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Selective attenuation of self-stimulation and sidman avoidance by baclofen: Dissociation from muscle relaxant effects
INTERNATIONAL
690
A131
SYMPOSIUM ON GABA
Al34
The effects of baclofen (BF) in rats were investigated on self-stimulation in several different experimental paradigms, and on Sidman avoidance. When bar-pressing baselines a"eraged W-100 bar-presses per min.. SF (3 mg/kg i.p.) had no significant effect on self-stimulation. However, when the current intensity in the same animals was lowered to yield baseline rates of approximately 20 bar-presses per min. SF significantly reduced self-stimulation at 1 and 3 mglkg. In Sidman avoidance, with baseline bar-pressing rates approximately 8 oer min. BF had no sienificant effect at 3 melke. I ". indicating that k's greater effect on self-stimulation at lower stimulation intensities was not simply a rate-dependent phenomenon. When several differenr current infensities were
presented
in the same
session,
BF selectively
re-
duced self-stimulation at lower current intensities. Latenties to initiate rewarding brain srimulatian in a shuttlebox test were elevated by BF more strongly than were latencies tu terminate stimulation. Thus. the attenuating effect of baclofen on self-stimulation cannot be atfributed solely to a performance deficit. 1t is suggested that BF reduces release of "eurotransmitter (possibly dopamine) which is involved in reward, but that higher brain stimulation intensities DYercome this effect.
A135 A CONvl?NIENT
Al32 BARBITURATE TOLERANCE AND DEPENDENCE INVESTIGATED IN TISSUE CULTURE. P. Mandel, B. Roth-Schechter and G. Tholey. Centre de Neurochimie du CNRS. 11 rue Humann, 67085 Strasbourg cedex - France Exposure of glial cells of primary culture and of a continuous cell line (clone NN) to pentobarbital (Pb) induces biochemical tolerance, as defined by the response of cellular respiration and glucose consumption challenging dose of Pb. The tolerance to Pb is accompanied by increased activities of hexokinase, glucose-'&phosphate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase and by a marked increase in the number of olial cell mitochondria. as observed in electron micrograph;. The results are inte;preted to indicate a compensation by glial cells to the continuous presence of Pb by an accelerated glucose uptake and metabolism, an accelerated metabolism of succinate, and an induction of mitochondrial neobiogenesis.
to a
FOR y-AMINOBUTYRIC
ACID
TRANSAMINASE.
Al36
Al33
prevented growth in rats fed 8 6% casein diet. The effect "8s barely apparent "hen GAB* was 1.5% of the diet,
and
but was
ASSAY
Setti S. Rengachwy and Bob In-yu Yang. Depart&t of Chemistry, University of Mi8souri, Kansas City, HO 64110. Although qa"y methods have been used for the determination.of y-aminobutryic acid transaminase (GAS&T) activity. most suffer various disadvantages such as the use of radio: active substrates, isolation of reaction products and disCo"ti""O"8 (or stop) assays. The spectrophotometric procedure of Jakoby (Methods in Enryrrology,v, 777 (1962)). coupling succinic srmieldehyde dehydrogenase (SSADH) to CAM-T is straightforward but the preparation of the dehydrogenase has been complicated. We have devised a facile technique for isolating SSAD" from a conm~rcielly available mixture of GABA-T and SSMIH. The enzyne mixture "as passed through a Sephadex G-25-40 column in 5M phosphate buffer, p" 7.2, to rewve interfering ions. The enzymes were then adsorbed onto a two-ml S-NADP-agarose column. CAM-T vas removed with 5omN phosphate buffer, pH 7.2. SSADH "a8 rhen specifically desorbed with IOr&,NADP in 50&f phqpphate buffer. p" 7.2. An 80% yield of SSADH was obtained. ContaminaLion by CAM-T and NAP'" oxidase was determined co be 0.2 and 0.04% respectively. The SSAD" so prepared was stable, e.g. reteining 89% of the acti"ity after 24 days, when scored in concentrated farm in the presence of glycerol plus bovine serum albumin or egg lysolecithin. Direcr determninationof CAM-T acti"ify. usi,,p. Iu-14ClGABA as the substrate and separating radiaa&"e S&-by ion exchange chromatography, yielded eransaminase activity comparable to that arrived at by the epecerophatonerric assay.
almost
a.5 great
with
3% as 4.5%
GAB&..
Increasing
dietary protein lessened the inhibitory effects of GAB*; in one study, when caaein "as 6, 12, 20, 40 or 60% of the diet growth was 10, aT, 51, 67 or 862, and food TO, 73 or 904, Of values for corresponding when GABA Y&S removed from the diet GABA.
intake 55, 63, controls wxttlollt growth and food
intake promptly increased to control rates. Plasma GAB* levels decreased as dietary protein increased (1.4 or 0.2 w~~les/ml in rats fed 6 or 60% casein). After feeding a 60% casein diet for 4 days GA&k-transaminase (GAB&T) activity reached B plateau at about *"ice that for rats eating 6% casein
(104 YS
55 nmo&~
glulmg
protein/15
min);
when
casein "as decreased to 6X, activity decliped. Such trcatments had no consistent effect On brain or kidney GAB&T. In rats fed 80% cnsein hepatic GABA-T was about twice that in rats ted o or 6% casein; total activity per liver was calculated to increase from 14 umoles glu (0% casein) to 106 wholes (80%). About 20% of injected 14C-GABA (2 mmolesl 100 8 rat) was converted to 14C02 in 6 h by rat5 fed 6% casein, as compared to 40% conversion by rats fed 20 or 60%
casein; urinary radioactivity "as not altered by dietary protein. Relationships between these effects of CAB* on food intake and grOYth and its function in the CNS are not clear. (Supported by "SPHS Grant AM 10747.)
SINGLE UNIT RECORDING STUDIES OF EFFECTS OF GABA AGONISTS AND ANTAGONISTS ON WPAMINERGIC NEURONS. B.L. Waszczak and J.R. Walters, NINCDS, NIH, Bethesda, MD 20205. The recent development of a series of GABA agonists make it possible to test various hypotheses concerning the function of GABA pathways in the central nervcw system. In pwtitular, it has been thought that GABAergic neurons may provide a tonic inhibitory influence upon dopaminergic neurons (Ag) and the venof the substantia ni ra (SN) pars compacta tral tegrnental area 9 AlO). There studies explore the effects of two related GABA agonists, muscimol (MUX) and 4,5,6,7tetrahydroisoxazolo-[5,4-cl-pyridin-3-01 (THIP), and the GABA antagonist, picrotoxin (PIC). upon the single unit activity of both populations of dopamine (DA) neurons. Unexpectedly, i.v. administration of MUSC and THIP caused doserelated increases ifa the firing of both Ag and A10 OA neurons. However, non-dopaminergic neurons of the SN pars reticulata were markedly inhibited by i.v. administration of While the inhibition of reticulata cell firing these drugs. was PIC-reversible, the stimulatory effects of MUSC and THIP upon DA neurons were not reversed by PIC. In contrast to their systemic actions, iontophoresis of MUSC and THIP slowed the firing of both DA and pars reticulata neurons although the DA cells were much less sensitive to these inhibitory effects. Therefore, despite the ability of MUSC and THIP to inhibit dopaminergic activity when applied directly, these drugs increase DA cell firing after i.v. treatment. Systemic administration of these agents does not appear to provide a useful pharmacologic or therapeutic means of inhibiting the activity of A9 or A10 dopaminergic neurons.
690
A131
SYMPOSIUM ON GABA
Al34
The effects of baclofen (BF) in rats were investigated on self-stimulation in several different experimental paradigms, and on Sidman avoidance. When bar-pressing baselines a"eraged W-100 bar-presses per min.. SF (3 mg/kg i.p.) had no significant effect on self-stimulation. However, when the current intensity in the same animals was lowered to yield baseline rates of approximately 20 bar-presses per min. SF significantly reduced self-stimulation at 1 and 3 mglkg. In Sidman avoidance, with baseline bar-pressing rates approximately 8 oer min. BF had no sienificant effect at 3 melke. I ". indicating that k's greater effect on self-stimulation at lower stimulation intensities was not simply a rate-dependent phenomenon. When several differenr current infensities were
presented
in the same
session,
BF selectively
re-
duced self-stimulation at lower current intensities. Latenties to initiate rewarding brain srimulatian in a shuttlebox test were elevated by BF more strongly than were latencies tu terminate stimulation. Thus. the attenuating effect of baclofen on self-stimulation cannot be atfributed solely to a performance deficit. 1t is suggested that BF reduces release of "eurotransmitter (possibly dopamine) which is involved in reward, but that higher brain stimulation intensities DYercome this effect.
A135 A CONvl?NIENT
Al32 BARBITURATE TOLERANCE AND DEPENDENCE INVESTIGATED IN TISSUE CULTURE. P. Mandel, B. Roth-Schechter and G. Tholey. Centre de Neurochimie du CNRS. 11 rue Humann, 67085 Strasbourg cedex - France Exposure of glial cells of primary culture and of a continuous cell line (clone NN) to pentobarbital (Pb) induces biochemical tolerance, as defined by the response of cellular respiration and glucose consumption challenging dose of Pb. The tolerance to Pb is accompanied by increased activities of hexokinase, glucose-'&phosphate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase and by a marked increase in the number of olial cell mitochondria. as observed in electron micrograph;. The results are inte;preted to indicate a compensation by glial cells to the continuous presence of Pb by an accelerated glucose uptake and metabolism, an accelerated metabolism of succinate, and an induction of mitochondrial neobiogenesis.
to a
FOR y-AMINOBUTYRIC
ACID
TRANSAMINASE.
Al36
Al33
prevented growth in rats fed 8 6% casein diet. The effect "8s barely apparent "hen GAB* was 1.5% of the diet,
and
but was
ASSAY
Setti S. Rengachwy and Bob In-yu Yang. Depart&t of Chemistry, University of Mi8souri, Kansas City, HO 64110. Although qa"y methods have been used for the determination.of y-aminobutryic acid transaminase (GAS&T) activity. most suffer various disadvantages such as the use of radio: active substrates, isolation of reaction products and disCo"ti""O"8 (or stop) assays. The spectrophotometric procedure of Jakoby (Methods in Enryrrology,v, 777 (1962)). coupling succinic srmieldehyde dehydrogenase (SSADH) to CAM-T is straightforward but the preparation of the dehydrogenase has been complicated. We have devised a facile technique for isolating SSAD" from a conm~rcielly available mixture of GABA-T and SSMIH. The enzyne mixture "as passed through a Sephadex G-25-40 column in 5M phosphate buffer, p" 7.2, to rewve interfering ions. The enzymes were then adsorbed onto a two-ml S-NADP-agarose column. CAM-T vas removed with 5omN phosphate buffer, pH 7.2. SSADH "a8 rhen specifically desorbed with IOr&,NADP in 50&f phqpphate buffer. p" 7.2. An 80% yield of SSADH was obtained. ContaminaLion by CAM-T and NAP'" oxidase was determined co be 0.2 and 0.04% respectively. The SSAD" so prepared was stable, e.g. reteining 89% of the acti"ity after 24 days, when scored in concentrated farm in the presence of glycerol plus bovine serum albumin or egg lysolecithin. Direcr determninationof CAM-T acti"ify. usi,,p. Iu-14ClGABA as the substrate and separating radiaa&"e S&-by ion exchange chromatography, yielded eransaminase activity comparable to that arrived at by the epecerophatonerric assay.
almost
a.5 great
with
3% as 4.5%
GAB&..
Increasing
dietary protein lessened the inhibitory effects of GAB*; in one study, when caaein "as 6, 12, 20, 40 or 60% of the diet growth was 10, aT, 51, 67 or 862, and food TO, 73 or 904, Of values for corresponding when GABA Y&S removed from the diet GABA.
intake 55, 63, controls wxttlollt growth and food
intake promptly increased to control rates. Plasma GAB* levels decreased as dietary protein increased (1.4 or 0.2 w~~les/ml in rats fed 6 or 60% casein). After feeding a 60% casein diet for 4 days GA&k-transaminase (GAB&T) activity reached B plateau at about *"ice that for rats eating 6% casein
(104 YS
55 nmo&~
glulmg
protein/15
min);
when
casein "as decreased to 6X, activity decliped. Such trcatments had no consistent effect On brain or kidney GAB&T. In rats fed 80% cnsein hepatic GABA-T was about twice that in rats ted o or 6% casein; total activity per liver was calculated to increase from 14 umoles glu (0% casein) to 106 wholes (80%). About 20% of injected 14C-GABA (2 mmolesl 100 8 rat) was converted to 14C02 in 6 h by rat5 fed 6% casein, as compared to 40% conversion by rats fed 20 or 60%
casein; urinary radioactivity "as not altered by dietary protein. Relationships between these effects of CAB* on food intake and grOYth and its function in the CNS are not clear. (Supported by "SPHS Grant AM 10747.)
SINGLE UNIT RECORDING STUDIES OF EFFECTS OF GABA AGONISTS AND ANTAGONISTS ON WPAMINERGIC NEURONS. B.L. Waszczak and J.R. Walters, NINCDS, NIH, Bethesda, MD 20205. The recent development of a series of GABA agonists make it possible to test various hypotheses concerning the function of GABA pathways in the central nervcw system. In pwtitular, it has been thought that GABAergic neurons may provide a tonic inhibitory influence upon dopaminergic neurons (Ag) and the venof the substantia ni ra (SN) pars compacta tral tegrnental area 9 AlO). There studies explore the effects of two related GABA agonists, muscimol (MUX) and 4,5,6,7tetrahydroisoxazolo-[5,4-cl-pyridin-3-01 (THIP), and the GABA antagonist, picrotoxin (PIC). upon the single unit activity of both populations of dopamine (DA) neurons. Unexpectedly, i.v. administration of MUSC and THIP caused doserelated increases ifa the firing of both Ag and A10 OA neurons. However, non-dopaminergic neurons of the SN pars reticulata were markedly inhibited by i.v. administration of While the inhibition of reticulata cell firing these drugs. was PIC-reversible, the stimulatory effects of MUSC and THIP upon DA neurons were not reversed by PIC. In contrast to their systemic actions, iontophoresis of MUSC and THIP slowed the firing of both DA and pars reticulata neurons although the DA cells were much less sensitive to these inhibitory effects. Therefore, despite the ability of MUSC and THIP to inhibit dopaminergic activity when applied directly, these drugs increase DA cell firing after i.v. treatment. Systemic administration of these agents does not appear to provide a useful pharmacologic or therapeutic means of inhibiting the activity of A9 or A10 dopaminergic neurons.