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Selection of control populations for clinical cerebrospinal fluid GABA investigations based on comparison with normal volunteers
INTERNATIONAL
700
Al93
acios,&W. ScottYarq III.Department Of Ih~nmcolcgy.JchnsHopkinsuliwrsityschml of Medicine, Salto,MD 21205 * technique involving the in vitrolakliq of mounted tissue sections has beenusedt0lccaliz.e GRBR and BZ receptors at the lightmicmsnpic level. 3H-wscinvlva5usedto label GABAreceptors and 3H-fltmitrazepam was usedto labelBZ receptors. ?he bindingof radioactive dmqs has allof the characteristics associated witha phanramlcgically validreceptor.For example, 3H%uscinolbirdiq had a KD of 6-13nt4, a& of atcat200 fm=l/hg tissue,and was displaced by CABAagonists ad antagonists but was not displaced by picmxoxinof WIBAuptakeinhibitors. I" the rat cerebellum, WIBA receptors were Vex-Y highlylocalized to the granulecelllayer,witha rmchsmallerlevelin the molecular layer. Negligible levelswerefoundin thewhitematter. & catrast, BZ receptors werehighlylocalized to the rrolecular are in ag-ntwith the nolayer. ?hesefindings tionthatthe highaffinity Cubabindingsiteis not uniquely linkedto the BZ receptor. .GABAand Bz receptors wererappedin detailin the rat brainand alsoexaminedin the himanbrain. lhe dLstribution of SZ receptors suggestmajorsitesof actionof thesedrugsin the brainand helpsexplainthephysiolcgical effectsof thesedrugs. S"ppxw?dby USEISgrantsMH25951, MMooo53. DAOO266, TWO2583and MHO7624.
Al94 NEUROANATOMICAL SUBSTRATES OF CARDIOVASCULAR POSSIBLE SlTES WHERE GABAERGIC FUNCTION: TRANSMISSION MAY BE IMPORTANT. Betty L. Hamilton. Dept. of Anatomy,Geargetown University Sch&s of MedIcme and Dentrstry, Washmgton, D.C. 20007. Various branstem kites have been shown to be important for medratmg changes m cardiovascular system function induced by GABA agonists and antagomst drugs. These w!ll be discussed m terms of their cellular morphology, afferent co”“ectm”s, and f?**erent cytoarchitectonrcs, transmltfers and fmally, therr e~acf stereoxtaxic locations in a living cat. Thrs will encompass both a review of the exlstlng hferature and some unpublished work from OUT laboratory. The generai framework far thrs presentation was based on computewed ala, tamograph,c scans of the head of a cat held in true stereotaxic position. These scans were correlated with both gross slices and histological sections f~ provide better coordinates far approachrng bran stem. part~ularly The mdividual areas medullary, st~uctuces sttereotaxically. m&de the ventral surface of the medulla oblangata, the chemosensitive areas of Mitchell (‘63), Schlsfke (‘67, ‘70). and Loeschske (‘70); the lateral reticular nucleus; nucleus ambiguus; nucleus of the t~acf~s solitarmus; dorsal nucleus of the vagus; nucleus raphe dorsahs and the periaqueducta, gray matter.
SYMPOSIUM
ON GABA
Al96 MUSCIMOL THERAPY or WP~KKINET,C EXWAPYWM~OAI DISORDERS Thomas N. Chase and Carol A. Tanninga, Experimental TheraFrutlcs Branch, NINCDS, Bethesda. Md. Anatomic and phariracologic evidence in the experimental aniolal suggest the participation of GABA neurons in the regulation of extrapyramidal motor function. In one hyperkinetic extrapjramidal disorder, Huntington's disease, there i)~echaracteristic reductions in striatal levels of GABA and in the activity of glutamic acid decarboxylasr. These observations prompted double-blind placebo-controlled trials of muscimol, a potent GABPmimetic, in otherwise untreated patients with Huntinqton's analog of GABA chorea or tardive dyskinesia. This semirigid structural was given orally, once daily, in doses ranging from 3 to 14 mg. NO consistent effect on choreatic movements in Huntingtonian patients was noted. Individuals with tardive dyskinesia. on the other hand, manifested a significant reduction in dyskinesias, with the peak response occurring about 2 hours after drug administration. Concomitant parkinsonian signs, when present, tended to worsen. Adverse reactions included sedation, diffuse myoclonir Jerking. and psychotomimetic effects. These results indicate that muscimol will have no practical value in the treatment of Huntington's disease 01‘tardive dyskinesia, but should encb*bge further clinical trials of drugs which potcntiate W\BA-mediated synaptic transmission in patients with extrapyramidal dysfunction.
Al97 SELECTION OF CONTqOL POPULATIONS FOR CLINICAL CERBBROSPINAL FLUID GABA INVESTIGATIONS BABED ON COMPARISON WIT" NORMAL VOLUNTEERS. Theo-
dore A. Hare, JaiuesH. Wood, N.". Bala "anyam, James C. Ballenger, Robert M. Post. Thomas Jefferson Univ., Phlla., PA, Univ. of Penn., Phila., PA. Vet. Admin. Ned. 6 Reg. Office cent., Wilmington, DE and NIGH, Bethesda, MD Altered cerebrospinal fluid (CSF) GABA levels have been implicated in various neurologic and psychiatric disorders, therefore, the establishment of the normal range of human CSF GABA is essential. CSF speciroenswire collected, stored and prepared for analysis under conditions which assure GABA data validity. The mean CSF GaBA concentrations determined by an ion-exchange/fluorometricmethod in 40 normal volunteers was 233t75 (SD1 pmol/ml. Mean CSF GABA level in females (N=l4) was 254f 54 ml/ml while that in males (N=261 was 222f 90 pmol/ml. These values compare favorably With the mean CSF GABA concentration of 233fSO pmol/ml obtained from 20 "control" patients with no evidence of organic neurologic or mental disease. The mean CSF GABA levels for the control females (N=S) and males (N=12) were 192272 and 236f92 pmol/ml, respectively. Thus, careful selection of both methodology and patients in clinical protocols ray yield acceptable control groups for data comparison.
A195 MOWLATION OF TRANSMISSIDN IN THE NAT PINEAL BY GLIAL GABA. Robert A. Nanlewski and knln Suria, Dept. of Phanacolagy. George Washingron Univ. Ned. Center, Washington, D.C. 20037. Previous studies have demonstrated that 3H-GABA is taken up exclusively by glial cells in the rat pineal gland. Ye have found that endogenous pines1 levels of G&I, ranging frm 105-450 pnoles/mg prot.,do not change at 1, 2 or 6 weeks after denervation. This further fndicates a nonneuronal locus for the plneal GABA system. GAD activity was found to be 176.4 pnoles/mg prot./min. Ethanolamine-D-sulphrte (4DD mg/kg; i.p.) elevated pineal GABA levels fran 238.4*47.7 to 3615.8+1063.7 pnoles/mg prot. GABA receptor bindiiigpropertfes w&e studied in bovine pineals. Two populations of sodium-independent, saturable GABA-bindirtg sites were observed. One site had an affinity of 27.5 nM fK& and a 6,jq,x of 0.821 pnoles/ng prot.,whereas the other had a K of 190.5 nN and a hx of 2.242 proles/mg prot. NorepPnephrine (NE) infusion (5 ug/kg/min.; i.v.;:1 hr) in the presence of ADAA (50 mg/kg; i.p., 4 hr) signifi; cantly elevated GASA levels fran 565.4505.7 to 1029.2+ 136.1 pnoles/mg prot. The funcri$nal role of pinyl gTia1 GA6A was then studied. GA6A (lo- M) plus NE (lo- H) significantly elevated rat pinea cyclic GMP fra 170.1+55.a. pnoles/ng prot. with WE alone to 477+126.4. Thus,pineal glial GA6A increases in response to iioradrenergicstfmulstion and patcntiates the NE-induced increase fn cGMP. Since ctnP is thought to inhibit the release of NE, glial GABA may play a m&latory role to augment presynaptic inhibition. (Supported by NIt4HGrant XROl-W-30024-03)
Al98 ~'HAWIACOLOGY01 GABA OUlPUi FROM nii CEREBRAL CCRTEX IN FKttLY MOVING KfiTS.F. Morons, F. Casamenti & G. Pepeu - Dept. of Pharmacology, University of Florence, Italy. G4BA, glutanlate(GL) and glutamine (GLN) were measured by a mass-fragnlentographicmethod in Ringer solution filling epidural cups of freely moving rats and in cortical cups of urethane anaesthetized rats. The samples were collected ever,! 20 min. The concentration in epidural cups of GABA, GL and GLN was respectively 0.6 t 0.08, 9.3 1 1.2 and I1 i 2.1 nmole/cm2/ min (6 rats-)and remained remarkably constant over a period of at least two hrs. Similar values were found in the anaesthetized rats. The addition of KC1 50 nM to the cortical cups was followed by a more than 20 times increase in GABA level and by a significant decrease in GL level. The i.c.v. administration of aminooxyacetic acid (30 ug) caused a 47 % increase in GABA level in the epidural cups but no change in blood GABA
level.
We
conclude that GABA and part of GL found in the cups are released from the underlying nervous tissue. Change in GAEA level in the epidural cups may therefore indicate changes in the activity of the GABAergic neurons. The effect of drugs affecting GABAergic mechanisms are presently investigated.
700
Al93
acios,&W. ScottYarq III.Department Of Ih~nmcolcgy.JchnsHopkinsuliwrsityschml of Medicine, Salto,MD 21205 * technique involving the in vitrolakliq of mounted tissue sections has beenusedt0lccaliz.e GRBR and BZ receptors at the lightmicmsnpic level. 3H-wscinvlva5usedto label GABAreceptors and 3H-fltmitrazepam was usedto labelBZ receptors. ?he bindingof radioactive dmqs has allof the characteristics associated witha phanramlcgically validreceptor.For example, 3H%uscinolbirdiq had a KD of 6-13nt4, a& of atcat200 fm=l/hg tissue,and was displaced by CABAagonists ad antagonists but was not displaced by picmxoxinof WIBAuptakeinhibitors. I" the rat cerebellum, WIBA receptors were Vex-Y highlylocalized to the granulecelllayer,witha rmchsmallerlevelin the molecular layer. Negligible levelswerefoundin thewhitematter. & catrast, BZ receptors werehighlylocalized to the rrolecular are in ag-ntwith the nolayer. ?hesefindings tionthatthe highaffinity Cubabindingsiteis not uniquely linkedto the BZ receptor. .GABAand Bz receptors wererappedin detailin the rat brainand alsoexaminedin the himanbrain. lhe dLstribution of SZ receptors suggestmajorsitesof actionof thesedrugsin the brainand helpsexplainthephysiolcgical effectsof thesedrugs. S"ppxw?dby USEISgrantsMH25951, MMooo53. DAOO266, TWO2583and MHO7624.
Al94 NEUROANATOMICAL SUBSTRATES OF CARDIOVASCULAR POSSIBLE SlTES WHERE GABAERGIC FUNCTION: TRANSMISSION MAY BE IMPORTANT. Betty L. Hamilton. Dept. of Anatomy,Geargetown University Sch&s of MedIcme and Dentrstry, Washmgton, D.C. 20007. Various branstem kites have been shown to be important for medratmg changes m cardiovascular system function induced by GABA agonists and antagomst drugs. These w!ll be discussed m terms of their cellular morphology, afferent co”“ectm”s, and f?**erent cytoarchitectonrcs, transmltfers and fmally, therr e~acf stereoxtaxic locations in a living cat. Thrs will encompass both a review of the exlstlng hferature and some unpublished work from OUT laboratory. The generai framework far thrs presentation was based on computewed ala, tamograph,c scans of the head of a cat held in true stereotaxic position. These scans were correlated with both gross slices and histological sections f~ provide better coordinates far approachrng bran stem. part~ularly The mdividual areas medullary, st~uctuces sttereotaxically. m&de the ventral surface of the medulla oblangata, the chemosensitive areas of Mitchell (‘63), Schlsfke (‘67, ‘70). and Loeschske (‘70); the lateral reticular nucleus; nucleus ambiguus; nucleus of the t~acf~s solitarmus; dorsal nucleus of the vagus; nucleus raphe dorsahs and the periaqueducta, gray matter.
SYMPOSIUM
ON GABA
Al96 MUSCIMOL THERAPY or WP~KKINET,C EXWAPYWM~OAI DISORDERS Thomas N. Chase and Carol A. Tanninga, Experimental TheraFrutlcs Branch, NINCDS, Bethesda. Md. Anatomic and phariracologic evidence in the experimental aniolal suggest the participation of GABA neurons in the regulation of extrapyramidal motor function. In one hyperkinetic extrapjramidal disorder, Huntington's disease, there i)~echaracteristic reductions in striatal levels of GABA and in the activity of glutamic acid decarboxylasr. These observations prompted double-blind placebo-controlled trials of muscimol, a potent GABPmimetic, in otherwise untreated patients with Huntinqton's analog of GABA chorea or tardive dyskinesia. This semirigid structural was given orally, once daily, in doses ranging from 3 to 14 mg. NO consistent effect on choreatic movements in Huntingtonian patients was noted. Individuals with tardive dyskinesia. on the other hand, manifested a significant reduction in dyskinesias, with the peak response occurring about 2 hours after drug administration. Concomitant parkinsonian signs, when present, tended to worsen. Adverse reactions included sedation, diffuse myoclonir Jerking. and psychotomimetic effects. These results indicate that muscimol will have no practical value in the treatment of Huntington's disease 01‘tardive dyskinesia, but should encb*bge further clinical trials of drugs which potcntiate W\BA-mediated synaptic transmission in patients with extrapyramidal dysfunction.
Al97 SELECTION OF CONTqOL POPULATIONS FOR CLINICAL CERBBROSPINAL FLUID GABA INVESTIGATIONS BABED ON COMPARISON WIT" NORMAL VOLUNTEERS. Theo-
dore A. Hare, JaiuesH. Wood, N.". Bala "anyam, James C. Ballenger, Robert M. Post. Thomas Jefferson Univ., Phlla., PA, Univ. of Penn., Phila., PA. Vet. Admin. Ned. 6 Reg. Office cent., Wilmington, DE and NIGH, Bethesda, MD Altered cerebrospinal fluid (CSF) GABA levels have been implicated in various neurologic and psychiatric disorders, therefore, the establishment of the normal range of human CSF GABA is essential. CSF speciroenswire collected, stored and prepared for analysis under conditions which assure GABA data validity. The mean CSF GaBA concentrations determined by an ion-exchange/fluorometricmethod in 40 normal volunteers was 233t75 (SD1 pmol/ml. Mean CSF GABA level in females (N=l4) was 254f 54 ml/ml while that in males (N=261 was 222f 90 pmol/ml. These values compare favorably With the mean CSF GABA concentration of 233fSO pmol/ml obtained from 20 "control" patients with no evidence of organic neurologic or mental disease. The mean CSF GABA levels for the control females (N=S) and males (N=12) were 192272 and 236f92 pmol/ml, respectively. Thus, careful selection of both methodology and patients in clinical protocols ray yield acceptable control groups for data comparison.
A195 MOWLATION OF TRANSMISSIDN IN THE NAT PINEAL BY GLIAL GABA. Robert A. Nanlewski and knln Suria, Dept. of Phanacolagy. George Washingron Univ. Ned. Center, Washington, D.C. 20037. Previous studies have demonstrated that 3H-GABA is taken up exclusively by glial cells in the rat pineal gland. Ye have found that endogenous pines1 levels of G&I, ranging frm 105-450 pnoles/mg prot.,do not change at 1, 2 or 6 weeks after denervation. This further fndicates a nonneuronal locus for the plneal GABA system. GAD activity was found to be 176.4 pnoles/mg prot./min. Ethanolamine-D-sulphrte (4DD mg/kg; i.p.) elevated pineal GABA levels fran 238.4*47.7 to 3615.8+1063.7 pnoles/mg prot. GABA receptor bindiiigpropertfes w&e studied in bovine pineals. Two populations of sodium-independent, saturable GABA-bindirtg sites were observed. One site had an affinity of 27.5 nM fK& and a 6,jq,x of 0.821 pnoles/ng prot.,whereas the other had a K of 190.5 nN and a hx of 2.242 proles/mg prot. NorepPnephrine (NE) infusion (5 ug/kg/min.; i.v.;:1 hr) in the presence of ADAA (50 mg/kg; i.p., 4 hr) signifi; cantly elevated GASA levels fran 565.4505.7 to 1029.2+ 136.1 pnoles/mg prot. The funcri$nal role of pinyl gTia1 GA6A was then studied. GA6A (lo- M) plus NE (lo- H) significantly elevated rat pinea cyclic GMP fra 170.1+55.a. pnoles/ng prot. with WE alone to 477+126.4. Thus,pineal glial GA6A increases in response to iioradrenergicstfmulstion and patcntiates the NE-induced increase fn cGMP. Since ctnP is thought to inhibit the release of NE, glial GABA may play a m&latory role to augment presynaptic inhibition. (Supported by NIt4HGrant XROl-W-30024-03)
Al98 ~'HAWIACOLOGY01 GABA OUlPUi FROM nii CEREBRAL CCRTEX IN FKttLY MOVING KfiTS.F. Morons, F. Casamenti & G. Pepeu - Dept. of Pharmacology, University of Florence, Italy. G4BA, glutanlate(GL) and glutamine (GLN) were measured by a mass-fragnlentographicmethod in Ringer solution filling epidural cups of freely moving rats and in cortical cups of urethane anaesthetized rats. The samples were collected ever,! 20 min. The concentration in epidural cups of GABA, GL and GLN was respectively 0.6 t 0.08, 9.3 1 1.2 and I1 i 2.1 nmole/cm2/ min (6 rats-)and remained remarkably constant over a period of at least two hrs. Similar values were found in the anaesthetized rats. The addition of KC1 50 nM to the cortical cups was followed by a more than 20 times increase in GABA level and by a significant decrease in GL level. The i.c.v. administration of aminooxyacetic acid (30 ug) caused a 47 % increase in GABA level in the epidural cups but no change in blood GABA
level.
We
conclude that GABA and part of GL found in the cups are released from the underlying nervous tissue. Change in GAEA level in the epidural cups may therefore indicate changes in the activity of the GABAergic neurons. The effect of drugs affecting GABAergic mechanisms are presently investigated.