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Gabaergic actions of thip in vivo and in vitro: A comparison with muscimol an GABA
European Journal o f Pharmacology, 65 (1980) 21--29 © Elsevier/North-Holland Biomedical Press
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GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO: A COMPARISON WITH MUSCIMOL AND GABA BARBARA L. WASZCZAK *, ROBERT E. HRUSKA and JUDITH R. WALTERS Experimental Therapeutics Branch, Physiological and Behavioral Neuropharmacology Units, National Institute o f Neurological and Communicative Disorders and Stroke, 9000 Rockville Pike, Bethesda, Maryland 20205, U.S.A.
Received 28 September 1979, revised MS received 7 January 1980, accepted 9 April 1980
B.L. WASZCZAK, R.E. HRUSKA and J.R. WALTERS, GABAergic actions o f THIP in vivo and in vitro: a comparison with muscimol and GABA, European J. Pharmacol. 65 (1980) 21--29. GABAmimetic actions of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP) were evaluated both in vivo and in vitro and compared with the actions of muscimol and GABA. The GABAergic potencies of these agents were assessed in vivo by measuring their ability to inhibit firing of rat substantia nigra pars reticulata neurons after systemic administration and iontophoretic applications, and in vitro by measuring their ability to inhibit 3H-GABA binding to rat cerebellar membranes. The effects of THIP were found to be similar to those of muscimol and GABA with regard to inhibition of reticulata cell firing and 3H-GABA binding. The order of potency was muscimol > GABA > THIP. The magnitude of the differences between drug potencies in iontophoretic studies closely paralleled their relative potencies in binding studies, with muscimol approximately 3 times more potent than GABA and 25-40 times more potent than THIP. After systemic (i.v.) administration, however, muscimot was only 3 times more potent than THIP in inhibiting reticulata cell firing, possibly because THIP passes the blood-brain barrier more readily. THIP
Muscimol
GABA
Single unit recording
1. I n t r o d u c t i o n T h e r e c e n t d e v e l o p m e n t o f a series o f 7 - a m i n o b u t y r i c acid ( G A B A ) a n a l o g u e s m a k e s it possible to e x p l o r e w h e t h e r or n o t v a r i o u s G A B A r e c e p t i v e sites possess d i f f e r e n t c o n f o r m a t i o n a l characteristics. T h e q u e s t i o n o f whether different conformations of GABA are p r e f e r r e d at d i f f e r e n t sites was raised b y several studies w h i c h revealed t h a t G A B A m i m e t i c drugs d i f f e r e d in t h e i r relative affinities a t G A B A u p t a k e sites, t r a n s p o r t sites ( K r o g s g a a r d - L a r s e n a n d J o h n s t o n , 1 9 7 5 ; J o h n s t o n et al., 1 9 7 8 ; L o d g e et al., 1978), sites labeled in G A B A r e c e p t o r b i n d i n g * Send correspondence to: Barbara Waszczak, Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, 9000 Rockville Pike, Bldg. 36, Rm. 5A31, Bethesda, Maryland 20205, U.S.A.
Microiontophoresis
Substantia nigra
studies ( K r o g s g a a r d - L a r s e n a n d J o h n s t o n , 1 9 7 8 ; K r o g s g a a r d - L a r s e n et al., 1 9 7 9 a ) , a n d sites m e d i a t i n g i o n t o p h o r e t i c a c t i o n s o f these compounds o n c a t spinal c o r d n e u r o n s { J o h n s t o n et al., 1 9 6 8 ; K r o g s g a a r d - L a r s e n et al., 1 9 7 5 ; K r o g s g a a r d - L a r s e n e t al., 1977). T h e rigid i s o x a z o l e derivatives o f G A B A n o w b e c o m i n g available s h o w p r o m i s e as pharmacologic tools for better characterizing a n d distinguishing s o m e o f these GABA r e c e p t i v e sites. O n e such c o m p o u n d , 4,5,6,7t e t r a h y d r o i s o x a z o l o - [ 5,4-c ]-pyridin-3-ol ( T H I P ) , a rigid, bicyclic i s o x a z o l e derivative o f G A B A , has r e c e n t l y b e e n distinguished from both GABA and muscimol by virtue of its greater relative s p e c i f i c i t y f o r i n h i b i t i n g 3H-GABA binding than for stimulating b e n z o d i a z e p i n e b i n d i n g ( K a r o b a t h e t al., 1 9 7 9 ; Maurer, 1979). In a d d i t i o n , it has b e e n suggested t h a t T H I P m i g h t be m o r e suited
22 than muscimol for use as a systemic GABAmimetic agent because it appears to penetrate the blood-brain barrier more readily than muscimol, it may be more stable in vivo, and it is less toxic than muscimol after systemic administration (Krogsgaard-Larsen et al., 1979b). Considering these potential advantages, the following studies were undertaken to determine whether THIP would act at GABA receptors within the central nervous system in a manner comparable to muscimol, and to compare and correlate t h e potencies of THIP, GABA, and muscimol both in vivo and in vitro. Accordingly, the effects of these agents upon the firing of substantia nigra pars reticulata neurons were studied in vivo, after both systemic (i.v.) administration and during iontophoretic application. In addition, the ability of each c o m p o u n d to inhibit brain 3H-GABA binding in vitro was determined.
2. Materials and methods
2.1. Single unit recording and iontophoresis techniques Male, Sprague-Dawley rats (Zivic-Miller Co., Allison Park, PA}, weighing 250-300 g, were anesthetized with chloral hydrate, 400 mg/kg, i.p., and m o u n t e d in a stereotaxic apparatus. Additional injections of chloral hydrate were administered as needed through a tail vein. Body temperature was maintained t h r o u g h o u t the recording period between 3638°C. Single unit recording and iontophoresis experiments were conducted according to standard techniques (Bunney et al., 1973b; Aghajanian and Bunney, 1977; Smith and Hoffer, 1978}. A small (3 mm) burr hole was drilled in the skull 2.0 m m lateral to lambda and 3.0 mm anterior to the lambdoid suture. An electrode was passed through the hole to the level of the substantia nigra with a hydraulic microdrive. Electrical signals were displayed on a oscilloscope screen and passed into a rate meter set to c o u n t pulses which
B.L. WASZCZAKET AL. fell within the selected amplitude time gates. Counts of unit activity were summed over either successive 5 or 10 sec intervals. Histogram plots of the rate of cell firing were produced by a strip chart recorder, and digital printouts of the number of counts per 5 or 10 sec period were also obtained. Single barrel electrodes, prepared according to techniques previously described (Tasaki et al., 1968), were used in recording extracellular, single unit activity of pars reticulata cells during cumulative (i.v.) dose-response studies. The electrodes were broken back to a tip diameter of approximately 1-2 t~m and were filled with 1% Pontamine Sky Blue (GURR, High Wycombe, Bucks, England) in 2 M N a C 1 . Electrode resistances varied between 3.8 and 6.0 megohms (measured at 135 Hz). A 5 min period of baseline activity was recorded before the first drug injection. Muscimol or THIP was then administered, i.v., through a tail vein. Successive doses were given at 2 min intervals so that the cumulative dose after each injection was twice the cumulative dose before the injection (i.e. individual doses of 0.05, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg/kg result in cumulative doses of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 mg/kg, respectively). The percent change from the baseline firing rate was calculated by comparing the average firing rate over successive 10 sec intervals during the 2 min period after each dose with the average baseline rate before drug. Only one cell per rat was monitored in studies in which drugs were administered systemically. Cumulative log dose-response curves were constructed, and EDs0 values for the inhibition of reticulata cell firing by muscimol and THIP were calculated for individual experiments by least squares linear regression analysis of probit values versus the log dose. The average EDs0 values for the two drugs were calculated and compared. In iontophoretic studies, 5-barrel micropipettes were used for both recording and ejecting drugs. Pipette tips were broken back to diameter of 4-5 ~m. The central barrel,
GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO used for recording, was filled with 1% Pontamine Blue solution (above). Side barrels contained: (1) THIP, 0.01 M in 0.2 M NaC1, pH 4.0; (2) GABA, 0.01 M in 0.2 M NaC1, pH 4.0; (3) muscimol, 0.01 M in 0.2 M NaC1, pH 4.0. The remaining side channel was filled with 4 M NaC1 and served as a balance channel. The resistance of the recording channel ranged between 3.5 and 8 megohms; resistances of the side channels averaged between 40 and 100 megohms. During periods of baseline activity, a negative retaining current of 10 nA was passed through each of the drug channels. A drug was iontophoresed from a particular channel by passing through it a positive ejecting current of appropriate magnitude to produce at least a 50% inhibition of cell firing within 60 sec. Results obtained with iontophoresis techniques are essentially of a qualitative nature. However, in order to compare the potencies of these drugs when iontophoresed with their potencies after systemic administration and in receptor binding studies, an a t t e m p t was made to quantify the iontophoretic responses. It has been assumed that during the early phase of iontophoresis of GABA and GABA agonists the rate of release increases approximately exponentially with time and is proportional to the current (I) applied (Hill and Simmonds, 1973). On the basis of this assumption ITs0 values were calculated for each of the 3 drugs and were used as an estimate of the iontophoretic potencies of the compounds tested. Ts0 values represent the time required to inhibit firing to 50% of the baseline rate. For each cell, the Ts0 value at a given current was derived from plots of the percent inhibition of firing vs time (in 5 sec intervals, plotted exponentially) after the ejecting current was turned on. In instances where more than one iontophoretic application of a drug was carried out, the mean ITs0 value for that drug was considered as a single observation. Average ITs0 values for each drug were calculated and compared. It should be understood, however, that differences in the transport numbers of the compounds studied
23
and factors such as local uptake and metabolism of the ejected agents, especially GABA, could represent a source of error in these studies. Frequently, more than one cell per rat was monitored in iontophoresis experiments.
2.2. Identification of cells The cells recorded in these studies were located in the pars reticulata region of the substantia nigra. Reticulata neurons could be tentatively identified during the recording period on the basis of their location and electrophysiological characteristics. All cells studied were located within the stereotaxic coordinates anterior, 1760-2580 t~m; lateral, 1.7-2.5 mm; ventral, --1.5 to --2.5 mm, according to KSnig and Klippel (1970). Since the pars reticulata lies ventral to the pars compacta region of the substantia nigra, reticulata cells were typically encountered after passing through a zone of pars compacta (dopamine) neurons whose firing rate, extracellular action potential shape and duration are characteristic, easily recognized, and well documented (Aghajanian and Bunney, 1977; Bunney et al., 1973a,b, G u y e n e t and Aghajanian, 1978; Waszczak et al., 1980). The electrophysiologic properties of the pars reticulata cells have also been previously described (Guyenet and Aghajanian, 1978). These neurons have firing rates ranging between 10 and 40 spikes/sec. Their extracellular action potentials are smooth, sharp, and biphasic with a duration of 0.5 to 0.7 msec (mean duration, 0.63 ± 0.02 msec, n = 20). In order to verify placement of the electrode within the pars reticulata, the location of the recording site was marked at the end of the experiment by passing a 10-20 nA current through the electrode for approximately 15 min. This caused a small a m o u n t of the Pontamine Blue dye to be deposited at the electrode tip. Rats were then perfused with 10% buffered formalin phosphate, pH 7.0 (Fisher Scientific Co., Fairlawn, NJ). Brains were removed, sectioned, m o u n t e d , and
24
B.L. WASZCZAK ET AL.
stained. The location of the blue spot identified the recording site.
2.3. Receptor binding techniques Neurotransmitter receptor binding of ~HGABA was measured by standard procedures {Zukin et al., 1974; Greenlee et al., 1978). The sodium-independent binding was assayed in rat cerebellum, obtained from SpragueDawley rats after sacrifice and rapid removal of the brain into ice-cold saline. The cerebellum was dissected, weighed, and homogenized with a Polytron in 20 volumes of 50 mM Triscitrate buffer (pH 7.1 at 4°C). The suspension was centrifuged at 50 000 × g for 10 min, the resultant supernatant discarded, and the homogenization and washing procedure
repeated twice. The final homogenate was frozen overnight, thawed, and washed 3 times again before use in the 3H-GABA assay. The specific binding was determined by subtracting from the total binding the nonspecific binding obtained in the presence of 1 mM unlabeled GABA. Under our conditions the specific binding of 3H-GABA was 122 -+ 15 fmole/mg protein using 4.8 + 0.8 nM ~H-GABA. Inhibition curves were obtained by adding increasing concentrations of GABA, muscimol, or THIP. The slope and ICs0 values (concentration required to inhibit 50% of the binding) were estimated by linear regression analyses of Hill plots for each experiment. The mean ICs0 values for 3 experiments were compared.
S.N. pars reticulata
MUSC
PIC
¢,,,,,,
'°:1
'
¢
................................
I
o @
THIP
PIC
,
,
3oo1 0-~ !
!
5 min Fig. 1. Effects of systemic muscimol and THIP upon single unit activity of substantia nigra pars reticulata neurons. Top panel: Effect of increasing i.v. doses of muscimol (MUSC) administered at 2 min intervals so that each injection doubled the previously administered cumulative dose (i.e. 0.05, 0.05, 0.1-, 0.2, 0.4, 0.8, 1.6 mg/kg; total cumulative dose, 3.2 mg/kg). Inhibitory effect of muscimol was reversed by picrotoxin (PIC 4.5 mg/kg, i.v.). Bottom panel: Effect of cumulative (see above) i.v. doses of THIP (0.2, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mg/kg; total dose, 51.2 mg/kg). Picrotoxin (PIC, 5.0 mg/kg i.v. ) reversed the inhibition.
G A B A E R G I C A C T I O N S O F THIP IN V I V O A N D IN V I T R O
25
2.4. Drugs Chloral hydrate was obtained from Sigma Chemical Co., St. Louis, MO; 7-aminobutyric acid (GABA) from Sigma Chemical Co., St. Louis, MO; muscimol from Regis Chemical Co., Morton Grove, IL; and 4,5,6,7-tetrahydroisoxazolo-[ 5,4-c]-pyridin-3-ol (THIP) from Dr. P. Krogsgaard-Larsen, Royal Danish School of Pharmacy, Department of Chemistry BC, Copenhagen, Denmark.
~ 70'
3. Results
3.1. Effects of systemic administration of THIP and muscimol upon reticulata cell unit activity When administered in a series of increasing i.v. doses, THIP produced dose-dependent decreases in the firing rates of substantia nigra pars reticulata neurons (fig. 1). The responses o f reticulata cells to i.v. THIP were qualitatively similar to their responses to i.v. muscimol, although muscimol was the more p o t e n t inhibitor. THIP produced its first significant decrease in firing after a total dose of 6.4 mg/kg (P < 0.005), while only 1.6 mg/kg muscimol were required to significantly inhibit firing (P < 0.05). All cells studied were completely inhibited at a total cumulative dose of 12.8 mg/kg muscimol, whereas firing was almost totally inhibited (4.4% of baseline) at 51.2 mg/kg and completely inhibited at 102.4 mg/kg THIP. The inhibitory actions of both drugs were reversible by subsequent i.v. administration of 4-5 mg/kg picrotoxin, a GABA antagonist (fig. 1). Cumulative log dose-response curves comparing the effects of THIP and muscimol upon the firing of reticulata neurons are shown in fig. 2. The calculated dose required to produce a 50% inhibition of firing (EDs0) was 14.4 ± 4.2 mg/kg for THIP and 5.1 -+ 1.0 mg/kg for muscimol (table 1). Thus, systemically administered THIP was approximately 3 times less p o t e n t than muscimol
0
0~3 ~.i 0:2 0.~4 018 1~6 3:2 ~4 12.8 25.6 51.2 102,4 mg/kg DOSE(log scale}
Fig. 2. Cumulative log d o s e - r e s p o n s e curves o f t h e e f f e c t s o f i.v. THIP (~. A; n = 9) and m u s c i m o ! (~ e; n = 10) u p o n the single unit activity o f s u b s t a n t i a nigra pars reticulata n e u r o n s . Drugs were a d m i n i s t e r e d i.v. in increasing i n c r e m e n t s so t h a t each dose d o u b l e d t h e previous cumulative dose. Average firing ra~e o f each cell a f t e r each dose was e x p r e s s e d as a p e r c e n t o f the baseline rate. Points r e p r e s e n t t h e average r e s p o n s e o f all cells a f t e r t h a t dose. Vertical bars r e p r e s e n t s t a n d a r d errors o f t h e m e a n s (S.E.M.). One cell was m o n i t o r e d per rat.
(P < 0.05) in inhibiting single unit activity of substantia nigra pars reticulata cells.
3.2. Effects o f microiontophoresis o f THIP, GABA, and muscimol upon activity of pars reticulata neurons THIP, like muscimol and GABA, was able to depress reticulata firing when iontophoretically applied. Equimolar solutions of THIP, GABA, and muscimol were iontophoresed in turn onto individual reticulata cells until a current was selected for each drug which caused at least a 50% inhibition of firing within i min (fig. 3). In some instances complete inhibitory responses to GABA and/or muscimol occurred immediately after the ejecting current was turned on, even at currents as low as 2.5 nA. In order to achieve a more gradual rate of inhibition, the nega-
26
B.L. WASZCZAK ET AL.
TABLE 1 Potencies of THIP, GABA, and muscimol (MUSC)were evaluated by comparing: (1) EDs0 values for inhibition of reticulata cell firing after cumulative i.v. administration of drugs, (2) ITs0 values (see text) for inhibition of cell firing during iontophoresis of THIP (n = 7), GABA (n = 7) and MUSC (n = 7), and (3) ICs0 values for inhibition of aH-GABA binding to rat cerebellar membranes by each compound. Potencies are also expressed relative to that of MUSC, the most effective inhibitor in all cases.
THIP GABA Muscimol
EDs0 (mg/kg)
EDs0 EDs0 (MUSC)
ITs0 (hA . sec)
ITs0 ITs0 (MUSC)
14.4_+4.21 -5.1 -+ 1.0
2.9 -1
98.2 -+ 43.91 6 . 5 + 3.0 2.6 -+ 1.4
37.8 2.5 1
ICs0 (M)
ICs0 ICs0 (MUSC)
2 6 . 4 _ + 1 . 1 x 10 -7 I 6.6_+1.5x 10 -8 2.4 -+ 0.5 × 10 -s
26.7 2.8 1
1 Significantly different from muscimol, P < 0.05 2 Significantly different from muscimol, P < 0.005.
tive retaining current through that barrel was turned off to permit only the passive leakage of drug from the pipette tip. In c a l c u l a t i o n s o f ITs0 v a l u e s in s u c h i n s t a n c e s , a value of 0.1 nA current was used. A comp a r i s o n o f t h e m e a n ITs0 v a l u e s f o r t h e 3 compounds studied revealed that the order of potency for inhibition of reticulata cell firing was muscimol > GABA > THIP ( t a b l e 1). W h i l e a p p r o x i m a t e in n a t u r e , t h e s e values suggest that muscimol was between 2
THIP lOnA
GABA 5nA
5hA
OnA
and 3 times more potent than GABA and between 35 and 40 times more potent than THIP.
3.3. Inhibition o f 3H-GABA THIP, GABA, and muscimol
by
I n h i b i t i o n c u r v e s f o r t h e i n h i b i t i o n o f 3HGABA binding to rat cerebellar membranes b y T H I P , G A B A , a n d i n u s c i m o l a r e s h o w n in fig. 4. T h e ICso v a l u e s f o r e a c h c o m p o u n d ,
120~
MUSC 2.SnA
binding
OnA
~
100
•
•
~.
~OOl
I
!
,
5 rain
!
Fig. 3. Inhibition of pars reticulata cell firing by iontophoretieally applied THIP, GABA, and museimol (MUSC). Equimolar solutions (0.01 M in 0.2 M NaC1) of the drugs were ejected from a 5-barrel micropipette at currents suffielent to produce at least a 50% inhibition of firing within one rain. Bars represent the duration of the designated ejection current. Ordinate: spikes/5 sec. Calibration bar: 5 rain.
...
~
~ I~1 ~
~0
~
~0
m
~~ 0 ~ 0
-11
Mosc,MoL\
-1~
_;
_;
,
,
-7 -6 LOG [INHIBITOR], M
-5
-4
Fi~. 4. I n h i b i t i o n o f s o d i u m - i n d e p e n d e n t b i n d i n g o f 3 H - G A B A t o rat cerebe]lar m e m b r a n e s b y T H e ,
G A B A , and museimo]. Points p r e ~ n t o f 3 experiments.
the average
GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO
estimated from the Hill plots of the data, are compared in table 1. Muscimol was the most p o t e n t inhibitor. GABA and THIP were 2.8 and 27 times less potent, respectively, than muscimol. The Hill slopes for GABA, muscimol, and THIP were quite similar {--0.872, --0.895 and --0.787, respectively). The similarity of the Hill slopes suggests homogeneity of the binding sites.
4. Discussion Previous studies have demonstrated that muscimol, administered systematically, and both GABA and muscimol, applied iontophoretically, could completely inhibit the firing of rat substantia nigra pars reticulata neurons (Waszczak et al., 1978; Waszczak et al., 1980). Since the substantia nigra is the termination site of a major GABAergic projection from the striatum and globus pallidus ( F o n n u m et al., 1974; Kataoka et al., 1974; Kim et al., 1971; Ribak et al., 1976), the sensitivity of the reticulata neurons to exogenous GABA may parallel a normal sensitivity of these cells to endogenously released GABA. Thus, the inhibitory response of nigral pars reticulata cells to GABAmimetic agents was viewed as a physiologically relevant test system for studying the GABAmimetic actions of THIP in the central nervous system and for comparing the potencies of THIP, GABA, and muscimol in vivo. Furthermore, by determining the effects of these agents on cell firing after i.v. administration and iontophoretic application it was possible to compare the difference between their direct actions and their effectiveness after systemic administration. These studies demonstrate that i.v. administration of THIP, like muscimol, results in dose-related decreases in the firing rates of substantia nigra pars reticulata neurons. The inhibition was reversible by i.v. administration of the GABA antagonist, picrotoxin. THIP also markedly inhibited reticulata cell firing when applied iontophoretically. Although the
27
effects of THIP, both systemic and iontophoretic, occurred at doses higher than those required for muscimol, the nature of the inhibitory responses to both drugs and the reversibility by a GABA antagonist were similar. A comparison of the effects of these drugs reveals that muscimol exhibits the greatest p o t e n c y for inhibiting reticulata cell firing and 3H-GABA binding. Results of iontophoresis and binding studies, in which GABA was included, indicate that GABA was second in order of potency and THIP was the least p o t e n t of the 3 agents. The effects of systemic administration of GABA were not considered because rapid neuronal and glial uptake of that fraction of GABA which crosses the blood-brain barrier apparently prevents most or all of the drug from reaching reticulata neurons. The relative potencies of these agents in vivo, as assessed by iontophoresis studies, correlated closely with their relative potencies in in vitro binding studies. Indeed, muscimol was nearly 3 times more effective than GABA in inhibiting both 3H-GABA binding and reticulata • cell firing (table 1). Similarly, muscimol was approximately 26 times more potent than THIP in inhibiting ~H-GABA binding and a b o u t 38 times more p o t e n t in inhibiting reticulata cell activity. Although it may be unrealistic to a t t e m p t to precisely quantitate iontophoretic responses by these techniques or to generalize the results to populations of neurons, the degree of similarity between the in vivo and in vitro measures of potency lends some support to the feasibility of the approach. When the membranes were washed extensively and frozen prior to the assay by techniques similar to those utilized in the present studies, Krogsgaard-Larsen et al. (1979a) also obtained ICs0 values for the inhibition of ~HGABA binding by THIP, GABA, and muscimol which were in the range of those reported here. Our results and their findings agree with respect to the order of p o t e n c y of the compounds as well as the magnitude of difference
28 between their potencies. In addition, Arnt et al. (1979), who have com pa r ed the in vivo potencies o f THIP and muscimol by measuring contralateral turning behavior after unilateral injection o f these drugs into the substantia nigra, have also r e p o r t e d relative differences in p o t e n c y very similar to the differences observed during iontophoresis of these agents o n to reticulata cells. R e c en t studies have suggested that THIP differs from muscimol and GABA in its ability to affect benzodiazepine binding (Karobath et al., 1979; Maurer, 1979). Although all 3 c o m p o u n d s could displace 3H-GABA or 3H-muscimol from their binding sites, THIP was unique in that it did n o t stimulate benzodiazepine binding to rat cortical membranes. These findings and others (Karobath and Sperk, 1979) suggested that different GABA r e c e p t o r sites may exist in brain with differing affinities for agonists. Since in the current studies the potencies of the 3 c o m p o u n d s in iontophoresis experiments correlated well with their potencies as inhibitors of 3H-GABA binding, our results suggest that the receptors which mediated the changes in reticulata cell firing were probably unlike those associated with the benzodiazepine binding site. Rather, the sites responsible for the changes in cell firing appear comparable to GABA binding sites in the c o r t e x (Krogsgaard-Larsen and J ohns t on, 1978) and cerebellum {present study) and the sites involved in the i o n t o p h o r e t i c effects of these c o m p o u n d s on cat spinal cord neurons (Johnston et al., 1968; Krogsgaard-Larsen et al., 1975; Krogsgaard-Larsen et al., 1977). While the i o n t o p h o r e t i c actions of these agents on reticulata cells closely paralleled their potencies in the binding studies, the systemic effects of muscimol and THIP differed somewhat from their effects when iontophoresed. Though muscimol was m or e effective than THIP in inhibiting reticulata cell firing after both i.v. administration and iontophoretic application, the margin of difference in their potencies was much less p r o n o u n c e d when the drugs were given i.v. (table 1). The
B.L. WASZCZAK ET AL. narrowing of the difference in their effectiveness following systemic administration most likely reflects a relatively greater ability of THIP to pass the blood-brain barrier. Thus, although THIP is a considerably weaker GABA agonist than muscimol in vitro and when applied directly o n t o receptive neuronal elements, its GABAmimetic p o t e n c y within the central nervous system after systemic administration appears much closer to that of muscimol. This observation, together with the possibilities t hat THIP might be less toxic than muscimol and may interact selectively at one t ype of GABA site in the brain, suggest t hat THIP might be useful as a therapeutic agent in certain clinical disorders, such as Huntington's disease and schizophrenia, which m ay benefit from enhanced GABAmediated neurotransmission.
Acknowledgements We wish to thank Dr. P. Krogsgaard-Larsen for his generous gift of the THIP used in these studies. We also gratefully acknowledge the expert technical assistance of Mr. Raymond R. Vane.
References Aghajanian, G.K. and B.S. Bunney, 1977, Dopamine 'autoreceptors': Pharmacological characterization by microiontophoretic single cell recording studies, Naunyn-Schmiedeb. Arch. Pharmacol. 297, 1. Arnt, J., J. Scheel-Kriiger, G. Magetund and P. Krogsgaard-Larsen, 1979, Muscimol and related GABA receptor agonists: The potency of GABAergic drugs in vivo determined after intranigral injection, J. Pharm. Pharmacol. 31,306~ Bunney, B.S., G.K. Aghajanian and R.H. Roth, 1973a, Comparison of effects Of L-dopa, amphetamine and apomorphine on firing rate of rat dopamineric neurons, Natur~ N~w Biol. 245, 123. Bunney, B.S., J.R. Waiters, R.H. Roth and G.K. Aghajanian, 1973b, Dopaminergic neurons: Effect of antipsychotic drugs and amphetamine on single cell activity, J. Pharmacol. Exp. Ther. 185,560. Fonnum, F., I. Grofova, E. Rinvik, J. StormMathisen and F. Walberg, 1974, Origin and distribution of glutamate decarboxylase in substantia nigra of the cat, Brain Res. 71, 77.
GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO Greenlee, D.V., P.C. Van Ness and R.W. Olsen, 1978, Gamma°aminobutyric acid binding in mammalian brain : Receptor-like specificity of sodiumindependent sites, J. Neurochem. 31,933. Guyenet, P.G. and G.K. Aghajanian, 1978, Antidromic identification of dopaminergic and other output neurons of the rat substantia nigra, Brain Res. 150, 69. Hill, R.G. and M.A. Simmonds, 1973, A method for comparing the potencies of 7oaminobutyric acid antagonists on single cortical neurons using microiontophoretic techniques, Br. J. Pharmacol. 48, 1. Johnston, G.A.R., D.R. Curtis, W.C. De Groat and A.W. Duggan, 1968, Central actions of ibotenic acid and muscimol, Biochem. Pharmacol. 17, 2488. Johnston, G.A.R., S.M.E. Kennedy and D. Lodge, 1978, Muscimol uptake, release and binding in rat brain slices, J. Neurochem. 31, 1519. Karobath, M., P. Placheta, M. Lippitsch and P. Krogsgaard-Larsen, 1979, Is stimulation of benzodiazepine receptor binding mediated by a novel GABA receptor?, Nature 278,748. Karobath, M. and G. Sperk, 1979, Stimulation of benzodiazepine receptor binding by 7-aminobutyric acid, Proc. Natl. Acad. $ci. 76, 100~. Kataoka, K., I.J. Bak, R. Hassler, J.S. Kim and A. Wagner, 1974, L-glutamate decarboxylase and choline acetyltransferase activity in the substantia nigra and the striatum after surgical interruption of the striatonigral fibers of the baboon, Exp. Brain Res. 19,217. Kim, J.S., I.J. Bak, R. Hassler and Y. Okada, 1971, Role of 7-aminobutyric acid (GABA) in the extrapyramidal motor system. 2. Some evidence for the existence of a type of GABA rich strionigral neurons, Exp. Brain Res. 14, 95. KSnig, J.F.R. and R.A. Klippel, 1970, The Rat Brain: A Stereotaxic Atlas (Kreiger, Huntington, New York). Krogsgaard-Larsen, P., H. Hjeds, D.R. Curtis, D. Lodge and G.A.R. Johnston, 1979a, Dihydromuscimol, thiomuscimol and related heterocyclic compounds as GABA analogues, J. Neurochem. 32, 1717. Krogsg~ard-Larsen, P., T. Honore and K. Thyssen, 1979b, GABA receptor agonists: Design and structure-activity studies, in: GABA-Neurotransmitters. Pharmacochemical, Biochemical and Pharmacological Aspects, eds. P. Krogsgaard-Larsen,
29
J. Scheel-Kriiger and H. Kofod (Munksgaard, Copenhagen) p. 201. Krogsgaard-Larsen, P. and G.A.R. Johnston, 1975, Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem. 25,797. Krogsgaard-Larsen, P. and G.A.R. Johnston, 1978, Structure-activity studies on the inhibition of GABA binding to rat brain membranes by muscimol and related compounds, J. Neurochem. 30, 1377. Krogsgaard-Larsen, P., G.A.R. Johnston, D.R. Curtis, C.J.A. Game and R.M. McCulloch, 1975, Structure and biological activity of a series of conformationally restricted analogues of GABA, J. Neurochem. 25,803. Krogsgaard-Larsen, P., G.A.R. Johnston, D. Lodge and D.R. Curtis, 1977, A new class of GABA agonist, Nature 268, 53. Lodge, D., D.R. Curtis and G.A.R. Johnston, 1978, Does uptake limit action of GABA agonists in vivo? Experiments with muscimol, isoguvacine and THIP in cat spinal cord, J. Neurochem. 31, 1525. Maurer, R., 1979, The GABA agonist THIP, a muscimol analogue, does not interfere with the benzoo diazepine binding site on rats cortical membranes, Neurosci. Letters 12, 65. Ribak, E.C., J.E. Vaughn, K. Saito, R. Barber and E. Roberts, 1976, Immunocytochemical localization of glutamate decarboxylase in rat substantia nigra, Brain Res. 116,287. Smith, B.M. and B.J. Hoffer, 1978, A gated, high voltage iontophoresis system with accurate current monitoring, Electroenceph. Clin. Neurophysiol. 44,398. Tasaki, K., U. Tsukahara, S. Ito, M.J. Wayner and W.Y. Yu, 1968, A simple, direct and rapid method for filling microelectrodes, Physiol. Behav. 3, 1009. Waszczak, B.L., N. Eng and J.R. Walters, 1980, Effects of muscimoi and picrotoxin on single unit activity of substantia nigra neurons, Brain Res. 188,185. Waszczak, B.L., J.M. Lakoski and J.R. Walters, 1978, Effects of muscimol upon the activity of substantia nigra pars reticulata neurons, Soc. Neurosci. Abstr. 4 , 4 3 6 . Zukin, S.R., A.B. Young and S.H. Snyder, 1974, Gamma-aminobutyric acid binding to receptor sites in the rat central nervous system, Proc. Nat. Acad. Sci. U.S.A. 71, 4802.
21
GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO: A COMPARISON WITH MUSCIMOL AND GABA BARBARA L. WASZCZAK *, ROBERT E. HRUSKA and JUDITH R. WALTERS Experimental Therapeutics Branch, Physiological and Behavioral Neuropharmacology Units, National Institute o f Neurological and Communicative Disorders and Stroke, 9000 Rockville Pike, Bethesda, Maryland 20205, U.S.A.
Received 28 September 1979, revised MS received 7 January 1980, accepted 9 April 1980
B.L. WASZCZAK, R.E. HRUSKA and J.R. WALTERS, GABAergic actions o f THIP in vivo and in vitro: a comparison with muscimol and GABA, European J. Pharmacol. 65 (1980) 21--29. GABAmimetic actions of 4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP) were evaluated both in vivo and in vitro and compared with the actions of muscimol and GABA. The GABAergic potencies of these agents were assessed in vivo by measuring their ability to inhibit firing of rat substantia nigra pars reticulata neurons after systemic administration and iontophoretic applications, and in vitro by measuring their ability to inhibit 3H-GABA binding to rat cerebellar membranes. The effects of THIP were found to be similar to those of muscimol and GABA with regard to inhibition of reticulata cell firing and 3H-GABA binding. The order of potency was muscimol > GABA > THIP. The magnitude of the differences between drug potencies in iontophoretic studies closely paralleled their relative potencies in binding studies, with muscimol approximately 3 times more potent than GABA and 25-40 times more potent than THIP. After systemic (i.v.) administration, however, muscimot was only 3 times more potent than THIP in inhibiting reticulata cell firing, possibly because THIP passes the blood-brain barrier more readily. THIP
Muscimol
GABA
Single unit recording
1. I n t r o d u c t i o n T h e r e c e n t d e v e l o p m e n t o f a series o f 7 - a m i n o b u t y r i c acid ( G A B A ) a n a l o g u e s m a k e s it possible to e x p l o r e w h e t h e r or n o t v a r i o u s G A B A r e c e p t i v e sites possess d i f f e r e n t c o n f o r m a t i o n a l characteristics. T h e q u e s t i o n o f whether different conformations of GABA are p r e f e r r e d at d i f f e r e n t sites was raised b y several studies w h i c h revealed t h a t G A B A m i m e t i c drugs d i f f e r e d in t h e i r relative affinities a t G A B A u p t a k e sites, t r a n s p o r t sites ( K r o g s g a a r d - L a r s e n a n d J o h n s t o n , 1 9 7 5 ; J o h n s t o n et al., 1 9 7 8 ; L o d g e et al., 1978), sites labeled in G A B A r e c e p t o r b i n d i n g * Send correspondence to: Barbara Waszczak, Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, 9000 Rockville Pike, Bldg. 36, Rm. 5A31, Bethesda, Maryland 20205, U.S.A.
Microiontophoresis
Substantia nigra
studies ( K r o g s g a a r d - L a r s e n a n d J o h n s t o n , 1 9 7 8 ; K r o g s g a a r d - L a r s e n et al., 1 9 7 9 a ) , a n d sites m e d i a t i n g i o n t o p h o r e t i c a c t i o n s o f these compounds o n c a t spinal c o r d n e u r o n s { J o h n s t o n et al., 1 9 6 8 ; K r o g s g a a r d - L a r s e n et al., 1 9 7 5 ; K r o g s g a a r d - L a r s e n e t al., 1977). T h e rigid i s o x a z o l e derivatives o f G A B A n o w b e c o m i n g available s h o w p r o m i s e as pharmacologic tools for better characterizing a n d distinguishing s o m e o f these GABA r e c e p t i v e sites. O n e such c o m p o u n d , 4,5,6,7t e t r a h y d r o i s o x a z o l o - [ 5,4-c ]-pyridin-3-ol ( T H I P ) , a rigid, bicyclic i s o x a z o l e derivative o f G A B A , has r e c e n t l y b e e n distinguished from both GABA and muscimol by virtue of its greater relative s p e c i f i c i t y f o r i n h i b i t i n g 3H-GABA binding than for stimulating b e n z o d i a z e p i n e b i n d i n g ( K a r o b a t h e t al., 1 9 7 9 ; Maurer, 1979). In a d d i t i o n , it has b e e n suggested t h a t T H I P m i g h t be m o r e suited
22 than muscimol for use as a systemic GABAmimetic agent because it appears to penetrate the blood-brain barrier more readily than muscimol, it may be more stable in vivo, and it is less toxic than muscimol after systemic administration (Krogsgaard-Larsen et al., 1979b). Considering these potential advantages, the following studies were undertaken to determine whether THIP would act at GABA receptors within the central nervous system in a manner comparable to muscimol, and to compare and correlate t h e potencies of THIP, GABA, and muscimol both in vivo and in vitro. Accordingly, the effects of these agents upon the firing of substantia nigra pars reticulata neurons were studied in vivo, after both systemic (i.v.) administration and during iontophoretic application. In addition, the ability of each c o m p o u n d to inhibit brain 3H-GABA binding in vitro was determined.
2. Materials and methods
2.1. Single unit recording and iontophoresis techniques Male, Sprague-Dawley rats (Zivic-Miller Co., Allison Park, PA}, weighing 250-300 g, were anesthetized with chloral hydrate, 400 mg/kg, i.p., and m o u n t e d in a stereotaxic apparatus. Additional injections of chloral hydrate were administered as needed through a tail vein. Body temperature was maintained t h r o u g h o u t the recording period between 3638°C. Single unit recording and iontophoresis experiments were conducted according to standard techniques (Bunney et al., 1973b; Aghajanian and Bunney, 1977; Smith and Hoffer, 1978}. A small (3 mm) burr hole was drilled in the skull 2.0 m m lateral to lambda and 3.0 mm anterior to the lambdoid suture. An electrode was passed through the hole to the level of the substantia nigra with a hydraulic microdrive. Electrical signals were displayed on a oscilloscope screen and passed into a rate meter set to c o u n t pulses which
B.L. WASZCZAKET AL. fell within the selected amplitude time gates. Counts of unit activity were summed over either successive 5 or 10 sec intervals. Histogram plots of the rate of cell firing were produced by a strip chart recorder, and digital printouts of the number of counts per 5 or 10 sec period were also obtained. Single barrel electrodes, prepared according to techniques previously described (Tasaki et al., 1968), were used in recording extracellular, single unit activity of pars reticulata cells during cumulative (i.v.) dose-response studies. The electrodes were broken back to a tip diameter of approximately 1-2 t~m and were filled with 1% Pontamine Sky Blue (GURR, High Wycombe, Bucks, England) in 2 M N a C 1 . Electrode resistances varied between 3.8 and 6.0 megohms (measured at 135 Hz). A 5 min period of baseline activity was recorded before the first drug injection. Muscimol or THIP was then administered, i.v., through a tail vein. Successive doses were given at 2 min intervals so that the cumulative dose after each injection was twice the cumulative dose before the injection (i.e. individual doses of 0.05, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 mg/kg result in cumulative doses of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 mg/kg, respectively). The percent change from the baseline firing rate was calculated by comparing the average firing rate over successive 10 sec intervals during the 2 min period after each dose with the average baseline rate before drug. Only one cell per rat was monitored in studies in which drugs were administered systemically. Cumulative log dose-response curves were constructed, and EDs0 values for the inhibition of reticulata cell firing by muscimol and THIP were calculated for individual experiments by least squares linear regression analysis of probit values versus the log dose. The average EDs0 values for the two drugs were calculated and compared. In iontophoretic studies, 5-barrel micropipettes were used for both recording and ejecting drugs. Pipette tips were broken back to diameter of 4-5 ~m. The central barrel,
GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO used for recording, was filled with 1% Pontamine Blue solution (above). Side barrels contained: (1) THIP, 0.01 M in 0.2 M NaC1, pH 4.0; (2) GABA, 0.01 M in 0.2 M NaC1, pH 4.0; (3) muscimol, 0.01 M in 0.2 M NaC1, pH 4.0. The remaining side channel was filled with 4 M NaC1 and served as a balance channel. The resistance of the recording channel ranged between 3.5 and 8 megohms; resistances of the side channels averaged between 40 and 100 megohms. During periods of baseline activity, a negative retaining current of 10 nA was passed through each of the drug channels. A drug was iontophoresed from a particular channel by passing through it a positive ejecting current of appropriate magnitude to produce at least a 50% inhibition of cell firing within 60 sec. Results obtained with iontophoresis techniques are essentially of a qualitative nature. However, in order to compare the potencies of these drugs when iontophoresed with their potencies after systemic administration and in receptor binding studies, an a t t e m p t was made to quantify the iontophoretic responses. It has been assumed that during the early phase of iontophoresis of GABA and GABA agonists the rate of release increases approximately exponentially with time and is proportional to the current (I) applied (Hill and Simmonds, 1973). On the basis of this assumption ITs0 values were calculated for each of the 3 drugs and were used as an estimate of the iontophoretic potencies of the compounds tested. Ts0 values represent the time required to inhibit firing to 50% of the baseline rate. For each cell, the Ts0 value at a given current was derived from plots of the percent inhibition of firing vs time (in 5 sec intervals, plotted exponentially) after the ejecting current was turned on. In instances where more than one iontophoretic application of a drug was carried out, the mean ITs0 value for that drug was considered as a single observation. Average ITs0 values for each drug were calculated and compared. It should be understood, however, that differences in the transport numbers of the compounds studied
23
and factors such as local uptake and metabolism of the ejected agents, especially GABA, could represent a source of error in these studies. Frequently, more than one cell per rat was monitored in iontophoresis experiments.
2.2. Identification of cells The cells recorded in these studies were located in the pars reticulata region of the substantia nigra. Reticulata neurons could be tentatively identified during the recording period on the basis of their location and electrophysiological characteristics. All cells studied were located within the stereotaxic coordinates anterior, 1760-2580 t~m; lateral, 1.7-2.5 mm; ventral, --1.5 to --2.5 mm, according to KSnig and Klippel (1970). Since the pars reticulata lies ventral to the pars compacta region of the substantia nigra, reticulata cells were typically encountered after passing through a zone of pars compacta (dopamine) neurons whose firing rate, extracellular action potential shape and duration are characteristic, easily recognized, and well documented (Aghajanian and Bunney, 1977; Bunney et al., 1973a,b, G u y e n e t and Aghajanian, 1978; Waszczak et al., 1980). The electrophysiologic properties of the pars reticulata cells have also been previously described (Guyenet and Aghajanian, 1978). These neurons have firing rates ranging between 10 and 40 spikes/sec. Their extracellular action potentials are smooth, sharp, and biphasic with a duration of 0.5 to 0.7 msec (mean duration, 0.63 ± 0.02 msec, n = 20). In order to verify placement of the electrode within the pars reticulata, the location of the recording site was marked at the end of the experiment by passing a 10-20 nA current through the electrode for approximately 15 min. This caused a small a m o u n t of the Pontamine Blue dye to be deposited at the electrode tip. Rats were then perfused with 10% buffered formalin phosphate, pH 7.0 (Fisher Scientific Co., Fairlawn, NJ). Brains were removed, sectioned, m o u n t e d , and
24
B.L. WASZCZAK ET AL.
stained. The location of the blue spot identified the recording site.
2.3. Receptor binding techniques Neurotransmitter receptor binding of ~HGABA was measured by standard procedures {Zukin et al., 1974; Greenlee et al., 1978). The sodium-independent binding was assayed in rat cerebellum, obtained from SpragueDawley rats after sacrifice and rapid removal of the brain into ice-cold saline. The cerebellum was dissected, weighed, and homogenized with a Polytron in 20 volumes of 50 mM Triscitrate buffer (pH 7.1 at 4°C). The suspension was centrifuged at 50 000 × g for 10 min, the resultant supernatant discarded, and the homogenization and washing procedure
repeated twice. The final homogenate was frozen overnight, thawed, and washed 3 times again before use in the 3H-GABA assay. The specific binding was determined by subtracting from the total binding the nonspecific binding obtained in the presence of 1 mM unlabeled GABA. Under our conditions the specific binding of 3H-GABA was 122 -+ 15 fmole/mg protein using 4.8 + 0.8 nM ~H-GABA. Inhibition curves were obtained by adding increasing concentrations of GABA, muscimol, or THIP. The slope and ICs0 values (concentration required to inhibit 50% of the binding) were estimated by linear regression analyses of Hill plots for each experiment. The mean ICs0 values for 3 experiments were compared.
S.N. pars reticulata
MUSC
PIC
¢,,,,,,
'°:1
'
¢
................................
I
o @
THIP
PIC
,
,
3oo1 0-~ !
!
5 min Fig. 1. Effects of systemic muscimol and THIP upon single unit activity of substantia nigra pars reticulata neurons. Top panel: Effect of increasing i.v. doses of muscimol (MUSC) administered at 2 min intervals so that each injection doubled the previously administered cumulative dose (i.e. 0.05, 0.05, 0.1-, 0.2, 0.4, 0.8, 1.6 mg/kg; total cumulative dose, 3.2 mg/kg). Inhibitory effect of muscimol was reversed by picrotoxin (PIC 4.5 mg/kg, i.v.). Bottom panel: Effect of cumulative (see above) i.v. doses of THIP (0.2, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6 mg/kg; total dose, 51.2 mg/kg). Picrotoxin (PIC, 5.0 mg/kg i.v. ) reversed the inhibition.
G A B A E R G I C A C T I O N S O F THIP IN V I V O A N D IN V I T R O
25
2.4. Drugs Chloral hydrate was obtained from Sigma Chemical Co., St. Louis, MO; 7-aminobutyric acid (GABA) from Sigma Chemical Co., St. Louis, MO; muscimol from Regis Chemical Co., Morton Grove, IL; and 4,5,6,7-tetrahydroisoxazolo-[ 5,4-c]-pyridin-3-ol (THIP) from Dr. P. Krogsgaard-Larsen, Royal Danish School of Pharmacy, Department of Chemistry BC, Copenhagen, Denmark.
~ 70'
3. Results
3.1. Effects of systemic administration of THIP and muscimol upon reticulata cell unit activity When administered in a series of increasing i.v. doses, THIP produced dose-dependent decreases in the firing rates of substantia nigra pars reticulata neurons (fig. 1). The responses o f reticulata cells to i.v. THIP were qualitatively similar to their responses to i.v. muscimol, although muscimol was the more p o t e n t inhibitor. THIP produced its first significant decrease in firing after a total dose of 6.4 mg/kg (P < 0.005), while only 1.6 mg/kg muscimol were required to significantly inhibit firing (P < 0.05). All cells studied were completely inhibited at a total cumulative dose of 12.8 mg/kg muscimol, whereas firing was almost totally inhibited (4.4% of baseline) at 51.2 mg/kg and completely inhibited at 102.4 mg/kg THIP. The inhibitory actions of both drugs were reversible by subsequent i.v. administration of 4-5 mg/kg picrotoxin, a GABA antagonist (fig. 1). Cumulative log dose-response curves comparing the effects of THIP and muscimol upon the firing of reticulata neurons are shown in fig. 2. The calculated dose required to produce a 50% inhibition of firing (EDs0) was 14.4 ± 4.2 mg/kg for THIP and 5.1 -+ 1.0 mg/kg for muscimol (table 1). Thus, systemically administered THIP was approximately 3 times less p o t e n t than muscimol
0
0~3 ~.i 0:2 0.~4 018 1~6 3:2 ~4 12.8 25.6 51.2 102,4 mg/kg DOSE(log scale}
Fig. 2. Cumulative log d o s e - r e s p o n s e curves o f t h e e f f e c t s o f i.v. THIP (~. A; n = 9) and m u s c i m o ! (~ e; n = 10) u p o n the single unit activity o f s u b s t a n t i a nigra pars reticulata n e u r o n s . Drugs were a d m i n i s t e r e d i.v. in increasing i n c r e m e n t s so t h a t each dose d o u b l e d t h e previous cumulative dose. Average firing ra~e o f each cell a f t e r each dose was e x p r e s s e d as a p e r c e n t o f the baseline rate. Points r e p r e s e n t t h e average r e s p o n s e o f all cells a f t e r t h a t dose. Vertical bars r e p r e s e n t s t a n d a r d errors o f t h e m e a n s (S.E.M.). One cell was m o n i t o r e d per rat.
(P < 0.05) in inhibiting single unit activity of substantia nigra pars reticulata cells.
3.2. Effects o f microiontophoresis o f THIP, GABA, and muscimol upon activity of pars reticulata neurons THIP, like muscimol and GABA, was able to depress reticulata firing when iontophoretically applied. Equimolar solutions of THIP, GABA, and muscimol were iontophoresed in turn onto individual reticulata cells until a current was selected for each drug which caused at least a 50% inhibition of firing within i min (fig. 3). In some instances complete inhibitory responses to GABA and/or muscimol occurred immediately after the ejecting current was turned on, even at currents as low as 2.5 nA. In order to achieve a more gradual rate of inhibition, the nega-
26
B.L. WASZCZAK ET AL.
TABLE 1 Potencies of THIP, GABA, and muscimol (MUSC)were evaluated by comparing: (1) EDs0 values for inhibition of reticulata cell firing after cumulative i.v. administration of drugs, (2) ITs0 values (see text) for inhibition of cell firing during iontophoresis of THIP (n = 7), GABA (n = 7) and MUSC (n = 7), and (3) ICs0 values for inhibition of aH-GABA binding to rat cerebellar membranes by each compound. Potencies are also expressed relative to that of MUSC, the most effective inhibitor in all cases.
THIP GABA Muscimol
EDs0 (mg/kg)
EDs0 EDs0 (MUSC)
ITs0 (hA . sec)
ITs0 ITs0 (MUSC)
14.4_+4.21 -5.1 -+ 1.0
2.9 -1
98.2 -+ 43.91 6 . 5 + 3.0 2.6 -+ 1.4
37.8 2.5 1
ICs0 (M)
ICs0 ICs0 (MUSC)
2 6 . 4 _ + 1 . 1 x 10 -7 I 6.6_+1.5x 10 -8 2.4 -+ 0.5 × 10 -s
26.7 2.8 1
1 Significantly different from muscimol, P < 0.05 2 Significantly different from muscimol, P < 0.005.
tive retaining current through that barrel was turned off to permit only the passive leakage of drug from the pipette tip. In c a l c u l a t i o n s o f ITs0 v a l u e s in s u c h i n s t a n c e s , a value of 0.1 nA current was used. A comp a r i s o n o f t h e m e a n ITs0 v a l u e s f o r t h e 3 compounds studied revealed that the order of potency for inhibition of reticulata cell firing was muscimol > GABA > THIP ( t a b l e 1). W h i l e a p p r o x i m a t e in n a t u r e , t h e s e values suggest that muscimol was between 2
THIP lOnA
GABA 5nA
5hA
OnA
and 3 times more potent than GABA and between 35 and 40 times more potent than THIP.
3.3. Inhibition o f 3H-GABA THIP, GABA, and muscimol
by
I n h i b i t i o n c u r v e s f o r t h e i n h i b i t i o n o f 3HGABA binding to rat cerebellar membranes b y T H I P , G A B A , a n d i n u s c i m o l a r e s h o w n in fig. 4. T h e ICso v a l u e s f o r e a c h c o m p o u n d ,
120~
MUSC 2.SnA
binding
OnA
~
100
•
•
~.
~OOl
I
!
,
5 rain
!
Fig. 3. Inhibition of pars reticulata cell firing by iontophoretieally applied THIP, GABA, and museimol (MUSC). Equimolar solutions (0.01 M in 0.2 M NaC1) of the drugs were ejected from a 5-barrel micropipette at currents suffielent to produce at least a 50% inhibition of firing within one rain. Bars represent the duration of the designated ejection current. Ordinate: spikes/5 sec. Calibration bar: 5 rain.
...
~
~ I~1 ~
~0
~
~0
m
~~ 0 ~ 0
-11
Mosc,MoL\
-1~
_;
_;
,
,
-7 -6 LOG [INHIBITOR], M
-5
-4
Fi~. 4. I n h i b i t i o n o f s o d i u m - i n d e p e n d e n t b i n d i n g o f 3 H - G A B A t o rat cerebe]lar m e m b r a n e s b y T H e ,
G A B A , and museimo]. Points p r e ~ n t o f 3 experiments.
the average
GABAERGIC ACTIONS OF THIP IN VIVO AND IN VITRO
estimated from the Hill plots of the data, are compared in table 1. Muscimol was the most p o t e n t inhibitor. GABA and THIP were 2.8 and 27 times less potent, respectively, than muscimol. The Hill slopes for GABA, muscimol, and THIP were quite similar {--0.872, --0.895 and --0.787, respectively). The similarity of the Hill slopes suggests homogeneity of the binding sites.
4. Discussion Previous studies have demonstrated that muscimol, administered systematically, and both GABA and muscimol, applied iontophoretically, could completely inhibit the firing of rat substantia nigra pars reticulata neurons (Waszczak et al., 1978; Waszczak et al., 1980). Since the substantia nigra is the termination site of a major GABAergic projection from the striatum and globus pallidus ( F o n n u m et al., 1974; Kataoka et al., 1974; Kim et al., 1971; Ribak et al., 1976), the sensitivity of the reticulata neurons to exogenous GABA may parallel a normal sensitivity of these cells to endogenously released GABA. Thus, the inhibitory response of nigral pars reticulata cells to GABAmimetic agents was viewed as a physiologically relevant test system for studying the GABAmimetic actions of THIP in the central nervous system and for comparing the potencies of THIP, GABA, and muscimol in vivo. Furthermore, by determining the effects of these agents on cell firing after i.v. administration and iontophoretic application it was possible to compare the difference between their direct actions and their effectiveness after systemic administration. These studies demonstrate that i.v. administration of THIP, like muscimol, results in dose-related decreases in the firing rates of substantia nigra pars reticulata neurons. The inhibition was reversible by i.v. administration of the GABA antagonist, picrotoxin. THIP also markedly inhibited reticulata cell firing when applied iontophoretically. Although the
27
effects of THIP, both systemic and iontophoretic, occurred at doses higher than those required for muscimol, the nature of the inhibitory responses to both drugs and the reversibility by a GABA antagonist were similar. A comparison of the effects of these drugs reveals that muscimol exhibits the greatest p o t e n c y for inhibiting reticulata cell firing and 3H-GABA binding. Results of iontophoresis and binding studies, in which GABA was included, indicate that GABA was second in order of potency and THIP was the least p o t e n t of the 3 agents. The effects of systemic administration of GABA were not considered because rapid neuronal and glial uptake of that fraction of GABA which crosses the blood-brain barrier apparently prevents most or all of the drug from reaching reticulata neurons. The relative potencies of these agents in vivo, as assessed by iontophoresis studies, correlated closely with their relative potencies in in vitro binding studies. Indeed, muscimol was nearly 3 times more effective than GABA in inhibiting both 3H-GABA binding and reticulata • cell firing (table 1). Similarly, muscimol was approximately 26 times more potent than THIP in inhibiting ~H-GABA binding and a b o u t 38 times more p o t e n t in inhibiting reticulata cell activity. Although it may be unrealistic to a t t e m p t to precisely quantitate iontophoretic responses by these techniques or to generalize the results to populations of neurons, the degree of similarity between the in vivo and in vitro measures of potency lends some support to the feasibility of the approach. When the membranes were washed extensively and frozen prior to the assay by techniques similar to those utilized in the present studies, Krogsgaard-Larsen et al. (1979a) also obtained ICs0 values for the inhibition of ~HGABA binding by THIP, GABA, and muscimol which were in the range of those reported here. Our results and their findings agree with respect to the order of p o t e n c y of the compounds as well as the magnitude of difference
28 between their potencies. In addition, Arnt et al. (1979), who have com pa r ed the in vivo potencies o f THIP and muscimol by measuring contralateral turning behavior after unilateral injection o f these drugs into the substantia nigra, have also r e p o r t e d relative differences in p o t e n c y very similar to the differences observed during iontophoresis of these agents o n to reticulata cells. R e c en t studies have suggested that THIP differs from muscimol and GABA in its ability to affect benzodiazepine binding (Karobath et al., 1979; Maurer, 1979). Although all 3 c o m p o u n d s could displace 3H-GABA or 3H-muscimol from their binding sites, THIP was unique in that it did n o t stimulate benzodiazepine binding to rat cortical membranes. These findings and others (Karobath and Sperk, 1979) suggested that different GABA r e c e p t o r sites may exist in brain with differing affinities for agonists. Since in the current studies the potencies of the 3 c o m p o u n d s in iontophoresis experiments correlated well with their potencies as inhibitors of 3H-GABA binding, our results suggest that the receptors which mediated the changes in reticulata cell firing were probably unlike those associated with the benzodiazepine binding site. Rather, the sites responsible for the changes in cell firing appear comparable to GABA binding sites in the c o r t e x (Krogsgaard-Larsen and J ohns t on, 1978) and cerebellum {present study) and the sites involved in the i o n t o p h o r e t i c effects of these c o m p o u n d s on cat spinal cord neurons (Johnston et al., 1968; Krogsgaard-Larsen et al., 1975; Krogsgaard-Larsen et al., 1977). While the i o n t o p h o r e t i c actions of these agents on reticulata cells closely paralleled their potencies in the binding studies, the systemic effects of muscimol and THIP differed somewhat from their effects when iontophoresed. Though muscimol was m or e effective than THIP in inhibiting reticulata cell firing after both i.v. administration and iontophoretic application, the margin of difference in their potencies was much less p r o n o u n c e d when the drugs were given i.v. (table 1). The
B.L. WASZCZAK ET AL. narrowing of the difference in their effectiveness following systemic administration most likely reflects a relatively greater ability of THIP to pass the blood-brain barrier. Thus, although THIP is a considerably weaker GABA agonist than muscimol in vitro and when applied directly o n t o receptive neuronal elements, its GABAmimetic p o t e n c y within the central nervous system after systemic administration appears much closer to that of muscimol. This observation, together with the possibilities t hat THIP might be less toxic than muscimol and may interact selectively at one t ype of GABA site in the brain, suggest t hat THIP might be useful as a therapeutic agent in certain clinical disorders, such as Huntington's disease and schizophrenia, which m ay benefit from enhanced GABAmediated neurotransmission.
Acknowledgements We wish to thank Dr. P. Krogsgaard-Larsen for his generous gift of the THIP used in these studies. We also gratefully acknowledge the expert technical assistance of Mr. Raymond R. Vane.
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