European Journal of Pharmacology, 57 (1979) 115--125
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© Elsevier/North-Holland Biomedical Press
IS GLYCINE AN INHIBITORY SYNAPTIC TRANSMITTER IN THE SUBSTANTIA NIGRA? THOMAS A. JAMES and MICHAEL S. STARR
Department of Pharmacology The School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN lAX, England Received 12 January 1979, revised MS received 20 March 1979, accepted 24 April 1979
T.A. JAMES and M.S. STARR, Is glycine an inhibitory synaptic transmitter in the substantia nigra?, European J. Pharmacol. 57 (1979) 115--125. Nigral tissue accumulated 14C-glycine by an energy-, temperature- and sodium-dependent mechanism; the transport process was inhibited by small neutral amino acids and had an apparent Km of 143 pM and Vmax of 787 nmol/g/min. Re-release of accumulated 14C-glycine was initially extremely rapid (40% in first 5 rain) and subsequently accelerated by +40 mM K ÷ in a Ca2+-dependent manner. Intranigral kainate (0.25 pg) lowered the levels of striatal DA (63%) and nigral GABA (25%) ipsilaterally, but not nigral glycine. Injections of glycine or strychnine (10--100 pg) into one SN induced slow ipsiversive or contraversive turning respectively. The evidence for glycine as a neurotransmitter in SN is discussed. Substantia nigra
Glycine
1. Introduction The substantia nigra (SN) contains a population of short axon interneurones in the pars reticulata. These were first visualised with Golgi staining by Ramon y Cajal (1904) and more recently confirmed by other workers (Gulley and Wood, 1971; Hajdu et al., 1973; Bak et al., 1975). Cajal's original illustrations clearly show terminals of these Golgi stained cells impinging on the long dendritic processes of dopaminergic pars compacta neurones at what are currently believed to be functionally inhibitory synaptic junctions (see Dray and Straughan, 1976; Fahn, 1976). Indeed, the existence of tonically active inhibitory intrinsic fibres in the SN would provide a convenient ex_planation of how intranigrall.y administered GABA receptor agonists, instead of directly inhibiting the nigrostriatal dopamine (DA) neurones, may indirectly activate them by suppressing interneuronal firing (Dray et al., 1977; Di Chiara et al., 1978;
Martin and Haubrich, 1978;-MacNeil et al., 1978). The identity of the interneuronal inhibitory transmitter is unknown. Glycine was recently proposed as a possible candidate by Ch~ramy et al. (1978), but the supportive evidence is scanty and indirect. For example, it is known that t h e SN contains modest amounts of glycine, although the amino acid is fairly uniformly distributed throughout the brain (Perry et al., 1971). Okada and Hassler (1973) reported that SN slices were capable of taking up the exogenous amino acid, while Young and Snyder (1973) equated the strong binding of strychnine by midbrain tissue with an abundance of glycine receptors in this region of the brain. Although these binding studies were not directed specifically at the SN, later experiments provided evidence of the receptivity of SN neurones to this amino acid. Intranigrally administered glycine depressed the electrical activity of all types of nigral unit (Crossman et al., 1973; Dray and Gonye,
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1975; Dray et al., 1976), and diminished the o u t p u t of DA from the dopaminergic terminals in the corresponding striatum (Ch6ramy et al., 1978). These responses were allayed by strychnine. Little is known, however, of the processes of glycine uptake and release in SN, or of the behavioural consequences of microinjecting glycine or glycine receptor antagonists discretely into this nucleus. The present study seeks to remedy this deficit and assesses the current evidence for the transmitter status of glycine in the SN.
2. Materials and methods
2.1. Uptake experiments Experiments were performed using male Wistar albino rats (A.J. Tuck and Son, Rayleigh, Essex) weighing approximately 200 g. Animals were killed by cervical dislocation and their brains rapidly removed into icecold Krebs bicarbonate solution. Nigras were dissected o u t and divided into 0.2 mm thick sections using a mechanical tissue chopper. Tissue slices were placed in 25 ml conical flasks containing 10 ml Krebs solution, with or without added drugs, then gassed continuously with 5% CO2 in oxygen during 10 or 15 min preincubation at the appropriate temperature. ~4C-glycine was then added (0.1 or 0.57 pM, see text) and uptake determined after various times. At the end of each incubation slices were recovered, washed, lightly blotted and weighed on a torsion balance. Tissues were dissolved in 0.1 ml Soluene 350 (Packard), neutralised with 2 drops glacial acetic acid and prepared for liquid scintillation spectrometry by the addition of 2 ml 2-ethoxyethanol and 10 ml butyl PBD (0.5% w/v in toluene). Uptake was expressed as dpm/g wet wt. tissue: dpm/ml medium (T/M ratio). In the experiments to measure the kinetic parameters of glycine uptake nigras were cut into 0.2 mm × 0.2 mm cubes and incubated at 37°C for 10 min in the presence of glycine
T.A. JAMES, M.S. STARR
concentrations ranging from 10 -~ to 10 -3 M. Recovery of tissue was made by filtration under negative pressure onto preweighed paper discs. In order to determine the effect of extracellular Na ÷ concentration on 14C-glycine uptake slices of SN were preincubated and incubated in modified Krebs media, in which the sodium bicarbonate had been replaced by Tris/HC1 buffer (25 mM, pH 7.2) and the sodium chloride content varied by substituting iso-osmolar choline chloride. For comparative purposes the uptakes of several other nigral neurotransmitter candidates were studied. These included v-aminobutyric acid (GABA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT). Uptakes were determined after incubation for different times at 37°C with a substrate concentration of 10-TM. Aminooxyacetic acid (AOAA, 10 -s M) was included to eliminate the catabolism of GABA, while NA was protected against oxidation by ascorbic acid and disodium edetate (each at 10 mg/1).
2.2. Release experiments Slices of SN (0.2 mm thick) were preloaded with 14C-glycine by incubation with amino acid (10-~M) for 3 0 m i n at 37°C. Extracellular radioactivity was removed by washing the tissues for 1 min in fresh solution and the slices transferred randomly to each of the eight chambers (0.5 ml capacity) of a perspex perfusion bath. Perfusion was carried out with normal or modified Krebs solution (see text) at the rate of 0.75 ml/min from continuously oxygenated reservoirs at 37 ° C. The perfusates were collected routinely every 5 min for the first 3 0 m i n by an automatic fraction collector, and then every 2 min for a further 2 0 m i n . The effect of elevated K ÷ concentration on '4C-glycine outflow was studied by exposing the slices briefly to a high K ÷ medium (+40 mM KC1) at 38--42 min after the onset of perfusion. Residual tissue radioactivity was counted and the efflux of 14C-glycine calculated as the
GLYCINE IN THE SUBSTANTIANIGRA fractional rate coefficient ×100 (% min-l). Potassium-evoked release was calculated as the percentage increase in 14C liberated during the six collection periods after the onset of stimulation, compared to the six prestimulus perfusates.
2.3. Intranigral injections of kainic acid Kainic acid was dissolved in physiological saline and injected stereotaxically (0.25 #g in 0.5 pl) into the right SN of rats lightly anaesthetised with halothane (1% in oxygen), according to the following coordinates: A 2.0, L 2.0 and D +2.5 (KSnig and Klippel, 1963). One week later the animals were sacrificed and their brains removed and frozen in a solid CO2/methanol mixture within 1 rain of death. The corpora striata and nigras of both hemispheres were dissected from partially thawed brains and assayed respectively for dopamine (Shellenberger and Gordon, 1971) and free amino acids (Snodgrass and Iversen, 1973) using established fluorimetric and radiometric procedures. Results were expressed as nmol DA/g wet wt. and pmol amino acid/g wet wt.
2.4. Behavioural experiments Rats were lightly anaesthetised with halothane and injected stereotaxically with glycine or strychnine (1--100 pg in 0.5 ttl physiological saline) into one nigra (coordinates A 2.0-2.4, L 2.0 and D +2.5; KSnig and Klippel, 1963). After 1 0 m i n recovery from the anaesthetic appeared complete and the rats were placed in an open field and observed for signs of postural asymmetry, directional circling behaviour and other stereotyped behaviour (e.g. sniffing, gnawing, licking, etc.). The number of complete revolutions made by each rat during the 10--30 min post-injection period was scored and expressed as a cumulative total. Drug-induced turning on the spot was easily distinguished from the normal exploratory movements exhibited by non-, sham- or saline-injected controls.
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2.5. Materials All routine laboratory chemicals were of analytical purity except for 2-ethoxyethanol and toluene which were of scintillator grade. Aminooxyacetic acid and strychnine hydrochloride were purchased from Sigma Chemical Co., St. Louis, U.S.A. L-[7-3H(N)]-noradrenaline (2.2 Ci/mmol) was obtained from New England Nuclear, while 5-hydroxy [G-3H] tryptamine creatinine sulphate (10.7 Ci! mmol), 4-amino-n-[G-3H] butyric acid (27.2 Ci/mmol) and [U-14C] glycine ( l l 4 m C i / mmol) were obtained from The Radiochemical Centre, Amersham.
3. Results
3.1. Time course of 14C-glycine uptake The accumulation of 14C-glycine (10 -7 M) by thin slices of SN is illustrated in fig. 1. Glycine uptake was quantitatively small and gave tissue: medium (T/M) ratios of 7.7, 10.9 and 13.2 following incubation for 15, 30 and 45 min respectively. By comparison the avid accumulation of 3H-GABA, also at 10 -7 M and in the additional presence of aminooxyacetic acid (10 -s M), was linear with time and reached a T/M ratio in excess of 150 after 45 min. Two other putative nigral monoamine neurotransmitters were also studied, noradrenaline and 5-hydroxytryptamine. These, too, were taken up by SN slices and yielded T/M ratios of 7.0 and 56.9 respectively at 45 min (fig. 1), which roughly parallelled their endogenous tissue contents (Dray and Straughan, 1976).
3.2. Effect o f temperature on 14C-glycine uptake The temperature
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© Elsevier/North-Holland Biomedical Press
IS GLYCINE AN INHIBITORY SYNAPTIC TRANSMITTER IN THE SUBSTANTIA NIGRA? THOMAS A. JAMES and MICHAEL S. STARR
Department of Pharmacology The School of Pharmacy, University of London, 29/39 Brunswick Square, London WCIN lAX, England Received 12 January 1979, revised MS received 20 March 1979, accepted 24 April 1979
T.A. JAMES and M.S. STARR, Is glycine an inhibitory synaptic transmitter in the substantia nigra?, European J. Pharmacol. 57 (1979) 115--125. Nigral tissue accumulated 14C-glycine by an energy-, temperature- and sodium-dependent mechanism; the transport process was inhibited by small neutral amino acids and had an apparent Km of 143 pM and Vmax of 787 nmol/g/min. Re-release of accumulated 14C-glycine was initially extremely rapid (40% in first 5 rain) and subsequently accelerated by +40 mM K ÷ in a Ca2+-dependent manner. Intranigral kainate (0.25 pg) lowered the levels of striatal DA (63%) and nigral GABA (25%) ipsilaterally, but not nigral glycine. Injections of glycine or strychnine (10--100 pg) into one SN induced slow ipsiversive or contraversive turning respectively. The evidence for glycine as a neurotransmitter in SN is discussed. Substantia nigra
Glycine
1. Introduction The substantia nigra (SN) contains a population of short axon interneurones in the pars reticulata. These were first visualised with Golgi staining by Ramon y Cajal (1904) and more recently confirmed by other workers (Gulley and Wood, 1971; Hajdu et al., 1973; Bak et al., 1975). Cajal's original illustrations clearly show terminals of these Golgi stained cells impinging on the long dendritic processes of dopaminergic pars compacta neurones at what are currently believed to be functionally inhibitory synaptic junctions (see Dray and Straughan, 1976; Fahn, 1976). Indeed, the existence of tonically active inhibitory intrinsic fibres in the SN would provide a convenient ex_planation of how intranigrall.y administered GABA receptor agonists, instead of directly inhibiting the nigrostriatal dopamine (DA) neurones, may indirectly activate them by suppressing interneuronal firing (Dray et al., 1977; Di Chiara et al., 1978;
Martin and Haubrich, 1978;-MacNeil et al., 1978). The identity of the interneuronal inhibitory transmitter is unknown. Glycine was recently proposed as a possible candidate by Ch~ramy et al. (1978), but the supportive evidence is scanty and indirect. For example, it is known that t h e SN contains modest amounts of glycine, although the amino acid is fairly uniformly distributed throughout the brain (Perry et al., 1971). Okada and Hassler (1973) reported that SN slices were capable of taking up the exogenous amino acid, while Young and Snyder (1973) equated the strong binding of strychnine by midbrain tissue with an abundance of glycine receptors in this region of the brain. Although these binding studies were not directed specifically at the SN, later experiments provided evidence of the receptivity of SN neurones to this amino acid. Intranigrally administered glycine depressed the electrical activity of all types of nigral unit (Crossman et al., 1973; Dray and Gonye,
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1975; Dray et al., 1976), and diminished the o u t p u t of DA from the dopaminergic terminals in the corresponding striatum (Ch6ramy et al., 1978). These responses were allayed by strychnine. Little is known, however, of the processes of glycine uptake and release in SN, or of the behavioural consequences of microinjecting glycine or glycine receptor antagonists discretely into this nucleus. The present study seeks to remedy this deficit and assesses the current evidence for the transmitter status of glycine in the SN.
2. Materials and methods
2.1. Uptake experiments Experiments were performed using male Wistar albino rats (A.J. Tuck and Son, Rayleigh, Essex) weighing approximately 200 g. Animals were killed by cervical dislocation and their brains rapidly removed into icecold Krebs bicarbonate solution. Nigras were dissected o u t and divided into 0.2 mm thick sections using a mechanical tissue chopper. Tissue slices were placed in 25 ml conical flasks containing 10 ml Krebs solution, with or without added drugs, then gassed continuously with 5% CO2 in oxygen during 10 or 15 min preincubation at the appropriate temperature. ~4C-glycine was then added (0.1 or 0.57 pM, see text) and uptake determined after various times. At the end of each incubation slices were recovered, washed, lightly blotted and weighed on a torsion balance. Tissues were dissolved in 0.1 ml Soluene 350 (Packard), neutralised with 2 drops glacial acetic acid and prepared for liquid scintillation spectrometry by the addition of 2 ml 2-ethoxyethanol and 10 ml butyl PBD (0.5% w/v in toluene). Uptake was expressed as dpm/g wet wt. tissue: dpm/ml medium (T/M ratio). In the experiments to measure the kinetic parameters of glycine uptake nigras were cut into 0.2 mm × 0.2 mm cubes and incubated at 37°C for 10 min in the presence of glycine
T.A. JAMES, M.S. STARR
concentrations ranging from 10 -~ to 10 -3 M. Recovery of tissue was made by filtration under negative pressure onto preweighed paper discs. In order to determine the effect of extracellular Na ÷ concentration on 14C-glycine uptake slices of SN were preincubated and incubated in modified Krebs media, in which the sodium bicarbonate had been replaced by Tris/HC1 buffer (25 mM, pH 7.2) and the sodium chloride content varied by substituting iso-osmolar choline chloride. For comparative purposes the uptakes of several other nigral neurotransmitter candidates were studied. These included v-aminobutyric acid (GABA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT). Uptakes were determined after incubation for different times at 37°C with a substrate concentration of 10-TM. Aminooxyacetic acid (AOAA, 10 -s M) was included to eliminate the catabolism of GABA, while NA was protected against oxidation by ascorbic acid and disodium edetate (each at 10 mg/1).
2.2. Release experiments Slices of SN (0.2 mm thick) were preloaded with 14C-glycine by incubation with amino acid (10-~M) for 3 0 m i n at 37°C. Extracellular radioactivity was removed by washing the tissues for 1 min in fresh solution and the slices transferred randomly to each of the eight chambers (0.5 ml capacity) of a perspex perfusion bath. Perfusion was carried out with normal or modified Krebs solution (see text) at the rate of 0.75 ml/min from continuously oxygenated reservoirs at 37 ° C. The perfusates were collected routinely every 5 min for the first 3 0 m i n by an automatic fraction collector, and then every 2 min for a further 2 0 m i n . The effect of elevated K ÷ concentration on '4C-glycine outflow was studied by exposing the slices briefly to a high K ÷ medium (+40 mM KC1) at 38--42 min after the onset of perfusion. Residual tissue radioactivity was counted and the efflux of 14C-glycine calculated as the
GLYCINE IN THE SUBSTANTIANIGRA fractional rate coefficient ×100 (% min-l). Potassium-evoked release was calculated as the percentage increase in 14C liberated during the six collection periods after the onset of stimulation, compared to the six prestimulus perfusates.
2.3. Intranigral injections of kainic acid Kainic acid was dissolved in physiological saline and injected stereotaxically (0.25 #g in 0.5 pl) into the right SN of rats lightly anaesthetised with halothane (1% in oxygen), according to the following coordinates: A 2.0, L 2.0 and D +2.5 (KSnig and Klippel, 1963). One week later the animals were sacrificed and their brains removed and frozen in a solid CO2/methanol mixture within 1 rain of death. The corpora striata and nigras of both hemispheres were dissected from partially thawed brains and assayed respectively for dopamine (Shellenberger and Gordon, 1971) and free amino acids (Snodgrass and Iversen, 1973) using established fluorimetric and radiometric procedures. Results were expressed as nmol DA/g wet wt. and pmol amino acid/g wet wt.
2.4. Behavioural experiments Rats were lightly anaesthetised with halothane and injected stereotaxically with glycine or strychnine (1--100 pg in 0.5 ttl physiological saline) into one nigra (coordinates A 2.0-2.4, L 2.0 and D +2.5; KSnig and Klippel, 1963). After 1 0 m i n recovery from the anaesthetic appeared complete and the rats were placed in an open field and observed for signs of postural asymmetry, directional circling behaviour and other stereotyped behaviour (e.g. sniffing, gnawing, licking, etc.). The number of complete revolutions made by each rat during the 10--30 min post-injection period was scored and expressed as a cumulative total. Drug-induced turning on the spot was easily distinguished from the normal exploratory movements exhibited by non-, sham- or saline-injected controls.
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2.5. Materials All routine laboratory chemicals were of analytical purity except for 2-ethoxyethanol and toluene which were of scintillator grade. Aminooxyacetic acid and strychnine hydrochloride were purchased from Sigma Chemical Co., St. Louis, U.S.A. L-[7-3H(N)]-noradrenaline (2.2 Ci/mmol) was obtained from New England Nuclear, while 5-hydroxy [G-3H] tryptamine creatinine sulphate (10.7 Ci! mmol), 4-amino-n-[G-3H] butyric acid (27.2 Ci/mmol) and [U-14C] glycine ( l l 4 m C i / mmol) were obtained from The Radiochemical Centre, Amersham.
3. Results
3.1. Time course of 14C-glycine uptake The accumulation of 14C-glycine (10 -7 M) by thin slices of SN is illustrated in fig. 1. Glycine uptake was quantitatively small and gave tissue: medium (T/M) ratios of 7.7, 10.9 and 13.2 following incubation for 15, 30 and 45 min respectively. By comparison the avid accumulation of 3H-GABA, also at 10 -7 M and in the additional presence of aminooxyacetic acid (10 -s M), was linear with time and reached a T/M ratio in excess of 150 after 45 min. Two other putative nigral monoamine neurotransmitters were also studied, noradrenaline and 5-hydroxytryptamine. These, too, were taken up by SN slices and yielded T/M ratios of 7.0 and 56.9 respectively at 45 min (fig. 1), which roughly parallelled their endogenous tissue contents (Dray and Straughan, 1976).
3.2. Effect o f temperature on 14C-glycine uptake The temperature
