Brain Research, 175 (1979) 145-149 O Elsevier/North-HollandBiomedicalPress
145
Opiate receptors in the pigeon optic tectum
D. FELIX, H. HENKE and U. FRANGI Brain Research Institute, University of Ziirich, CH-8029 Ziirich (Switzerland)
(Accepted June 7th, 1979)
Specific opiate receptors and their physiological significance have been demonstrated in various structures of the central nervous systeml°,1z,1s. When applied microiontophoretically, morphine was found to cause either an increase or a decrease of spontaneous activity, the latter showed stereospecificity, and was blocked by the specific opiate antagonist naloxone~,6-s,16,17. Much data point to an involvement of putative neurotransmitters in the action of opiates. The pigeon optic tectum with its anatomically5, biochemically4 and electrophysiologically1,19 well described structure offers an attractive model to study such interaction between different neuronal systems. In the present investigation the determination of specific opiate receptor binding and the effects of morphine, met-enkephalin and naloxone on synaptically and chemically induced excitation were examined. Preliminary results of this study have been reported brieflyg. For the receptor binding studies, pigeons (Columba livia) were decapitated and the tecta and membranes prepared as described11, le. The assay procedure was adapted from Pert et al. 15. [ZH]Etorphine (30 Ci/mmol) or [o-AlaZ]-met-enkephalin (15 Ci/mmol) both from the Radiochemical Centre, Amersham, were used as ligands. The unspecific binding was determined with 10-6 M naloxone (Endo Labs.). No sodium was added to the incubation mixture. The electrophysiological experiments were performed on 13 pigeons, anaesthetized with Equithesin (0.3 ml/100 g, i.m.), paralyzed by Pavulon (Pancuromii bromidum) and artificially ventilated by unidirectional airflow of a O2-CO2 mixture. For electrical stimulation, a bipolar steel electrode was placed on the contralateral optic nerve and single rectangular pulses (0.5-1.0 mA intensity, 0.1-0.3 msec duration) were delivered. Spontaneous and evoked action potentials were recorded extracellularly using conventional techniques. The channels of a 5-barreled micropipette contained the following compounds to be ejected microiontophoretically: L-monosodium glutamate (1 M, pH 7.5), morphine.HC1 (50 mM, pH 5.0), naloxone.HCl (50 mM in 0.165 M NaCI, pH 5.0) and met-enkephalin (5 mM, pH 5.0). Dye marks using fast green (FCF) were made at one or more recording locations. Specific opiate binding sites were found in the tectum (Fig. 1). The kinetic constants for the ligand etorphine and [o-Ala2]-met-enkephalin in this order were:
146
@
Etorphine
binding
in p i g e o n
tectum 300
1000 800 "
U'~
Ot
600 -i~
t
RD : 190 pM Brnax: 173 fmol/mg prof.
E i
400 2O0
,8o
1
nM
®
E
200
Met-Enkephalin
E v
% -lOO ~
280
~
380
bound (fmol/mg prot.)
b i n d i n g in p i g e o n t e c t u m
1000
-50
800
- 40
A
El
-30
Lo E 600
E v
KD : 5.6 nM Bmax : 350 fmo[ / mg prot
o~ ~ 400
0
\
200
o12
o14
o16
1o
i
i
T
i
,
1--
f
o.8
1.o
o
lOO
200
300
400
'J
1
nM
bound (fmollmg prot)
Fig. 1. Double reciprocal and Scatchard plots of etorphine (A) and met-enkephalin (B) binding to crude tectal membrane preparations. Points represent the mean ± S.E.M. o f 3-4 separate experiments.
dissocation constant (Ka) 5.6 ~k 1.1/0.19 :~ 0.01 nM, and maximal binding (Bronx) 350 ± 44/172 ~ 12 fmol/mg protein. Microiontophoretically applied morphine (30-100 nA) reduced the spontaneous firing rate on 85 ~ of tested cells. The spike size was continuously recorded to assure that the depression was not due to a local anesthetic effect (Fig. 2A). The excitation by glutamate was depressed or abolished by morphine on all cells tested (Fig. 2B). Following the morphine ejection, an increased glutamate-sensitivity could be observed
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145
Opiate receptors in the pigeon optic tectum
D. FELIX, H. HENKE and U. FRANGI Brain Research Institute, University of Ziirich, CH-8029 Ziirich (Switzerland)
(Accepted June 7th, 1979)
Specific opiate receptors and their physiological significance have been demonstrated in various structures of the central nervous systeml°,1z,1s. When applied microiontophoretically, morphine was found to cause either an increase or a decrease of spontaneous activity, the latter showed stereospecificity, and was blocked by the specific opiate antagonist naloxone~,6-s,16,17. Much data point to an involvement of putative neurotransmitters in the action of opiates. The pigeon optic tectum with its anatomically5, biochemically4 and electrophysiologically1,19 well described structure offers an attractive model to study such interaction between different neuronal systems. In the present investigation the determination of specific opiate receptor binding and the effects of morphine, met-enkephalin and naloxone on synaptically and chemically induced excitation were examined. Preliminary results of this study have been reported brieflyg. For the receptor binding studies, pigeons (Columba livia) were decapitated and the tecta and membranes prepared as described11, le. The assay procedure was adapted from Pert et al. 15. [ZH]Etorphine (30 Ci/mmol) or [o-AlaZ]-met-enkephalin (15 Ci/mmol) both from the Radiochemical Centre, Amersham, were used as ligands. The unspecific binding was determined with 10-6 M naloxone (Endo Labs.). No sodium was added to the incubation mixture. The electrophysiological experiments were performed on 13 pigeons, anaesthetized with Equithesin (0.3 ml/100 g, i.m.), paralyzed by Pavulon (Pancuromii bromidum) and artificially ventilated by unidirectional airflow of a O2-CO2 mixture. For electrical stimulation, a bipolar steel electrode was placed on the contralateral optic nerve and single rectangular pulses (0.5-1.0 mA intensity, 0.1-0.3 msec duration) were delivered. Spontaneous and evoked action potentials were recorded extracellularly using conventional techniques. The channels of a 5-barreled micropipette contained the following compounds to be ejected microiontophoretically: L-monosodium glutamate (1 M, pH 7.5), morphine.HC1 (50 mM, pH 5.0), naloxone.HCl (50 mM in 0.165 M NaCI, pH 5.0) and met-enkephalin (5 mM, pH 5.0). Dye marks using fast green (FCF) were made at one or more recording locations. Specific opiate binding sites were found in the tectum (Fig. 1). The kinetic constants for the ligand etorphine and [o-Ala2]-met-enkephalin in this order were:
146
@
Etorphine
binding
in p i g e o n
tectum 300
1000 800 "
U'~
Ot
600 -i~
t
RD : 190 pM Brnax: 173 fmol/mg prof.
E i
400 2O0
,8o
1
nM
®
E
200
Met-Enkephalin
E v
% -lOO ~
280
~
380
bound (fmol/mg prot.)
b i n d i n g in p i g e o n t e c t u m
1000
-50
800
- 40
A
El
-30
Lo E 600
E v
KD : 5.6 nM Bmax : 350 fmo[ / mg prot
o~ ~ 400
0
\
200
o12
o14
o16
1o
i
i
T
i
,
1--
f
o.8
1.o
o
lOO
200
300
400
'J
1
nM
bound (fmollmg prot)
Fig. 1. Double reciprocal and Scatchard plots of etorphine (A) and met-enkephalin (B) binding to crude tectal membrane preparations. Points represent the mean ± S.E.M. o f 3-4 separate experiments.
dissocation constant (Ka) 5.6 ~k 1.1/0.19 :~ 0.01 nM, and maximal binding (Bronx) 350 ± 44/172 ~ 12 fmol/mg protein. Microiontophoretically applied morphine (30-100 nA) reduced the spontaneous firing rate on 85 ~ of tested cells. The spike size was continuously recorded to assure that the depression was not due to a local anesthetic effect (Fig. 2A). The excitation by glutamate was depressed or abolished by morphine on all cells tested (Fig. 2B). Following the morphine ejection, an increased glutamate-sensitivity could be observed
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~ ,-.- ~~'~ r~ 0
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~
--~'.p.~ ~ ~ 0 ~ ~,..--'
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