THE CENTRAL ACTION OF GAMMA-BUTYROLACTONE AND GAMMA-HYDROXYBUTYRATE

THE

CENTRAL

ACTION

AND (II)

OF

GAMMA-BUTYROLACTONE

GAMMA-HYDROXYBUTYRATE

THE EFFECT OF GHB AND RELATED AGENTS ON THE CORTICAL DENDRITIC RESPONSES

TAKASHI BAN AND MASAKAZU HOJO Department of Pharmacology, Facultyof Medicine, KyotoUniversity, Sakyo-ku, Kyoto Receivedfor publicationAugust27, 1968

The biphasic mode of action of gamma-butyrolactone (GBL) and ,gamma-hydroxybuty rate (GHB) in the central nervous system has been confirmed in the previous report (1) . The former compound elevates the reticular arousal stimulation threshold for the mani festation of the hippocampal theta waves without significantly affecting the increased mani festation of the fast waves in the rabbit's EEG. At the same time , the thalamic recruit ing response is usually depressed or sometimes is slightly augmented . In order to observe the mode of action on the brain structures more in detail, the effects of both compounds on the cortical dendritic responses (DR) caused by the electrical stimulation of the corti cal surface are studied in the cats. The small doses of GHB augment the DR , while the large doses inhibit it. Based on the obtained results the dose-response relationship of GHB and its related compounds is discussed. On the other hand, GBL proved to be inactive in this respect . Correla tion of the effects between GHB and gamma-aminobutyric acid (GABA), the latter of which is now accepted as the inhibitory transmitter in the several parts of the central nervous system, is also investigated. Roth et al. (2, 3) have already postulated that GHB is the pharmacologically active metabolite derived from gamma-butyrolactone in the body. METHODS Female or male cats weighing 1.8 to 4.2 kg were anesthetized with ether inhalation or with intraperitoneal injection of 40 to 50 mg/kg of o-chloralose or 30 mg/kg of pento barbital sodium. After the tracheal intubation the anesthetized cats with ether were made as encephaleisole preparation and were maintained on the artificial respiration . All the anesthetized animals were fixed of their heads on the stereotaxic instrument of Todai-Noken type. The contact surface of the animal body with frame bars of the instrument was , when required, anesthetized with the topical application of xylocaine hydrochloride . Constant level of the nnecthee;a wnwac attn;nvr1 1-.vthe S7unnlementnrv inieetinn of nentnl.a, h;tal nr  

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co-chloralose into the femoral vein via a previously inserted polyethylene tube. The trepanation of the skull was followed by the exposure of the required cortical areas by reiing the dura mater and by the placement of the bipolar platinum stimulation electrodes about 0.3 mm in diameter and about 1 mm apart from each other at the tip on the left marginal gyrus or sometimes on the left middle suprasylvian gyrus. The mono polar silver recording electrode about 1 mm in diameter at the tip was placed on either cortical surface at the site 2 to 3 mm apart from the stimulating site. An indifferent electrode used was a screw fixed on the skull bone covering the frontal sinus. The square waves used as the electrical stimuli were delivered from the electronic stimulator (Nihon Koden, MSE-3 type) and the stimulation condition 0.01-x-0.1 millisecond in duration, one per two seconds in frequency and 10 to 25 V in intensity was adjusted by trans mitting the electric current through isolater and diode to reduce the stimulation artifact. The cortical evoked potentiale were amplified through an amplifier (Nihon Koden, RB-2 type) at the time constant of 0.3 second, and were further fed into the medical computer (Sanei Sokki, MC' 401 type).' The average potential displayed every 30 recordings was photographed. In some of the experiments the evoked potentials were recorded at the decreased time constant on the ink-writing paper at the speed of, 0.25 mm/sec. Before the injection ' of either test agent the spontaneous variation of the individual potentials within tGe range of 10% was previously confirmed by recording the average potential for 3 to 5 minutes at least three times.' The test agents used were GHB (25% aqueous 'solution of sodium gamma-hydroxy butyrate), sodium n-propionate and sodium n-butyrate (30% aqueous solution), which were diluted with physiological saline for the topical application. In addition, ethanol, n-propanol, n-butanol' and GBL were prepared 100% aqueous solution as a stock. GABA used was 5 % solution. They were diluted with physiological saline before use. In the part I experiments the 'semi-circle shaped filter paper with a diameter of 5.5 mm', soaked previously in the various concentrations of either test agents, was applied locally at the site' of the cortex about one mm in the proximity of the potential-inducing site for 8 minutes and the responses of the DR potential during the application of the filter paper were studied. The local application of the filter paper soaked with physiological saline for 8 minutes produced the change of the DR responses by 0.97±0.149 folds of the previous levels. Therefore, the change of the potential in amplitude exceeding the range of 0.61 to 1.29 were iregarded to .be significant. In the p wit, II experiments one or two drops of the various concentrations of the test agents were applied directly in the proximity at the potential-inducing site and 5 minutes thereafter the applicaltion site was washed with physiological saline. In addition, GHB was also injected intravenously. RESULTS

I.

Effectsof GHB and relatedagents on the DR The DR in the marginal and suprasylvian gyri caused by electrical stimulation varied considerably according to the experimental conditions such as the level of anesthesia and

stimulation intensity. The difference of these conditions seemed to affect the drug action on the DR (4). The DR of our own choice produced by stimulation of the cortical surface was the graded monophasic wave with 10 to 20 msec in peak latency and 200 to 400 PV in surface negativity (Figs. 1, 2 and 5). This wave was recorded at first by Adrian (5) and was identified by Chang (6) and Bishop et al. (7) to represent an activity of the apical dendrite. GHB in the concentrations from 0.03 to 10% (2.38 to 794 mm), sodium butyrate in the concentrations from 0.03 to 30% (2.71 to 2,710 mm) and propanol in the concentrations from 0.03 to 100% (4.98 mm to 16,600 mm) were applied topically to the marginal or suprasylvian gyrus for 8 minutes. The maximal effects of either agent observed during the topical application time-length were expressed as the relative ratio to the mean potentials before the drug application. Thus, the dose-response relationships of the respective three test agents are shown in Fig. 3. Each point represents the mean relative

Fir 1. Augmenting effects of GHB and propanol on the dendritic potentials. A : From left figure, before and 2 minutes and 5 minutes after 0.3% GHB application. B : From left figure, before and 7 minutes and 10 minutes after 3% propanol application. The right side figures show the recovery (also in Fig. 2). Time mark indicates 10 msec (also in Figs. 2 and 5). Negativity is upward (also in Figs. 2 and 5).

FiG. 2. Inhibiting A : From application.

effects of GHB left, before and

B : From left, before application.

and Na butyrate 5 minutes and

and 6 minutes

on the dendritic potentials. 30 minutes after 5% GHB

and 30 minutes

after

30% Na butyrate

FIG. 3.

A.

Dose

response

curve

of

GHB.

B.

Dose

response

curve

of

Na

C.

Dose

response

curve

of

propanol.

Ordinate tion

of

: The the

Abscissa

average

agents. : Dose

relative

Standard of

the

butyrate.

agents

change

errors in

of

the

of

the

means

height are

of

DR

represented

by by

the the

topical I-shaped

applica bars.

percent.

changes of the DR in three to seven animals. GHB produced a biphasic effect, aug mentation of the DR in the small doses and inhibition in the large doses. The peak augmentation caused by the topical application of 0.3% GHB was the increase by about 1.2 folds. On the other hand, the topical application of sodium butyrate in lower doses inhibited slightly the DR and the inhibition caused by the same procedure of 30% of sodium butyrate was about 67%. The local application of the filter paper soaked in the more hypertonic saline solution (3-4 M) produced an inhibition of the DR by 40% in average from the previous level. The topical application of 0.1 to 10% propanol produced a slight augmentation and the peak effect obtained by the application of 3% solution was 1.16 folds. However, the increased concentrations of 30 to 100% produced a slight inhibition. Figs. 1 and 2 show the augmentation of the DR caused by the topical application of 0.3% GHB and 3% n-propanol as well as the inhibition of the DR caused by the same pro cedure of 5% GHB and 30% sodium butyrate. Either effect manifested within several minutes after the topical application and was usually reversible. However, the augmen tation caused by propanol and the inhibition caused by GHB or high concentration of sodium butyrate recovered often in 20 to 30 minutes. In contrast to the effect of GABA,

FIG. 4. The percentage of the number change exceeded the control saline

of the level.

preparations

Upward columns : The percentage of the preparations the significant augmentation of the DR. Downward columns : The percentage of showed the significant inhibition of the DR. Column within the 0%/ level lines indicates

of which which

the

showed

the

preparations

which

that

all the preparations

remained to show the insignificant change of the DR. Abscissa : Dose range of GHB, Na butyrate and propanol.

the topical application of GHB never produced a potential reversal, that is, turning of the negative potential to the positive one. Fig. 4 shows the percentage of the number of animals which responded to the concen trations of the test agent with a significant augmentation of the DR (upward column) and with a significant inhibition (downward column). Such a measure seems to supplement the drug effects expressed by the mean dose-response curve which is usually much less acute than that for the real dose-response curves for the individual objects (8). As shown in this figure, the topical application of 0.03 to 0.1 % and 0.2 to 1.0% of GHB produced an aug mentation of the DR in 20 and 31% of the animals. The same procedure of 0.2 to 1.0% and 2.0 to 10.0% GHB produced an inhibitoin of the DR in 9 and 39% of the animals, respectively. The same procedure of 0.03 to 10% and 30% soduim butyrate produced the inhibition of the DR in about 10 and 67% of the animals, while that of 0.3 to 1.0% sodium butyrate produced the slight augmentation. The topical application of 0.3 to 1.0% and 3.0 to 10.0% propanol produced an augmentation of the DR in 15% of the animals. However, 3 to 10%, 30% and 100% propanol produced an inhibition of the DR in 5, 17 and 67% of the animals, respectively. The effect of the topical application of sodium propionate, ethanol, butanol, choline chloride and GBL on the DR was also studied in the respectively two or three animals.

`+

1 . LLlLf \J LYl.llVJV

The concentrations of sodium propionate and choline chloride used were 0.03, 0.1, 0.3, 1.0, 3, 10 and 30%. In addition to the respective concentrations, the other agents were used as 100% concentration. A significant inhibition of the DR was observed by the to pical application of 30% sodium propionate, 100% ethanol, 100% butanol and choline chloride in the concentrations above 10%. A slight affection of the DR produced by the low concentrations of the respective agents was not more marked than the effects of GHB, propanol and sodium butyrate. Ten per cent GBL solution stored relatively long at the room temperature exhibited a strong acidity with pH 2.5. The topical application of 1 to 10% of this GBL preparation produced a significant augmentation of the DR in 8 of 20 animals and a significant inhibition in three animals. The topical application of 2.7 to 9% NaCI solution, of which pH was adjusted to 2.5 by adding 6 NHCI solution, produced a slight inhibition of the DR. The relatively marked effect of the stored GBL preparation was likely to derive from the formation of organic acids by the decomposition of lactone. II.

Effectsof GHB on the reversedpotential of the DR in responseto the topicalapplicationof GABA The topical application of GABA on the cortical surface produced the manifestation of the high amplitude slow waves in the cortical EEG and the increased appearance of the positive phases of the spindle waves as well as the reversal of the negative phases of the evoked potentials including the DR (4, 9-15). The effects were confirmed in the present experiments. In order to observe the effects of the topical pretreatment of the cortical surface with GHB on the reversal of the DR potential caused by the same procedure of GABA, the effective concentration of GABA to produce the potential reversal lasting for 10 to 30 minutes was obtained by the application of 0.5 to 5% solution of GABA. At 40 minutes to one and half hours after the application when the DR potential recovered to the previous level, the same concentration of GABA was applied locally at the sites with or without pretreatment with the topical application of GHB. When the pretreatment with GHB produced a marked change of the DR, GABA was applied topically 20 to 30 minutes thereafter. However, when the pretreatment with GHB did not affect the DR, GABA was applied 10 minutes thereafter. As shown in Table 1, the duration of the po tential reversal, that is, the time periods of the persistence of the potential reversal, caused by the repeated application of GABA did not present clear-cut reproducibility. Espe cially in the anesthetized cats with sodium pentobarbital the duration of the potential re versal caused by the second application was significantly shorter than that caused by the first application. However, such a sign of the tachyphylaxis was rarely observed in the TABLE 1.

Relative

induced

change by

sents

the

than

three

the

of

second

average animals.

the

duration

application

change

and

of of

the

the

GABA.

standard

potential Each deviation

reversal figure in

repre more

Fir 5. Reversing effects of GABA on the dendric potentials and the influence of GHB (chloralosed cat). From upper and left figures before and 5 minutes and 25 minutes after 3% GABA application. Lower figures : 7 minutes after the second application of GABA (the same concentration as before), 18 minutes after 3% GHB application and 8 minutes after the third application of the same concentration of GABA. The third application of GABA was performed 20 minutes after 3% GHB application.

anesthetized cats with chloralose and encephaleisolepreparation.

The similar sign of tachy

phylaxis more easily observed in the anesthetized cats than in the non-anesthetized cats was demonstrated by Murayama (16). The easiness of the development of the tachyphy laxis seems to differ according to the anesthetics used. The repeated application of GABA was usually followed by the decrease on peak height of the reversal positive potential, as shown in Fig. 5. No direct correlation between the decrease in peak height and duration of the reversed potential was demonstrated. The animals that responded to the topical application of GABA with the potential reversal lasting within 10 minutes did not usually respond to the second application. These animals were excluded from the results in Table 1. In the chloralosed cats the topical application of GABA at the cortical sites pretreated with GHB in the concentrations below that of GABA produced a significant prolongation of the duration of the potential reversal about 1.5 to 2 times of the previous duration in 3 of 5 animals. However, the prolongation was not dose-dependent. The pretreatment of GHB in the concentrations above that of GABA did not produce the similar prolonga tion. It was much likely that the effect of pretreatment with GHB on the potential reversal caused by GABA did not relate to the augmenting or inhibiting effect of GHB alone on the DR. Fig. 5 shows the potential reversal caused by the first and second topical applica tion of GABA and by the third application of GABA after the pretreatment with GABA. The prolonging effect of GHB on the potential reversal caused by the topical appli cation of GABA was also apparently demonstrated by the intravenous administration. The intravenous administration of 250 to 650 mg/kg of GHB prolonged the duration of the potential reversal caused by the topical application of GABA about 1.8 to 3.2 folds in all of three cats. However, the large intravenous doses such as 1,000 to 1,300 mg/kg pro duced the similar prolongation only about 1.45 folds. The evidence indicated that the

prolonging effect of GHB was not dose-dependent. Figs. 6-A-E, F-H, I-K and L-O show the effects of the topical application of GABA on the DR in the absence of the pretreatment with GHB and in the presence of the pretreatment with the intravenous injection of 50, 250 and 700 mg/kg of GHB repeated at the time-interval of 1.5 hours. The pretreatments of the animals with 50 mg/kg and 700 mg/kg of GHB, which alone have no effects or in hibited the DR, retarded slightly the recovery from the potential reversal of the DR caused by the topical application of GABA, while the pretreatment of the animals with 250 mg/kg of GHB which alone augmented the DR, retarded markedly the recovery from the poten

FIG.6. Effects of GHB and GABA on the DR. A : Before. B : 0-4 minutes after the first application of 3% GABA. C : 20-24 minutes after the first application of 3% GABA. D : 30-34 minutes after the first application of 3% GABA. E : 54-58 minutes after the first application of 3% GABA. F : 4-8 minutes after the i.v. administration of GHB 50 mg/kg (the first administration of GHB). G : 16-20 minutes after the second application of GABA which was performed 10 minutes after the first administration of GHB. H : 73-77 minutes after the second administration of GABA. 1 : 2-6 minutes after the i.v. administration of GHB 250 mg/kg (the second administration of GHB : 1.5 hours after the first administration of GHB). j : 37-41 minutes after the third application of GABA which was performed 10 minutes after the second administration of GHB. K : 73-77 minutes after the third application of GABA. L : 9-13 minutes after the i.v. administration of GHB 700 mg/kg (the third administration of GHB : 1.5 hours after the second administration of GHB). M : 23-27 minutes after the third administration of GHB. N : 5-9 minutes after the fourth application of GABA, which was performed 30 minutes after the third administration of GHB. O : 55-59 minutes after the fourth application of GABA. Calibration signal : 1 minute and 500 sV.

tial reversal of the DR. Though GHB in the intravenous doses above 1,000mg/kg al ways inhibited the DR, the effect of GHB in the intravenous doses of 250 to 650 mg/kg on the DR was the occasional augmentation and also the occasional inhibition. The pro longation of the duration of the DR potential reversalin responseto the topical application of GABAwas obtained by the topical as well as by the systemicadministration of GHB in the doses, which alone produced an augmentation or an inhibition of the DR. As shown in this figure, the intravenous injection of 700 mg/kg of GHB produced the flattening of the cortical EEG intermingled with the spiking waves. The appearance of the latter waves was somewhat depressed by the topical application of GABA. DISCUSSION The inherent difficultyin the quantitation of the drug effectson the dendrite potential consistsin the factors such as the spontaneous variation in amplitude, individual difference in the drug sensitivityand others. In the present experiments the effectsof the drugs on the DR were analyzed quantatively as the variation of the average response intergrated by use of a computer and thus obtained dose-responserelationship was examined to know the mode of action. The postsynapticnature of the dendritic potential has been postulated by many investigators (11, 12, 17, 18). However, the mode of biogenesisremains still to be settled in detail. Although Grundfest (4) and Purpura et al. (9, 11, 12) have regarded the DR as the summation of the two opposing excitatory and inhibitory postsynaptic potentials, their hypothesis has been bombarded relating to the effects of GABA on the DR (10, 13, 19). The biphasic effects of topically applied GHB on the DR, an aug mentation in the small dose and an inhibition in the large dose, may serve also for the clue to know the biogenesis of the potential. The topical application of GABA and related compounds produced the potential reversal of the DR , which was postulated by Purpura et al. (11, 12) to derive from the specific inhibition of EPSP. The activitiesof these agents are also shown to be conditioned by the dimension of the dipole as the Zwitterion and by the interpolar distance (11, 14). The similar conclusion has been presented regarding the depressant effect of these agents on the activity of single neuron (20). The augmentation of.the DR, postulated by Purpura et al. (11) to derive from the specific inhibition. of the IPSP, is not fully elucidated in the relationship between the chemical structure and the drug action. The augmentation of the DR was shown by Purpura et al. (11) to be. caused by w-amino acids more than 6 in carbon number and w-guadino acids more than four in carbon number as well as by w-guanidinoaceticacid derivatives such as creatine and creatinine having co-guanidinoacid structures with two carbons. Since the topical.application of GHB in low concentration produced the augmentation of the DR, the presence of the amino group in the chemical structure is indicated not essen tial for the augmenting effect. Therefore, the decarboxylated and dehydroxylated pro ducts, of GHB, n-propanol and sodium butyrate were further studied of their effects on the DR. The topical application of propanol in the low concentration augmented slightly

TABLE 2.

The

potential

effects height

of

propanol, in

molar

Na

butyrate

and

GHB

on

the

DR

concentration

the DR, while sodium butyrate in the high concentration inhibited considerably the DR. The comparison of the effects of propanol, sodium butyrate and GHB in Figs. 3-A, B and C at the molar ratio presents the Table 2. Provided that the interaction between the hydroxyl group and receptor results in the augmentation of the DR and that between the carboxyl group and a receptor results in the inhibition of the DR, the predicted pharma cological effects of both propanol and sodium butyrate interacting each independently with the receptors are classified into the figures of column III in Table 2. Compared with the effects of GHB in column IV, propanol and sodium butyrate proved to be actually less effective in both augmentation and inhibition. This evidence indicates that both groups in GHB mutually influence favorably for the interaction with the receptors. Further, the evidence supports the hypothesis devised by Purpura et al. (11) that the DR consists of the at least two mutually opposing mechanisms and is involved at least in the two types of receptor mechanism. The lack of butyric acid in the significant effect on the DR in lower concentration was already reported by Koshino (14) and Usuda (15). The compounds having a trimethyl nitrogen group in their chemical structures such as carnitine (11) and trimethyl-GABA (15) are reported to augment the DR. The topical application of the aliphatic alcohols such as ethanol and n-butanol and choline chloride did not exhibit a significant augmentation of the DR, which indicated that both hydroxyl group and trimethylnitrogen exert minor roles for the effect. The topical application of GBL, so far as the freshly prepared solution was used, even in the concentration of 100% did not affect the DR. However, GBL was more effective as an anesthetic agent than GHB (1). Roth et al. (2, 3) have postulated that GHB alone is pharmacologically active in vivo and GBL exerts the pharmacological effects by converting to GHB due to the activity of lactonase. According to the authors GBL produce more powerful effect in vivo than GHB in virtue of easy uptake by the tissues and less metabolic inactivation. Besides, the injection of GBL into the brain tissue or ventricle, where no activity of lacto nase is present, did not produce high amplitude slow waves in the EEG (3). Recently evidences to support the role of GABA as the inhibitory transmitter of the brain in the mammalian species are accumulating (21). The metabolic correlation of GHB with GABA as well as the pharmacological similarity and dissimilarity between both agents has been studied extensively since the original assumption of.Laborit et.al. (22, 23)

that GHB was a biosynthetic precursor of GABA. Fishbein and Bessman (24) showed that GHB converted to succinate or GABA by way of succinic semialdthyde in vitro. The administration of GHB to rats elevated the brain level of GABA (25). By studying the effect of GHB on, the spinal reflex Basil et al. (26) concluded that GHB as well as GABA affected the internuncial neurons in the spinal cord., On the other hand, Giarman et al. (27) could not find ;the significant change of the revel of brain GABA in the mouse that re ceived GBL, and .Walkenstein et al .(28) and Roth et 'al. (2) could not confirm the conver sion of GA-B,to.isuccinate. -The. characteristic effects of GHB on the EEG and behaviors were postulated-,,by Winters et al. (29) and Marcus et al, (30) to be somewhat similar to the generalized nonconvulsant epilepsy. These effects of GHB were assumed to be incompati ble with the inhibitory nature of the GABA effects. Therefore, these authors suggested that GHB might antagonize some effect of GABA (28) or the effects of GHB might relate to the interference with, the glucose metabolism (29). In the present experiments the topical application of GHB in the considerable concentration or the intravenous injection prolong ed the duration of the potential reversal in response to the topical application of GABA. If it is assumed that this potentiating effect is derived from the pharmacological or bioche mical potentiation of the effect of endogenous GABA or from the increased activity of the GABA neuron in ,the brain cortex, the shortening, effect of sodium pentobarbital on the duration of the potential reversal would indicate the decreased activity of GABA neuron. Thus the anesthetic effects is unlikely to be'associated with the increased activity of exoge nous or endogenous GABA, though. inhibitory, in nature. Roberts and Kuriyama (21) have shown that the elevation of the level of brain GABA in the animals that received GABA or aminoacetic acid is accompanied with the prevention of the electroshock and the beha vioral abnormality such as an ataxia and a motor incoordination. Since GHB is confirmed to produce the similar effects (1, 31), these . behavioral effects might be' produced by the increased activity of the GABA neuron in response to GHB. Winters et al. (26) have observ ed that the intravenous injection of 200 to 600 mg/kg of GHB facilitated the click response in the anterior sigmoid gyrus, midbrain reticular formation and nucleus centralis lateralis. In the present experiments the intravenous injection of 250 to 650 mg/kg of GHB produced an occasional augmentation of the DR, while the same procedure of the larger doses inhi bited it. The biphasic effect of GHB was also observed by the topical application, sugge sting that at least some parts of the augmentation and inhibition of the evoked potential by intravenous administration is induced by the direct action of GHB on the cortical levels. SUMMARY 1. The effects of the topical application of sodium gamma-hydroxybutyrate (GHB) and other related agents on the dendritic resopnse (DR) were studied on the cerebral cortex of encephaleisolepreparation of cats. The biphasic effects of GHB, that is, the augmenting effects in low concentration (0.1 to 0.5% solution) and the inhibitory effects in high con centration (more than 1.0% solution) were demonstrated. More than 10% solution of sodium n-butyrate manifested the inhibitory effect, while

0.1 to 10% solution of n-propanol manifested the slight augmenting effect. Choline chloride in the concentrations more than 10%, sodium n-propionate in the concentrations more than 30%, ethanol and n-butanol in both 100% solution exerted the inhibitory effect, while the solution of less concentration of the above agents and gamma butyrolactone even in the 100% solution were without effect on the DRs. 2. The potential reversal of the DRs in response to the topical application of 0.5 to 5% gamma-aminobutyric acid (GABA) occasionally showed a tendency of the develop ment of tachyphylaxis. This tendency was more pronounced in the cat anesthetized with sodium pentobarbital, less in chloralosed cat and in the anesthetized encelbhaleisole pre paration. Pretreatment with topical application of GHB in concentration less than GABA used prolonged the duration of this potential reversal in chloralosed cat. This potentiating ef fect was also demonstrated on cat with pretreatment with the intravenous administration of 250 to 650 mg/kg of GHB. However, GHB in large doses of either route did not show the marked potentiating effect dose-dependently. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18) 19) 20) 21) 22)

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