TY
K.
Gale*,
F.
Moronir
M.
Kumakura
and
A, Guidotti
Laboratory of PreclinicalPharmacology,National fnstitute of Mental Health, Saint Elizabeths Hospital, Washington,D.C. 20032 *Departmentof Pharmacology,Georgetown University, Washington, D.C, 20(107
Morphine injected into the substantianiqxa fSN) of rats failed to change DA metabolism in striatusn but blocked the haloperidol induced activation of striatal tyrosine hydroxylase (TX). This blockade was mediated by stimulationof opiate recept3rs in SN because it was antagoni2xXi by naltrexone, The binding of H-enkephalinamideto opiate receptors in SN was not altered after lesion of the niqrostriatalDA pathways with 6hydroxydopaminebut was decreased (approximately30%) after brain hemitransection. These results sugqest that intraniqralmorphine prevents the haloperidol induced activation of striatal TfIby modulating the release of endogenous transmittersor modulators on DA neurons. Afferent projections to substantia nigra (SN) include pathways Containing GABA, substance P and serotonin. These transmittershave been shown to affect both efectrophysioloqicaland neurochemical. function of efferent pathways from SN, including the nigrostriataldopamine (DA) projection ffor a review see Dray and Straughan, 19761. Recently, met-enkephalinhas been maasured ih SN (Yang et al., X978), and although the concentrationof this peptide in SB is not as high as that found in other nuclei of the basal ganglia, opiate receptors a~@ abundant in SN (Pollardet al., 19781. This suggests that endogenous opiate peptides may represent an important neurotransmittersystem in the regulationof the activity of SN neurons. At present, the only evideaee in the literaturewhich suggests a functional role for the opiate'receptarsin SN is the observation that Unilateral intraniqxal injection of morphine or beta-endorphininduces contralateralcircling behavior which can be reversed by naloxone (Iwamotoand Way, 1978). In order tm determine whether activation of these opiate receptors in 5% could have functional importance for the regulation of nigrostriatalDA neurons, we chose to study the effect of intranigrallyapplied morphine on the tyrosine hydroxylase ITHI activity in the DA terminals of striatum and to characterize the neuronal population of SN where the opiate receptors are located. After systemic or intrastriatalinjectionsof morphineI an increase in DA metabolism was found in rat striatum. However, 30 min. after injection of morphine directly into SN (in doses of 1,2,5,10 and 20 Ug of the f.90) salt, dissolved in 0,s ~1 saline) we found no change in striatal IF.TA content (Guidottiet al., 1979). These results suggest that unlike the opiate receptors in striatum, the opiate receptors in SN do not mediate the morphine-inducedstimulation of strkatal RA metabolismIn order to determine whether activation of opiate receptors in .W night exert an inhibitory effect on DA neuronsI we examined whether intrraniqraf morphine could inhibit the activation of striatal TN.induced by haloperidol. The action of haluperidolon striatal TH is mediated via the transsynaptisregulation of DA neurons by niqral afferents, including the striate-dgral axon8 containing GABA or substance P (Gale et al., 1978). We therefore expected that if the nigxal opiate receptors exert an inhibitory influence on DA neurons (eitherdirectly ox indirectly),then intraniqralmorphine would be able to antagonize the activation of striatal TN elicited by haloperidol, AS shown in Fig, 1, although fntranigralmorphine by itself did not change the affinity of striatal 9z-X for nMPa , it completely prevented the increase in affinity of striatal Ttrfor DMPE4 in animals trentttdw$th haloperifiol.This effect of intranigralmorphine was reversed by systemic (Fig+ I) or intranigral. injection (Table 1) of the opiate-receptoxantagonist, naltrexone, but was nat reversed by intranigral injection of bicuculline methiodide (Table 1) a EABA receptor antagonist. The specificityof the action of morphine was further sup&orted by experiments which demonstrated that the ability of muscimol to antagonize the haloperidol-induced
427
428
Preliminary Notes
activation of striatal TH was reversed by intranigral bicuculline methiodide but not by naltrexone (Table 1).
htranigral morpthe blockade
ofHabperii-inducedktivatii
of Striatal TH
l
Mmpkm
\lOvg intronigrd)
0.37 0 HdopriQ((lmg/kg i.P.1
Figure 1.
DOUBLE RECIPROCAL PMTS OF THE INITIAL VELOCITY OFl$RIATAL TH AGAINST VARIOUS CONCENTRATIONS OF DNPH4 IN THE PRESENCE OF 0.1 ml4 C-TYROSINB. TH activity was determined using a dopadecarboxylasecoupled enzymatic reaction to measure 14 CO2 formed from carboxyl labelled tyrosine according to the method of Eivkovic et al. (1974). Haloperidol was injected 30 min prior sacrifice, morphine sulfate 10 min before haloperidol and naltrexone 10 min after haloperidol. Each point represent the mean obtained from six animals. The standard error of the Km values was between 5 and 10%. Stereotaxic coordinates for the intranigral injections were the same as those used by Gale and Guidotti (1976). The volume The Km for TH of untreated rats was 0.83 mM. injected into SN was 0.5 W. Table 1.
Relationship Between Opiate Receptors and GABA Receptors in SN for The Control of Haloperidol-InducedActivation of Striatal TH A Intravenous Injection
B First Intranigral Injection
C Second Intranigral Injection Saline (Km of
Bicuculline Naltrexone TH for DNPH4; mM)
Striatal
SALINE
Saline Muscimol Morphine
.85 .80 .83
.83 .88 .87
.79 .87 .85
HALOPERIDOL
Saline Muscimol Morphine
.37* .85 .73
.32* .91 .30*
.42* .40* .78
A) Saline or haloperidol (0.3 umoles/kg) was administered 30 min prior to sacrifice. B) Saline (0.5 W), morphine sulfate (10 llgin 0.5 ~1) or muscimol (100 ng in 0.5 ~1) was injected 10 min before haloperidol; C) Saline (0.5 ~11, naltrexone (10 vg in 0.5 ~1) or bicuculline methiodide (500 ng in 0.5 ~1) was injected 10 min after haloperidol. TH measurements were done as described in the legend of Fig. 1. Values represent the mean of 6-8 animals. Standard errors were less than 15% of the mean. *Significantlydifferent (P
K.
Gale*,
F.
Moronir
M.
Kumakura
and
A, Guidotti
Laboratory of PreclinicalPharmacology,National fnstitute of Mental Health, Saint Elizabeths Hospital, Washington,D.C. 20032 *Departmentof Pharmacology,Georgetown University, Washington, D.C, 20(107
Morphine injected into the substantianiqxa fSN) of rats failed to change DA metabolism in striatusn but blocked the haloperidol induced activation of striatal tyrosine hydroxylase (TX). This blockade was mediated by stimulationof opiate recept3rs in SN because it was antagoni2xXi by naltrexone, The binding of H-enkephalinamideto opiate receptors in SN was not altered after lesion of the niqrostriatalDA pathways with 6hydroxydopaminebut was decreased (approximately30%) after brain hemitransection. These results sugqest that intraniqralmorphine prevents the haloperidol induced activation of striatal TfIby modulating the release of endogenous transmittersor modulators on DA neurons. Afferent projections to substantia nigra (SN) include pathways Containing GABA, substance P and serotonin. These transmittershave been shown to affect both efectrophysioloqicaland neurochemical. function of efferent pathways from SN, including the nigrostriataldopamine (DA) projection ffor a review see Dray and Straughan, 19761. Recently, met-enkephalinhas been maasured ih SN (Yang et al., X978), and although the concentrationof this peptide in SB is not as high as that found in other nuclei of the basal ganglia, opiate receptors a~@ abundant in SN (Pollardet al., 19781. This suggests that endogenous opiate peptides may represent an important neurotransmittersystem in the regulationof the activity of SN neurons. At present, the only evideaee in the literaturewhich suggests a functional role for the opiate'receptarsin SN is the observation that Unilateral intraniqxal injection of morphine or beta-endorphininduces contralateralcircling behavior which can be reversed by naloxone (Iwamotoand Way, 1978). In order tm determine whether activation of these opiate receptors in 5% could have functional importance for the regulation of nigrostriatalDA neurons, we chose to study the effect of intranigrallyapplied morphine on the tyrosine hydroxylase ITHI activity in the DA terminals of striatum and to characterize the neuronal population of SN where the opiate receptors are located. After systemic or intrastriatalinjectionsof morphineI an increase in DA metabolism was found in rat striatum. However, 30 min. after injection of morphine directly into SN (in doses of 1,2,5,10 and 20 Ug of the f.90) salt, dissolved in 0,s ~1 saline) we found no change in striatal IF.TA content (Guidottiet al., 1979). These results suggest that unlike the opiate receptors in striatum, the opiate receptors in SN do not mediate the morphine-inducedstimulation of strkatal RA metabolismIn order to determine whether activation of opiate receptors in .W night exert an inhibitory effect on DA neuronsI we examined whether intrraniqraf morphine could inhibit the activation of striatal TN.induced by haloperidol. The action of haluperidolon striatal TH is mediated via the transsynaptisregulation of DA neurons by niqral afferents, including the striate-dgral axon8 containing GABA or substance P (Gale et al., 1978). We therefore expected that if the nigxal opiate receptors exert an inhibitory influence on DA neurons (eitherdirectly ox indirectly),then intraniqralmorphine would be able to antagonize the activation of striatal TN elicited by haloperidol, AS shown in Fig, 1, although fntranigralmorphine by itself did not change the affinity of striatal 9z-X for nMPa , it completely prevented the increase in affinity of striatal Ttrfor DMPE4 in animals trentttdw$th haloperifiol.This effect of intranigralmorphine was reversed by systemic (Fig+ I) or intranigral. injection (Table 1) of the opiate-receptoxantagonist, naltrexone, but was nat reversed by intranigral injection of bicuculline methiodide (Table 1) a EABA receptor antagonist. The specificityof the action of morphine was further sup&orted by experiments which demonstrated that the ability of muscimol to antagonize the haloperidol-induced
427
428
Preliminary Notes
activation of striatal TH was reversed by intranigral bicuculline methiodide but not by naltrexone (Table 1).
htranigral morpthe blockade
ofHabperii-inducedktivatii
of Striatal TH
l
Mmpkm
\lOvg intronigrd)
0.37 0 HdopriQ((lmg/kg i.P.1
Figure 1.
DOUBLE RECIPROCAL PMTS OF THE INITIAL VELOCITY OFl$RIATAL TH AGAINST VARIOUS CONCENTRATIONS OF DNPH4 IN THE PRESENCE OF 0.1 ml4 C-TYROSINB. TH activity was determined using a dopadecarboxylasecoupled enzymatic reaction to measure 14 CO2 formed from carboxyl labelled tyrosine according to the method of Eivkovic et al. (1974). Haloperidol was injected 30 min prior sacrifice, morphine sulfate 10 min before haloperidol and naltrexone 10 min after haloperidol. Each point represent the mean obtained from six animals. The standard error of the Km values was between 5 and 10%. Stereotaxic coordinates for the intranigral injections were the same as those used by Gale and Guidotti (1976). The volume The Km for TH of untreated rats was 0.83 mM. injected into SN was 0.5 W. Table 1.
Relationship Between Opiate Receptors and GABA Receptors in SN for The Control of Haloperidol-InducedActivation of Striatal TH A Intravenous Injection
B First Intranigral Injection
C Second Intranigral Injection Saline (Km of
Bicuculline Naltrexone TH for DNPH4; mM)
Striatal
SALINE
Saline Muscimol Morphine
.85 .80 .83
.83 .88 .87
.79 .87 .85
HALOPERIDOL
Saline Muscimol Morphine
.37* .85 .73
.32* .91 .30*
.42* .40* .78
A) Saline or haloperidol (0.3 umoles/kg) was administered 30 min prior to sacrifice. B) Saline (0.5 W), morphine sulfate (10 llgin 0.5 ~1) or muscimol (100 ng in 0.5 ~1) was injected 10 min before haloperidol; C) Saline (0.5 ~11, naltrexone (10 vg in 0.5 ~1) or bicuculline methiodide (500 ng in 0.5 ~1) was injected 10 min after haloperidol. TH measurements were done as described in the legend of Fig. 1. Values represent the mean of 6-8 animals. Standard errors were less than 15% of the mean. *Significantlydifferent (P
